Patent application title: T CELL ACTIVATING BISPECIFIC ANTIGEN BINDING MOLECULES
Inventors:
IPC8 Class: AC07K1646FI
USPC Class:
1 1
Class name:
Publication date: 2016-07-21
Patent application number: 20160208019
Abstract:
The present invention generally relates to novel bispecific antigen
binding molecules for T cell activation and re-direction to specific
target cells. In addition, the present invention relates to
polynucleotides encoding such bispecific antigen binding molecules, and
vectors and host cells comprising such polynucleotides. The invention
further relates to methods for producing the bispecific antigen binding
molecules of the invention, and to methods of using these bispecific
antigen binding molecules in the treatment of disease.Claims:
1. A T cell activating bispecific antigen binding molecule comprising (i)
a first antigen binding moiety which is a Fab molecule capable of
specific binding to CD3, and which comprises at least one heavy chain
complementarity determining region (CDR) amino acid sequence selected
from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:
39 and at least one light chain CDR selected from the group of SEQ ID NO:
32, SEQ ID NO: 33, SEQ ID NO: 34; (ii) a second antigen binding moiety
capable of specific binding to Folate Receptor 1 (FolR1).
2. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first antigen binding moiety comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
3. The T cell activating bispecific antigen binding molecule of claim 1 or 2, additionally comprising (i) a third antigen binding moiety capable of specific binding to FolR1.
4. The T cell activating bispecific antigen binding molecule of claim 3, wherein the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences.
5. The T cell activating bispecific antigen binding molecule of claim 4, wherein the third antigen binding moiety is identical to the second antigen binding moiety.
6. The T cell activating bispecific antigen binding molecule of claim 3, wherein at least one of the second and third antigen binding moiety is a Fab molecule.
7. The T cell activating bispecific antigen binding molecule of claim 1, additionally comprising (i) an Fc domain composed of a first and a second subunit capable of stable association.
8. The T cell activating bispecific antigen binding molecule of claim 7, wherein the first antigen binding moiety and the second antigen binding moiety are each connected at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
9. The T cell activating bispecific antigen binding molecule of claim 7 or 8, wherein a third antigen binding moiety is connected at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety via a peptide linker.
10.-21. (canceled)
22. The T cell activating bispecific antigen binding molecule of claim 1, which binds to a human FolR1, a cynomolgus monkey FolR1 and a murine FolR1.
23. The T cell activating bispecific antigen binding molecule of claim 1, which binds to a human FolR1 and a cynomolgus monkey FolR1 and not a murine FolR1.
24. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
25. The T cell activating bispecific antigen binding molecule of claim 1, comprising not more than one antigen binding moiety capable of specific binding to CD3.
26. The T cell activating bispecific antigen binding molecule of claim 7, wherein the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule.
27. The T cell activating bispecific antigen binding molecule of claim 26, wherein the Fc domain is an IgG class immunoglobulin.
28. The T cell activating bispecific antigen binding molecule of claim 27, wherein the Fc domain is a human IgG.sub.1 or IgG.sub.4 Fc domain.
29. The T cell activating bispecific antigen binding molecule of claim 7 or 28, wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
30. The T cell activating bispecific antigen binding molecule of claim 29, wherein in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
31. The T cell activating bispecific antigen binding molecule of claim 7, wherein the Fc domain comprises at least one amino acid substitution that reduces binding to an Fc receptor and/or effector function, as compared to a native IgG.sub.1 Fc domain.
32. The T cell activating bispecific antigen binding molecule of claim 31, wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G (Kabat numbering).
33. (canceled)
34. The T cell activating bispecific antigen binding molecule of claim 31, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
35. The T cell activating bispecific antigen binding molecule of claim 1, wherein the T cell activating bispecific antigen binding molecule induces at least one of (a) proliferation of a human CD3 positive T cell in vitro; (b) human peripheral blood mononuclear cell mediated killing of a FolR1-expressing human tumor cell in vitro; (c) T cell-mediated killing of a FolR1-expressing human tumor cell in vitro; and (d) upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry.
36.-37. (canceled)
38. The T cell activating bispecific antigen binding molecule of claim 35, wherein the FolR1-expressing human tumor cell is a Hela, Skov-3, or a cell.
39. The T cell activating bispecific antigen binding molecule of claim 35 or 38, wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing human tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours.
40. The T cell activating bispecific antigen binding molecule of claim 39, wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours.
41. The T cell activating bispecific antigen binding molecule of claim 39, wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours.
42. The T cell activating bispecific antigen binding molecule of claim 39, wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours.
43. (canceled)
44. The T cell activating bispecific antigen binding molecule of claim 35, wherein the T cell is a CD4 positive T cell or a CD8 positive T cell.
45. The T cell activating bispecific antigen binding molecule of claim 1, wherein the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K.sub.D of about 5.36 pM to about 4 nM.
46. The T cell activating bispecific antigen binding molecule of claim 1, wherein the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K.sub.D of about 4 nM.
47. (canceled)
48. The T cell activating bispecific antigen binding molecule of claim 1, wherein the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of at least about 1000 nM.
49. (canceled)
50. The T cell activating bispecific antigen binding molecule of claim 1, wherein the T cell activating bispecific antigen binding molecule binds to a conformational epitope of a human FolR1 expressed on a human tumor cell.
51. The T cell activating bispecific antigen binding molecule of claim 1, wherein the T cell activating bispecific antigen binding molecule does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3).
52. The T cell activating bispecific antigen binding molecule of claim 1, wherein the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227).
53. The T cell activating bispecific antigen binding molecule of claim 1, wherein the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 and 229.
54.-103. (canceled)
104. An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of claim 1 or a fragment thereof.
105.-109. (canceled)
110. A polypeptide encoded by the polynucleotide of claim 104.
111. A vector comprising the polynucleotide of claim 104.
112. A host cell comprising the polynucleotide of claim 104 or the vector of claim 111.
113. A method of producing the T cell activating bispecific antigen binding molecule capable of specific binding to CD3 and a target cell antigen, comprising the steps of a) culturing the host cell of claim 112 under conditions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule.
114. A T cell activating bispecific antigen binding molecule produced by the method of claim 113.
115. A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of claim 1 and a pharmaceutically acceptable carrier.
116.-119. (canceled)
120. A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of claim 1 in a pharmaceutically acceptable form.
121. The method of claim 120, wherein said disease is cancer.
122. A method for inducing lysis of a FolR1.sup.+ target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of claim 1 in the presence of a CD3.sup.+ T cell.
123. (canceled)
Description:
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 12, 2015, is named P32186-US-Seq-Listing.txt and is 445,556 bytes in size.
CROSS REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to European Patent Application No. 14194147.6, filed on Nov. 20, 2014, the entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention generally relates to bispecific antigen binding molecules for activating T cells, in particular to bispecific antibodies targeting CD3 and Folate Receptor 1 (FolR1). In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.
BACKGROUND
[0004] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged.
[0005] An attractive way of achieving this is by inducing an immune response against the tumor, to make immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells. CTLs constitute the most potent effector cells of the immune system, however they cannot be activated by the effector mechanism mediated by the Fc domain of conventional therapeutic antibodies.
[0006] In this regard, bispecific antibodies designed to bind with one "arm" to a surface antigen on target cells, and with the second "arm" to an activating, invariant component of the T cell receptor (TCR) complex, have become of interest in recent years. The simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Hence, the immune response is re-directed to the target cells and is independent of peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs. In this context it is crucial that CTLs are only activated when a target cell is presenting the bispecific antibody to them, i.e. the immunological synapse is mimicked. Particularly desirable are bispecific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells.
[0007] FOLR1 is expressed on epithelial tumor cells of various origins, e.g., ovarian cancer, lung cancer, breast cancer, renal cancer, colorectal cancer, endometrial cancer. Several approaches to target FOLR1 with therapeutic antibodies, such as farletuzumab, antibody drug conjugates, or adoptive T cell therapy for imaging of tumors have been described (Kandalaft et al., J Transl Med. 2012 Aug. 3; 10:157. doi: 10.1186/1479-5876-10-157; van Dam et al., Nat Med. 2011 Sep. 18; 17(10):1315-9. doi: 10.1038/nm.2472; Clifton et al., Hum Vaccin. 2011 February; 7(2):183-90. Epub 2011 Feb. 1; Kelemen et al., Int J Cancer. 2006 Jul. 15; 119(2):243-50; Vaitilingam et al., J Nucl Med. 2012 July; 53(7); Teng et al., 2012 August; 9(8):901-8. doi: 10.1517/17425247.2012.694863. Epub 2012 Jun. 5. Some attempts have been made to target folate receptor-positive tumors with constructs that target the folate receptor and CD3 (Kranz et al., Proc Natl Acad Sci USA. Sep. 26, 1995; 92(20): 9057-9061; Roy et al., Adv Drug Deliv Rev. 2004 Apr. 29; 56(8):1219-31; Huiting Cui et al Biol Chem. Aug. 17, 2012; 287(34): 28206-28214; Lamers et al., Int. J. Cancer. 60(4):450 (1995); Thompson et al., MAbs. 2009 July-August; 1(4):348-56. Epub 2009 Jul. 19; Mezzanzanca et al., Int. J. Cancer, 41, 609-615 (1988). However, the approaches taken so far have many disadvantages. The molecules used thus far could not be readily and reliably produced as they require chemical cross linking. Similarly, hybrid molecules cannot be produced at large scale as human proteins and require the use of rat, murine or other proteins that are highly immunogenic when administered to humans and, thus, of limited therapeutic value. Further, many of the existing molecules retained FcgR binding.
[0008] Thus, there remains a need for novel, improved bispecific antibodies for targeted T cell mediated immunotherapy. The present invention provides bispecific antigen binding molecules designed for targeted T cell activation, particularly, bispecific antigen binding molecules suitable as effective and safe therapeutics that can be readily produced and dosed.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides a T cell activating bispecific antigen binding molecule comprising
[0010] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34; and
[0011] (ii) a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1).
[0012] In one embodiment, the T cell activating bispecific antigen binding molecule comprises a first antigen binding moiety that comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31. In one embodiment, the T cell activating bispecific antigen binding molecule additionally comprises (iii) a third antigen binding moiety capable of specific binding to FolR1. In one embodiment, the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one embodiment, the third antigen binding moiety is identical to the second antigen binding moiety. In one embodiment of the T cell activating bispecific antigen binding molecule of the above embodiments, at least one of the second and third antigen binding moiety is a Fab molecule. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments, additionally comprises an Fc domain composed of a first and a second subunit capable of stable association. In some embodiments, the first antigen binding moiety and the second antigen binding moiety are each connected at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In some embodiments, a third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, optionally via a peptide linker.
[0013] In one embodiment of the T cell activating bispecific antigen binding molecule of the above embodiments, the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In one embodiment, the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31. In one embodiment, the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65. In one embodiment, the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
[0014] In another embodiment, the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54. In one embodiment, the antigen binding moiety capable of specific binding to FolR1 comprises (a) a complementarity determining region heavy chain 1 (CDR-H1) amino acid sequences of SEQ ID NO: 8; (b) a CDR-H2 amino acid sequence of SEQ ID NO: 9; (c) a CDR-H3 amino acid sequence of SEQ ID NO: 50; (d) a complementarity determining region light chain 1 (CDR-L1) amino acid sequence of SEQ ID NO: 52; (e) a CDR-L2 amino acid sequence of SEQ ID NO: 53, and (f) a CDR-L3 amino acid sequence of SEQ ID NO: 54. In one embodiment, the antigen binding moiety capable of specific binding to FolR1 comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
[0015] In another embodiment, the antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In one embodiment, the antigen binding moiety capable of specific binding to FolR1 comprises (a) a complementarity determining region heavy chain 1 (CDR-H1) amino acid sequences of SEQ ID NO: 16; (b) a CDR-H2 amino acid sequence of SEQ ID NO: 275; (c) a CDR-H3 amino acid sequence of SEQ ID NO: 315; (d) a complementarity determining region light chain 1 (CDR-L1) amino acid sequence of SEQ ID NO: 32; (e) a CDR-L2 amino acid sequence of SEQ ID NO: 33, and (f) a CDR-L3 amino acid sequence of SEQ ID NO: 34. In one embodiment, the antigen binding moiety capable of specific binding to FolR1 comprises a variable heavy chain domain (VH) comprising an amino acid sequence of SEQ ID NO: 274 and a variable light chain domain (VL) comprising an amino acid sequence of SEQ ID NO: 31.
[0016] In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1 and a cynomolgus monkey FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1, a cynomolgus monkey FolR1 and a murine FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiments binds to a human FolR1, a cynomolgus monkey FolR1 and not a murine FolR1.
[0017] In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments comprises not more than one antigen binding moiety capable of specific binding to CD3. In one embodiment of the T cell activating bispecific antigen binding molecule, the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule. In one embodiment, the Fc domain is an IgG class immunoglobulin, specifically an IgG.sub.1 or IgG.sub.4, Fc domain. In one embodiment, the Fc domain is a human Fc domain.
[0018] In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. In one embodiment, in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. In one embodiment, the Fc domain comprises at least one amino acid substitution that reduces binding to an Fc receptor and/or effector function, as compared to a native IgG.sub.1 Fc domain. In one embodiment, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G (Kabat numbering). In one embodiment, the Fc receptor is an Fc.gamma. receptor. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
[0019] In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the T cell activating bispecific antigen binding molecule induces proliferation of a human CD3 positive T cell in vitro. In one embodiment, the T cell activating bispecific antigen binding molecule induces human peripheral blood mononuclear cell mediated killing of a FolR1-expressing human tumor cell in vitro. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of a FolR1-expressing human tumor cell in vitro. In one embodiment, the T cell is a CD8 positive T cell. In one embodiment, the FolR1-expressing human tumor cell is a Hela, Skov-3, HT-29, or HRCEpiC cell. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing human tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours.
[0020] In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments induces upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry. In one embodiment, the T cell is a CD4 positive T cell or a CD8 positive T cell. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds human FolR1 with an apparent K.sub.D of about 5.36 pM to about 4 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K.sub.D of about 4 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds murine FolR1 with an apparent K.sub.D of about 1.5 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of at least about 1000 nM.
[0021] In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments is specific for FolR1 and does not bind to FolR2 or FolR3. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments has an affinity (monovalent binding) of 1 .mu.M or greater. In one embodiment, the affinity is around 1.4 .mu.M for human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments has an avidity (bivalent binding) of about 1-100 nM or lower. In one embodiment, the avidity is about 10 nM or less. In one embodiment, the avidity is 10 nM.
[0022] In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to FolR1 expressed on a human tumor cell. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to a conformational epitope on human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3). In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227). In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 and 229.
[0023] In another aspect, the invention provides for a bispecific antibody comprising a) a first antigen-binding site that competes for binding to human FolR1 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 49 and a variable light chain domain of SEQ ID NO: 51; and b) a second antigen-binding site that competes for binding to human CD3 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31, wherein binding competition is measured using a surface plasmon resonance assay.
[0024] In another aspect, the invention provides for a bispecific antibody comprising a) a first antigen-binding site that competes for binding to human FolR1 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31; and b) a second antigen-binding site that competes for binding to human CD3 with a reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31, wherein binding competition is measured using a surface plasmon resonance assay.
[0025] In another aspect, the invention provides for a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to CD3, and a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1), wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human FolR1 as a first reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 49 and a variable light chain domain of SEQ ID NO: 51; and wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human CD3 as a second reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31.
[0026] In another aspect, the invention provides for a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to CD3, and a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1), wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human FolR1 as a first reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain (VL) of SEQ ID NO: 31; and wherein the T cell activating bispecific antigen binding molecule binds to the same epitope on human CD3 as a second reference antibody comprising a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain (VL) of SEQ ID NO: 31.
[0027] In another aspect, the invention relates to an antibody or an antigen-binding fragment thereof that competes for binding to human FolR1 with an antibody that comprises a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31, wherein binding competition is measured using a surface plasmon resonance assay.
[0028] In one aspect, the invention provides for a T cell activating bispecific antigen binding molecule, wherein the antigen binding molecule comprises a first, second, third, fourth and fifth polypeptide chain that form a first, a second and a third antigen binding moiety, wherein the first antigen binding moiety is capable of binding CD3 and the second and the third antigen binding moiety each are capable of binding Folate Receptor 1 (FolR1), wherein a) the first and the second polypeptide chain comprise, in amino (N)-terminal to carboxyl (C)-terminal direction, VLD1 and CLD1; b) the third polypeptide chain comprises, in N-terminal to C-terminal direction, VLD2 and CH1D2; c) the fourth polypeptide chain comprises, in N-terminal to C-terminal direction, VHD1, CH1D1, CH2D1 and CH3D1; d) the fifth polypeptide chain comprises VHD1, CH1D1, VHD2, CLD2, CH2D2 and CH3D2; wherein VLD1 is a first light chain variable domain, VLD2 is a second light chain variable domain, CLD1 is a first light chain constant domain, CLD2 is a second light chain constant domain, VHD1 is a first heavy chain variable domain, VHD2 is a second heavy chain variable domain, CH1D1 is a first heavy chain constant domain 1, CH1D2 is a second heavy chain constant domain 1, CH2D1 is a first heavy chain constant domain 2, CH2D2 is a second heavy chain constant domain 2, CH3D1 is a first heavy chain constant domain 3, and CH3D2 is a second heavy chain constant domain 3.
[0029] In one embodiment of the T cell activating bispecific antigen binding molecule, (i) the third polypeptide chain and VHD2 and CLD2 of the fifth polypeptide chain form the first antigen binding moiety capable of binding CD3; (ii) the first polypeptide chain and VHD1 and CH1D1 of the fourth polypeptide chain form the second binding moiety capable of binding to FolR1; and (iii) the second polypeptide chain and VHD1 and CH1D1 of the fifth polypeptide chain form the third binding moiety capable of binding to FolR1. In one embodiment, CH2D1, CH3D1, CH2D2 and CH3D2 form an Fc domain of an IgG class immunoglobulin. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. In one embodiment, CH3D2 comprises an amino acid residue having a larger side chain volume, which is positionable in a cavity within CH3D1. In one embodiment, the Fc domain comprises at least one amino acid substitution that reduces binding to an Fc receptor and/or effector function, as compared to a native IgG.sub.1 Fc domain. In one embodiment, each subunit of the Fc domain comprises three amino acid substitutions that reduce at least one of binding to an activating Fc receptor and effector function wherein said amino acid substitutions are L234A, L235A and P329G according to Kabat numbering. In one embodiment, the Fc receptor is an Fc.gamma. receptor. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces proliferation of a human CD3 positive T cell in vitro. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces human peripheral blood mononuclear cell mediated killing of a FolR1-expressing human tumor cell in vitro. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of a FolR1-expressing human tumor cell in vitro. In one such embodiment, the FolR1-expressing human tumor cell is a Hela, Skov-3, HT-29, or HRCEpiC cell. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours. In one of the above embodiments, the T cell activating bispecific antigen binding molecule induces upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry. In one such embodiments, the T cell is a CD4 positive T cell or a CD8 positive T cell. In one of the above embodiments, wherein the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K.sub.D of about 5.36 pM to about 4 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K.sub.D of about 4 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds murine FolR1 with an apparent K.sub.D of about 1.5 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of at least about 1000 nM. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds to FolR1 expressed on a human tumor cell. In one of the above embodiments, the T cell activating bispecific antigen binding molecule binds to a conformational epitope on human FolR1. In one of the above embodiments, the T cell activating bispecific antigen binding molecule does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3). In one of the above embodiments, the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227). In one of the above embodiments, the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 and 229. In one of the above embodiments, the T cell activating bispecific antigen binding molecule is a humanized or a chimeric molecule. In one of the above embodiments, VHD2 and CH1D1 are linked through a peptide linker.
[0030] In one of the above embodiments of the T cell activating bispecific antigen binding molecule, the first and second polypeptide chain comprise the amino acid sequence of SEQ ID NO:230. In one of the above embodiments of the T cell activating bispecific antigen binding molecule, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:86. In one of the above embodiments of the T cell activating bispecific antigen binding molecule, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:309. In one of the above embodiments of the T cell activating bispecific antigen binding molecule, the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO:308. In one of the above embodiments of the T cell activating bispecific antigen binding molecule, the first and second polypeptide chain comprise the amino acid sequence of SEQ ID NO:230; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:86; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:309; and the fifth polypeptide chain comprise the amino acid sequence of SEQ ID NO:308.
[0031] In one aspect, the invention provides for a T cell activating bispecific antigen binding molecule comprising the amino acid sequence of SEQ ID NO:308. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiment further comprises the amino acid sequence of SEQ ID NO:230 and of SEQ ID NO:86.
[0032] In one aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:308. In one aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:309.
[0033] In one aspect, the invention provides for a T cell activating bispecific antigen binding molecule comprising the amino acid sequence of SEQ ID NO:276. In one embodiment, the T cell activating bispecific antigen binding molecule of the above embodiment further comprises the amino acid sequence of SEQ ID NO:277 and of SEQ ID NO:35.
[0034] In one aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:277. In one aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:276.
[0035] In one aspect, the invention provides for an isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of the embodiments disclosed herein. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:169. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:246. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:247. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:97. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:198.
[0036] In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:287. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:288. In one embodiment, the invention provides for an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule comprising the nucleotide sequence of SEQ ID NO:289.
[0037] In one aspect, the invention provides for an isolated polypeptide encoded by the polynucleotide of the above embodiment. In another aspect, the invention provides for a vector, particularly an expression vector, comprising the polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of the embodiments disclosed herein. In another aspect, the invention provides for a host cell comprising a polynucleotide or a vector of any of the embodiments disclosed herein.
[0038] In one aspect, the invention provides for a method of producing the T cell activating bispecific antigen binding molecule capable of specific binding to CD3 and a target cell antigen, comprising the steps of a) culturing the host cell of the above embodiments under conditions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule.
[0039] In one aspect, the invention provides for T cell activating bispecific antigen binding molecule produced by the method of the above embodiment.
[0040] In one aspect, the invention provides for a pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of any one of the above embodiments and a pharmaceutically acceptable carrier. In one aspect, the invention provides for the T cell activating bispecific antigen binding molecule of any one of the above embodiments or the pharmaceutical composition of any of the above embodiments for use as a medicament.
[0041] In one aspect, the invention provides for the T cell activating bispecific antigen binding molecule of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments for use in the treatment of a disease in an individual in need thereof. In some embodiments, the disease is cancer. In one aspect, the invention provides for a use of the T cell activating bispecific antigen binding molecule of any one of the above embodiments for the manufacture of a medicament for the treatment of a disease in an individual in need thereof.
[0042] In one aspect, the invention provides for a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of any one of the above embodiments in a pharmaceutically acceptable form. In some embodiments, said disease is a cancer.
[0043] In one aspect, the invention provides for a method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of any one of the above embodiments in the presence of a T cell.
[0044] In one aspect, the invention provides for a the invention as described hereinbefore.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0046] FIGS. 1A-I illustrate exemplary configurations of the T cell activating bispecific antigen binding molecules (TCBs) of the invention. All constructs except the kappa-lambda format in (FIG. 1I) have P329G LALA mutations and comprise knob-into-hole Fc fragments with knob-into-hole modifications. (FIG. 1A) Illustration of the "FolR1 TCB 2+1 inverted (common light chain)". The FolR1 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. These constructs are not crossed and have three times the same VLCL light chain. (FIG. 1B) Illustration of the "FolR1 TCB 1+1 head-to-tail (common light chain)". These constructs are not crossed and have two times the same VLCL light chain. (FIG. 1C) Illustration of the "FolR1 TCB 1+1 classical (common light chain)". These constructs are not crossed and have two times the same VLCL light chain. (FIG. 1D) Illustration of the "FolR1 TCB 2+1 classical (common light chain)". The CD3 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. These constructs are not crossed and have three times the same VLCL light chain. (FIG. 1E) Illustration of the "FolR1 TCB 2+1 crossfab classical". These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. The CD3 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. (FIG. 1F) Illustration of the "FolR1 TCB 2+1 crossfab inverted". These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. The FolR1 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. (FIG. 1G) Illustration of the "FolR1 TCB 1+1 crossfab head-to-tail". These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. (FIG. 1H) Illustration of the "FolR1 TCB 1+1 crossfab classical". These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. FIG. 1I illustrates the CD3/FolR1 kappa-lambda antibody format. These constructs comprise a crossed common light chain VLCH1 and one crossed VHCL chain specific for CD3 and one crossed VHCL chain specific for FolR1.
[0047] FIGS. 2A-C depict graphs summarizing Binding of FoLR1 IgG binders to HeLa cells. Binding of newly generated FolR1 binders to FolR1 expressed on HeLa cells were determined by flow cytometry. Bound antibodies were detected with a fluorescently labeled anti-human secondary antibody.
[0048] FIGS. 3A-B depict graphs summarizing specificity of FolR1 binders for FolR1. Binding of FolR1 IgGs to HEK cells transiently transfected with either FolR1 or FolR2 was analyzed by flow cytometry to identify clones which bind specifically to FolR1 and not to FolR2. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
[0049] FIGS. 4A-B depict graphs summarizing cross-reactivity of FolR1 binders to cyFoLR1. Cross-reactivity of the FolR1 antibodies to cyno FolR1 was addressed on HEK cells transiently transfected with cyFolR1 by flow cytometry. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
[0050] FIG. 5 depicts a graph illustrating internalization of FolR1 TCBs after binding. Internalization of the four FolR1 TCBs after binding to FolR1 was tested on HeLa cells. Remaining FolR1 TCBs on the surface were detected with a fluorescently labeled anti-human secondary antibody after indicated time points of incubation at 37.degree. C. Percentage of internalization was calculated.
[0051] FIGS. 6A-E depict graphs summarizing binding of FolR1 IgGs to cells with different FolR1 expression levels. Binding of 9D11, 16D5 and Mov19 IgG to tumor cells with different FolR1 expression levels was analyzed by flow cytometry. DP47 IgG was included as isotype control and MKN-45 were included as FolR1 negative cell line. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
[0052] FIGS. 7A-L depict graphs summarizing T cell mediated killing of HT-29 and SKOV3 cells. FolR1 TCBs were used to test T cell mediated killing of HT-29 and SKOV3 tumor cells and upregulation of activation marker on T cells upon killing. (FIGS. 7A-D) T cell mediated killing of HT-29 and SKOV3 cells in the presence of 9D11 FolR1 TCB and 16D5 FolR1 TCB was measured by LDH release after 24 h and 48 h. DP47 TCB was included as negative control. After 48 h incubation upregulation of the activation marker CD25 and CD69 on CD8 T cells and CD4 T cells upon killing of SKOV3 (FIGS. 7E-H) or HT-29 (FIG. 7I-L) tumor cells was assessed by flow cytometry.
[0053] FIG. 8 depicts a graph showing absence of anti-FolR1 binding to erythrocytes. Erythrocytes were gated as CD235a positive population and binding of 9D11 IgG, 16D5 IgG, Mov19 IgG and DP47 IgG to this population was determined by flow cytometry. The antibodies were detected with a fluorescently labeled anti-human secondary antibody.
[0054] FIGS. 9A-D depict graphs summarizing activation marker upregulation in whole blood. CD25 and CD69 activation marker upregulation of CD4 T cells and CD8 T cells 24 h after addition of 9D11 FolR1 TCB, 16D5 FolR1 TCB, Mov19 FolR1 TCB and DP47 TCB was analyzed by flow cytometry.
[0055] FIG. 10 Binding of 9D11 TCB a-glyco variants to HeLa cells. Binding of 9D11 FolR1 TCB a-glyco variants to Hela cells was compared to binding of the original 9D11 TCB on HeLa cells. The antibodies were detected with a fluorescently labeled anti-human secondary antibody and binding was determined by flow cytometry.
[0056] FIGS. 11A-F depict graphs summarizing T cell mediated killing with 9D11 FolR1 TCB a-glyco variants of tumor cells. 9D11 FolR1 TCB a-glyco variants were used to test T cell mediated killing of (FIG. 11A-D) SKOV3, MKN-45 (as FolR1 negative control) and (FIG. 11E-F) HT-29 tumor cells in comparison to killing with the original 9D11 FolR1 TCB. As read-out LDH release after 24 h and 48 h was used.
[0057] FIGS. 12A-X depict graphs summarizing T cell mediated killing of primary epithelial cells. Primary epithelial cells with very low levels of FolR1 were used to test T cell mediated killing with 16D5 FolR1 TCB and 9D11 FolR1 TCB, DP47 TCB was included as a negative control and HT29 cells were included as positive control. (FIGS. 12A-H) LDH release of human retinal pigment (HRP), human renal cortical (HRC), human bronchial (HB) and of HT29 cells was determined after 24 h and 48 h. CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells upon killing of (FIGS. 12I-L) HRP, (FIGS. 12M-P) HRC, (FIGS. 12Q-T) HB and (FIGS. 12 U-X) HT29 was determined after 48 h by flow cytometry.
[0058] FIGS. 13A-C show a comparison of different TCB formats with 16D5. Four different TCB formats containing the FolR1 binder 16D5 were compared in FIG. 13A binding to HeLa cells, in FIG. 14 B T cell mediated killing of SKOV3 cells after 24 h and 48 h and in FIG. 14C CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells 48 h after killing.
[0059] FIGS. 14A-C depict a comparison of different TCB formats with 9D11. Three different TCB formats containing the FolR1 binder 9D11 were compared in A) binding to HeLa cells, in B) T cell mediated killing of SKOV3 cells after 24 h and 48 h and in C) CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells 48 h after killing.
[0060] FIG. 15 depicts a PK-profile of FOLR1 TCB in NOG mice for three different doses.
[0061] FIG. 16 illustrates an experimental protocol for efficacy study with FOLR1 TCB.
[0062] FIGS. 17A-B depict tumor growth curves. (FIG. 17A) Mean values and SEM of tumor volumes in the different treatment groups. (FIG. 17B) Tumor growth of single mice in all treatment groups. TGI (tumor growth inhibition) give the percentage of the Mean tumor volume compared to vehicle group.
[0063] FIG. 18 shows tumor weights at study termination.
[0064] FIGS. 19A-B show FACS analysis of tumor infiltrating T-cells at study day 32. (FIG. 19A) Tumor single cells suspensions were stained with anti-human CD3/CD4/CD8 and analyzed by flow cytometry. (FIG. 19B) Mean values and SEM of T-cell counts per mg tumor tissue in different treatment groups.
[0065] FIGS. 20A-B show FACS analysis for T-cell activation/degranulation and cytokine secretion at study day 32. CD4+(FIG. 20A) and CD8+(FIG. 20B) tumor infiltrating T-cells were stained for cytokines, activation and degranulation markers. Displayed are the mean values and SEM of T-cell counts per mg tumor tissue in different treatment groups.
[0066] FIGS. 21A-B show percent tumor lysis. SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 24 h (FIG. 21A) and 48 h (FIG. 21B) killing of tumor cells was determined by measuring LDH release.
[0067] FIGS. 22A-D show CD25 and CD69 upregulation on CD4 T cells. SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells (FIG. 22A-B) and CD8 T cells (FIG. 22C-D) was measured by flow cytometry.
[0068] FIGS. 23A-B show percent tumor lysis. T-cell killing of SKov-3 cells (medium FolR1) induced by 36F2 TCB, Mov19 TCB and 21A5 TCB after 24 h (FIG. 23A) and 48 h (FIG. 23B) of incubation (E:T=10:1, effectors human PBMCs).
[0069] FIGS. 24A-C show T-cell killing induced by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, 16D5 TCB 1+1 and 16D5 TCB HT of Hela (high FolR1) (FIG. 24A), Skov-3 (medium FolR1) (FIG. 24B) and HT-29 (low FolR1) (FIG. 24C) human tumor cells (E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB was included as non-binding control.
[0070] FIGS. 25A-C show upregulation of CD25 and CD69 on human CD8+(FIG. 25A, B) and CD4+(FIG. 25C), T cells after T cell-mediated killing of Hela cells (high FolR1) (FIG. 25A), SKov-3 cells (medium FolR1) (FIG. 25B) and HT-29 cells (low FolR1) (FIG. 25C) (E:T=10:1, 48 h incubation) induced by 36F2 TCB, 16D5 TCB and DP47 TCB (non-binding control).
[0071] FIGS. 26A-F show T-cell killing induced by 36F2 TCB, 16D5 TCB and DP47 TCB of human Renal Cortical Epithelial Cells (FIG. 26A, B), human Retinal Pigment Epithelial Cells (FIG. 26C, D) and HT-29 cells (FIG. 26E, F) cells after 24 h (FIG. 26A, C, E) and 48 h (FIG. 26B, D, F) of incubation (E:T=10:1, effectors human PBMCs).
[0072] FIG. 27 depicts a table summarizing quantification of FolR1 binding sites on various normal and cancer cells lines.
[0073] FIGS. 28A-B show binding of 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 expressed on Jurkat cells.
[0074] FIGS. 29A-B show T-cell killing of SKov-3 (medium FolR1) human tumor cells induced by 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA (FIG. 29A) and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 29B) (E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB was included as non-binding control.
[0075] FIG. 30A-B show T-cell killing of HT-29 (low FolR1) human tumor cells induced by 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA (FIG. 30A) and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 30B) (E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB was included as non-binding control.
[0076] FIGS. 31A-C show mean fluorescence intensity and tumor cell lysis.
[0077] FIGS. 32A-E shows binding of 36F2 TCB, 16D5 TCB and 16D5 HC/LC variants to human FolR1 expressed on Hela cells.
[0078] FIG. 33 shows binding of 36F2 TCB, 16D5 TCB and the two 16D5 affinity reduced variants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to human FolR1 on Hela cells.
[0079] FIGS. 34A-E show binding of 36F2 TCB, 16D5 TCB and 16D5 HC/LC variants to human FolR1 expressed on HT-29 cells.
[0080] FIGS. 35A-D show binding of intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB), 16D5 TCB and 36F2 TCB to HEK293T cells expressing either human or mouse FolR1 or FolR2.
[0081] FIG. 36A-F show T-cell killing of Hela (high FolR1 expression), SKov-3 (medium FolR1 expression) and HT-29 (low FolR1 expression) human tumor cells induced by intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB), 16D5 TCB and 36F2 TCB after 24 h (A-C) and 48 h (D-F) of incubation. Human PBMCs were used as effector cells (E:T=10:1).
[0082] FIG. 37A-F shows T-cell killing of Hela (high FolR1 expression), SKov-3 (medium FolR1 expression) and HT-29 (low FolR1 expression) human tumor cells induced by affinity reduced 16D5 variants (16D5-G49S/S93A TCB, 16D5-G49S/K53A TCB, 16D5 W96Y TCB, 16D5 W96Y/D52E TCB), 16D5 TCB and 36F2 TCB after 24 h (FIG. 38A-C) and 48 h (FIG. 38D-F) of incubation. Human PBMCs were used as effector cells (E:T=10:1).
[0083] FIG. 38A-F show T-cell killing of primary human cells from retinal pigment epithelium and renal cortical epithelium induced by affinity reduced 16D5 variants (16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB), 16D5 TCB, 36F2 TCB and the intermediate FolR1 binder 14B1 TCB was assessed after 24 h (FIG. 39A-C) and 48 h (FIG. 39D-F) of incubation (E:T=10:1, effectors human PBMCs). HT-29 cells (low FolR1 expression) were included as control cell line and DP47 TCB served as non-binding control.
[0084] FIGS. 39A-B show single dose PK of FOLR1 TCB constructs in female NOG mice.
[0085] FIGS. 40A-G show in vivo efficacy of FOLR1 TCB constructs (16D5, 16D5 G49S/S93A and 16D5 W96Y/D52E) after human PBMC transfer in Hela-bearing NOG mice.
[0086] FIG. 41 shows that Farletuzumab (dark green, second from the top) and Mov19 (grey, top) are able to bind on huFolR1 that is captured on 16D5, demonstrating that the 16D5 series binders recognize an epitope distinct from that recognized by either Farletuzumab or Mov19.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0087] Terms are used herein as generally used in the art, unless otherwise defined in the following.
[0088] As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, e.g. fragments, thereof.
[0089] The term "bispecific" means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises at least two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
[0090] The term "valent" as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term "monovalent binding to an antigen" denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule.
[0091] An "antigen binding site" refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.
[0092] As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. In another embodiment an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: .alpha., .delta., .epsilon., .gamma., or .mu.. Useful light chain constant regions include any of the two isotypes: .kappa. and .lamda..
[0093] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope," and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins referred to as antigens herein, e.g., FolR1 and CD3, can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular embodiment the antigen is a human protein. Where reference is made to a specific protein herein, the term encompasses the "full-length", unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants. Exemplary human proteins useful as antigens include, but are not limited to: FolR1 (Folate receptor alpha (FRA); Folate binding protein (FBP); human FolR1 UniProt no.: P15328; murine FolR1 UniProt no.: P35846; cynomolgus FolR1 UniProt no.: G7PR14) and CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for the cynomolgus [Macaca fascicularis] sequence). The T cell activating bispecific antigen binding molecule of the invention binds to an epitope of CD3 or a target cell antigen that is conserved among the CD3 or target antigen from different species. In certain embodiments the T cell activating bispecific antigen binding molecule of the invention binds to CD3 and FolR1, but does not bind to FolR2 (Folate receptor beta; FRB; human FolR2 UniProt no.: P14207) or FolR3 (Folate receptor gamma; human FolR3 UniProt no.: P41439).
[0094] By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein is less than about 10% of the binding of the antigen binding moiety to the antigen as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety, has a dissociation constant (K.sub.D) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8M to 10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13 M).
[0095] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K.sub.D), which is the ratio of dissociation and association rate constants (k.sub.off and k.sub.on, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).
[0096] "Reduced binding", for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.
[0097] "T cell activation" as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein.
[0098] A "target cell antigen" as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma. In particular "target cell antigen" refers to Folate Receptor 1.
[0099] As used herein, the terms "first" and "second" with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the T cell activating bispecific antigen binding molecule unless explicitly so stated.
[0100] A "Fab molecule" refers to a protein consisting of the VH and CH1 domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin.
[0101] The term "Fab molecules having identical VLCL light chains" as used therein refers to binders that share one light chain but still have separate specificities, e.g., can bind CD3 or FolR1. In some embodiments the T-cell activating bispecific molecules comprise at least two Fab molecules having identical VLCL light chains. The corresponding heavy chains are remodeled and confer specific binding to the T cell activating bispecific antigen CD3 and the target cell antigen FolR1, respectively.
[0102] By "fused" is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.
[0103] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.
[0104] By a "crossover" Fab molecule (also termed "Crossfab") is meant a Fab molecule wherein either the variable regions or the constant regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region and the heavy chain constant region, and a peptide chain composed of the heavy chain variable region and the light chain constant region. For clarity, in a crossover Fab molecule wherein the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule. An antibody that comprises one or more CrossFabs is referred to herein as "CrossMab."
[0105] In contrast thereto, by a "conventional" Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant regions (VH-CH1), and a light chain composed of the light chain variable and constant regions (VL-CL).
[0106] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called .alpha. (IgA), .delta. (IgD), .epsilon. (IgE), .gamma. (IgG), or .mu. (IgM), some of which may be further divided into subtypes, e.g. .gamma..sub.1 (IgG.sub.1), .gamma..sub.2 (IgG.sub.2), .gamma..sub.3 (IgG.sub.3), .gamma..sub.4 (IgG.sub.4), .alpha..sub.1 (IgA.sub.1) and .alpha..sub.2 (IgA.sub.2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (.kappa.) and lambda (.lamda.), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
[0107] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.
[0108] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab').sub.2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab').sub.2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
[0109] The term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
[0110] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.
[0111] The term "hypervariable region" or "HVR", as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the complementarity determining regions (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
TABLE-US-00001 TABLE A CDR Definitions.sup.1 CDR Kabat Chothia AbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58 50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-32 24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97 .sup.1Numbering of all CDR definitions in Table A is according to the numbering conventions set forth by Kabat et al. (see below). .sup.2"AbM" with a lowercase "b" as used in Table A refers to the CDRs as defined by Oxford Molecular's "AbM" antibody modeling software.
[0112] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system. The polypeptide sequences of the sequence listing are not numbered according to the Kabat numbering system. However, it is well within the ordinary skill of one in the art to convert the numbering of the sequences of the Sequence Listing to Kabat numbering.
[0113] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.
[0114] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called .alpha., .delta., .epsilon., .gamma., and .mu., respectively.
[0115] The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. A "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
[0116] A "modification promoting the association of the first and the second subunit of the Fc domain" is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.
[0117] The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
[0118] As used herein, the terms "engineer, engineered, engineering", are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
[0119] The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G.sub.329, P329G, or Pro329Gly.
[0120] As used herein, term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
[0121] By an "isolated" polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
[0122] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
[0123] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.
[0124] By "isolated" nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator. By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).
[0125] The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
[0126] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
[0127] The terms "host cell", "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
[0128] An "activating Fc receptor" is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include Fc.gamma.RIIIa (CD16a), Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI (CD89).
[0129] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT publication no. WO 2012/130831).
[0130] An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
[0131] A "therapeutically effective amount" of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
[0132] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
[0133] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
[0134] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
[0135] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, T cell activating bispecific antigen binding molecules of the invention are used to delay development of a disease or to slow the progression of a disease.
[0136] The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that induces a biological activity of a native polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, including engineered antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a polypeptide may comprise contacting a polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.
[0137] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
[0138] All references, publication, patents and patent applications disclosed herein are hereby incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0139] The T cell activating bispecific antigen binding molecule of the invention is bispecific, i.e. it comprises at least two antigen binding moieties capable of specific binding to two distinct antigenic determinants, i.e. to CD3 and to FolR1. According to the invention, the antigen binding moieties are Fab molecules (i.e. antigen binding domains composed of a heavy and a light chain, each comprising a variable and a constant region). In one embodiment said Fab molecules are human. In another embodiment said Fab molecules are humanized. In yet another embodiment said Fab molecules comprise human heavy and light chain constant regions.
[0140] The T cell activating bispecific antigen binding molecule of the invention is capable of simultaneous binding to the target cell antigen FolR1 and CD3. In one embodiment, the T cell activating bispecific antigen binding molecule is capable of crosslinking a T cell and a FolR1 expressing target cell by simultaneous binding to the target cell antigen FolR1 and CD3. In an even more particular embodiment, such simultaneous binding results in lysis of the FolR1 expressing target cell, particularly a FolR1 expressing tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell. In other embodiments, such simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the T cell activating bispecific antigen binding molecule to CD3 without simultaneous binding to the target cell antigen FolR1 does not result in T cell activation.
[0141] In one embodiment, the T cell activating bispecific antigen binding molecule is capable of re-directing cytotoxic activity of a T cell to a FolR1 expressing target cell. In a particular embodiment, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and and/or specificity of the T cell.
[0142] Particularly, a T cell according to some of the embodiments of the invention is a cytotoxic T cell. In some embodiments the T cell is a CD4.sup.+ or a CD8.sup.+ T cell, particularly a CD8.sup.+ T cell.
[0143] The T cell activating bispecific antigen binding molecule of the invention comprises at least one antigen binding moiety capable of binding to CD3 (also referred to herein as an "CD3 antigen binding moiety" or "first antigen binding moiety"). In a particular embodiment, the T cell activating bispecific antigen binding molecule comprises not more than one antigen binding moiety capable of specific binding to CD3. In one embodiment the T cell activating bispecific antigen binding molecule provides monovalent binding to CD3. In a particular embodiment CD3 is human CD3 or cynomolgus CD3, most particularly human CD3. In a particular embodiment the CD3 antigen binding moiety is cross-reactive for (i.e. specifically binds to) human and cynomolgus CD3. In some embodiments, the first antigen binding moiety is capable of specific binding to the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence; UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for the cynomolgus [Macaca fascicularis] sequence).
[0144] In some embodiments, the CD3 antigen binding moiety comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
[0145] In one embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
[0146] In one embodiment the CD3 antigen binding moiety comprises a variable heavy chain comprising an amino acid sequence of: SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of: SEQ ID NO: 31.
[0147] In one embodiment the CD3 antigen binding moiety comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.
[0148] The T cell activating bispecific antigen binding molecule of the invention comprises at least one antigen binding moiety capable of binding to the target cell antigen FolR1 (also referred to herein as an "FolR1 binding moiety" or "second" or "third" antigen binding moiety). In one embodiment, the antigen binding moiety capable of binding to the target cell antigen FolR1 does not bind to FolR2 or FolR3. In a particular embodiment the FolR1 antigen binding moiety is cross-reactive for (i.e. specifically binds to) human and cynomolgus FolR1. In certain embodiments, the T cell activating bispecific antigen binding molecule comprises two antigen binding moieties capable of binding to the target cell antigen FolR1. In a particular such embodiment, each of these antigen binding moieties specifically binds to the same antigenic determinant. In an even more particular embodiment, all of these antigen binding moieties are identical. In one embodiment the T cell activating bispecific antigen binding molecule comprises not more than two antigen binding moieties capable of binding to FolR1.
[0149] The FolR1 binding moiety is generally a Fab molecule that specifically binds to FolR1 and is able to direct the T cell activating bispecific antigen binding molecule to which it is connected to a target site, for example to a specific type of tumor cell that expresses FolR1.
[0150] In one aspect the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0151] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34; and
[0152] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1).
[0153] In one embodiment the first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0154] In one embodiment the T cell activating bispecific antigen binding molecule additionally comprises
[0155] (iii) a third antigen binding moiety which is a Fab molecule capable of specific binding to FolR1.
[0156] In one such embodiment the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one such embodiment the third antigen binding moiety is identical to the second antigen binding moiety.
[0157] In one embodiment the T cell activating bispecific antigen binding molecule of any of the above embodiments additionally comprises an Fc domain composed of a first and a second subunit capable of stable association.
[0158] In one embodiment the first antigen binding moiety and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
[0159] In one embodiment the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, optionally via a peptide linker.
[0160] In a further particular embodiment, not more than one antigen binding moiety capable of specific binding to CD3 is present in the T cell activating bispecific antigen binding molecule (i.e. the T cell activating bispecific antigen binding molecule provides monovalent binding to CD3).
[0161] T Cell Activating Bispecific Antigen Binding Molecule with a Common Light Chain
[0162] The inventors of the present invention generated a bispecific antibody wherein the binding moieties share a common light chain that retains the specificity and efficacy of the parent monospecific antibody for CD3 and can bind a second antigen (e.g., FolR1) using the same light chain. The generation of a bispecific molecule with a common light chain that retains the binding properties of the parent antibody is not straight-forward as the common CDRs of the hybrid light chain have to effectuate the binding specificity for both targets. In one aspect the present invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to CD3 and the other one of which is a Fab molecule capable of specific binding to FolR1, wherein the first and the second Fab molecule have identical VLCL light chains. In one embodiment said identical light chain (VLCL) comprises the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. In one embodiment said identical light chain (VLCL) comprises SEQ ID NO. 35.
[0163] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0164] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0165] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
[0166] In one such embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, the heavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
[0167] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0168] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0169] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0170] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0171] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0172] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
[0173] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0174] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises the heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and the light chain CDR amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34;
[0175] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises the heavy chain complementarity determining region (CDR) amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 275 and SEQ ID NO: 315, and the light chain CDR amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.
[0176] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0177] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31;
[0178] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 274 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0179] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0180] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0181] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0182] In a further embodiment, the antigen binding moiety that is specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31 or variants thereof that retain functionality.
[0183] In one embodiment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.
[0184] In one embodiment the T cell activating bispecific antigen binding molecule additionally comprises
[0185] (iii) a third antigen binding moiety (which is a Fab molecule) capable of specific binding to FolR1.
[0186] In one such embodiment the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one such embodiment the third antigen binding moiety is identical to the second antigen binding moiety.
[0187] Hence in one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0188] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0189] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
[0190] (iii) a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.
[0191] In one such embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, the heavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
[0192] In one embodiment the present invention provides a T cell activating bispecific antigen binding molecule comprising
[0193] (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0194] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0195] (iii) a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 15 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0196] Thus, in one embodiment, the invention relates to bispecific molecules wherein at least two binding moieties have identical light chains and corresponding remodeled heavy chains that confer the specific binding to the T cell activating antigen CD3 and the target cell antigen FolR1, respectively. The use of this so-called `common light chain` principle, i.e. combining two binders that share one light chain but still have separate specificities, prevents light chain mispairing. Thus, there are less side products during production, facilitating the homogenous preparation of T cell activating bispecific antigen binding molecules.
[0197] The components of the T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations are depicted in FIGS. 1A-I and are further described below.
[0198] In some embodiments, said T cell activating bispecific antigen binding molecule further comprises an Fc domain composed of a first and a second subunit capable of stable association. Below exemplary embodiments of T cell activating bispecific antigen binding molecule comprising an Fc domain are described.
[0199] T Cell Activating Bispecific Antigen Binding Molecule with a Crossover Fab Fragment
[0200] The inventors of the present invention generated a second bispecific antibody format wherein one of the binding moieties is a crossover Fab fragment. In one aspect of the invention a monovalent bispecific antibody is provided, wherein one of the Fab fragments of an IgG molecule is replaced by a crossover Fab fragment. Crossover Fab fragments are Fab fragments wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Bispecific antibody formats comprising crossover Fab fragments have been described, for example, in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and WO2013/026831. In a particular embodiment, the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged. Such modification prevent mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the T cell activating bispecific antigen binding molecule of the invention in recombinant production. In a particular crossover Fab molecule useful for the T cell activating bispecific antigen binding molecule of the invention, the variable regions of the Fab light chain and the Fab heavy chain are exchanged. In another crossover Fab molecule useful for the T cell activating bispecific antigen binding molecule of the invention, the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
[0201] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0202] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0203] (ii) a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
[0204] In one such embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, the heavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59, the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQ ID NO:65.
[0205] In one embodiment, the second antigen binding moiety is a conventional Fab molecule.
[0206] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0207] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0208] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
[0209] In one embodiment, the second antigen binding moiety is a conventional Fab molecule.
[0210] In a further embodiment, the antigen binding moiety that is specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64 or variants thereof that retain functionality.
[0211] In one embodiment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64.
[0212] In one embodiment the T cell activating bispecific antigen binding molecule additionally comprises
[0213] (iii) a third antigen binding moiety capable of specific binding to FolR1.
[0214] In one embodiment, the third antigen binding moiety is a conventional Fab molecule. In one embodiment, the third antigen binding moiety is a crossover Fab molecule.
[0215] In one such embodiment the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one such embodiment the third antigen binding moiety is identical to the second antigen binding moiety.
[0216] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0217] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0218] (ii) a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
[0219] (iii) a third antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65.
[0220] In one such embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, the heavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59, the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQ ID NO:65.
[0221] In one embodiment, the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
[0222] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0223] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0224] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
[0225] (iii) a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 64.
[0226] In one embodiment, the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
[0227] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0228] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0229] (ii) a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
[0230] In one such embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
[0231] In one embodiment, the second antigen binding moiety is a conventional Fab molecule. In one embodiment, the second antigen binding moiety is a crossover Fab molecule.
[0232] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0233] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0234] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
[0235] In one embodiment, the second antigen binding moiety is a conventional Fab molecule. In one embodiment, the second antigen binding moiety is a crossover Fab molecule.
[0236] In a further embodiment, the antigen binding moiety that is specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 51 or variants thereof that retain functionality.
[0237] In one embodiment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 51.
[0238] In one embodiment the T cell activating bispecific antigen binding molecule additionally comprises
[0239] (iii) a third antigen binding moiety capable of specific binding to FolR1.
[0240] In one embodiment, the third antigen binding moiety is a conventional Fab molecule. In one embodiment, the second antigen binding moiety is a crossover Fab molecule.
[0241] In one such embodiment the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one such embodiment the third antigen binding moiety is identical to the second antigen binding moiety.
[0242] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0243] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;
[0244] (ii) a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 49 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
[0245] (iii) a third antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1) comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at least one light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54.
[0246] In one such embodiment the CD3 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
[0247] In one embodiment, the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
[0248] In one embodiment the T cell activating bispecific antigen binding molecule comprises
[0249] (i) a first antigen binding moiety which is a crossover Fab molecule capable of specific binding to CD3 comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31.
[0250] (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
[0251] (iii) a third antigen binding moiety which is a Fab molecule capable of specific binding to Folate Receptor 1 (FolR1) comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 49 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 51.
[0252] In one embodiment, the second antigen binding moiety and the third antigen binding moiety are both a conventional Fab molecule.
[0253] Thus, in one embodiment, the invention relates to bispecific molecules wherein two binding moieties confer specific binding to FolR1 and one binding moiety confers specificity to the T cell activating antigen CD3. One of the heavy chains is modified to ensure proper pairing of the heavy and light chains, thus eliminating the need for a common light chain approach. The presence of two FolR1 binding sites enables appropriate engagement with the target antigen FolR1 and the activation of T cells.
[0254] The components of the T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations are depicted in FIGS. 1A-I and are further described below.
[0255] In some embodiments, said T cell activating bispecific antigen binding molecule further comprises an Fc domain composed of a first and a second subunit capable of stable association. Below exemplary embodiments of T cell activating bispecific antigen binding molecule comprising an Fc domain are described.
[0256] T Cell Activating Bispecific Antigen Binding Molecule Formats
[0257] As depicted above and in FIGS. 1A-I, in one embodiment the T cell activating bispecific antigen binding molecules comprise at least two Fab fragments having identical light chains (VLCL) and having different heavy chains (VHCL) which confer the specificities to two different antigens, i.e. one Fab fragment is capable of specific binding to a T cell activating antigen CD3 and the other Fab fragment is capable of specific binding to the target cell antigen FolR1.
[0258] In another embodiment the T cell activating bispecific antigen binding molecule comprises at least two antigen binding moieties (Fab molecules), one of which is a crossover Fab molecule and one of which is a conventional Fab molecule. In one such embodiment the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
[0259] These components of the T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations are depicted in FIGS. 1A-I.
[0260] In some embodiments, the first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In one such embodiment the first and second antigen binding moiety both are Fab fragments and have identical light chains (VLCL). In another such embodiment the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule.
[0261] In one embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In one such embodiment the first and second antigen binding moiety both are Fab fragments and have identical light chains (VLCL). In another such embodiment the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.
[0262] In other embodiments, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a particular such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In one such embodiment the first and second antigen binding moiety both are Fab fragments and have identical light chains (VLCL). In another such embodiment the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.
[0263] The antigen binding moieties may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non-immunogenic peptide linkers include, for example, (G.sub.4S).sub.n (SEQ ID NO: 300), (SG.sub.4).sub.n (SEQ ID NO: 301), (G.sub.4S).sub.n (SEQ ID NO: 300) or G.sub.4(SG.sub.4).sub.n (SEQ ID NO: 302) peptide linkers. "n" is generally a number between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding moiety to each other is (G.sub.4S).sub.2 (SEQ ID NO: 303). An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second antigen binding moiety is EPKSC(D)-(G.sub.4S).sub.2 (SEQ ID NOS 304 and 305). Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where an antigen binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.
[0264] It has been found by the inventors of the present invention that T cell activating bispecific antigen binding molecule comprising two binding moieties specific for the target cell antigen FolR have superior characteristics compared to T cell activating bispecific antigen binding molecule comprising only one binding moiety specific for the target cell antigen FolR.
[0265] Accordingly, in certain embodiments, the T cell activating bispecific antigen binding molecule of the invention further comprises a third antigen binding moiety which is a Fab molecule capable of specific binding to FolR. In one such embodiment the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one such embodiment the third antigen binding moiety is identical to the second antigen binding moiety (i.e. they comprise the same amino acid sequences).
[0266] In one embodiment, the first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first, a second and a third antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In one such embodiment the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL). In another such embodiment the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second and third antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the third antigen binding moiety may additionally be fused to each other.
[0267] Accordingly, in certain embodiments, the T cell activating bispecific antigen binding molecule of the invention comprises five polypeptide chains that form a first, a second and a third antigen binding moiety wherein the first antigen binding moiety is capable of binding CD3 and the second and the third antigen binding moiety each are capable of binding Folate Receptor 1 (FolR1). The first and the second polypeptide chain comprise, in amino (N)-terminal to carboxyl (C)-terminal direction, a first light chain variable domain (VLD1) and a first light chain constant domain (CLD1). The third polypeptide chain comprises, in N-terminal to C-terminal direction, second light chain variable domain (VLD2) and a second heavy chain constant domain 1 (CH1D2). The fourth polypeptide chain comprises, in N-terminal to C-terminal direction, a first heavy chain variable domain (VHD1), a first heavy chain constant domain 1 (CH1D1), a first heavy chain constant domain 2 (CH2D1) and a first heavy chain constant domain 3 (CH3D1). The fifth polypeptide chain comprises VHD1, CH1D1, a second heavy chain variable domain (VHD2), a second light chain constant domain (CLD2), a second heavy chain constant domain 2 (CH2D2) and a second heavy chain constant domain 3 (CH3D2). The third polypeptide chain and VHD2 and CLD2 of the fifth polypeptide chain form the first antigen binding moiety capable of binding CD3. The second polypeptide chain and VHD1 and CH1D1 of the fifth polypeptide chain form the third binding moiety capable of binding to FolR1. The first polypeptide chain and VHD1 and CH1D1 of the fourth polypeptide chain form the second binding moiety capable of binding to FolR1.
[0268] In another embodiment, the second and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first, a second and a third antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second and third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the third antigen binding moiety. In one such embodiment the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL). In another such embodiment the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule and the second and third antigen binding moiety capable of specific binding to FolR is a conventional Fab molecule. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.
[0269] The antigen binding moieties may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the antigen binding moieties are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG.sub.1 hinge region.
[0270] In one embodiment the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule. In a particular embodiment the immunoglobulin molecule is an IgG class immunoglobulin. In an even more particular embodiment the immunoglobulin is an IgG.sub.1 subclass immunoglobulin. In another embodiment the immunoglobulin is an IgG.sub.4 subclass immunoglobulin. In a further particular embodiment the immunoglobulin is a human immunoglobulin. In other embodiments the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.
[0271] In a particular embodiment said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, wherein the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL), wherein the first antigen binding moiety capable of specific binding to CD3 comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.
[0272] In a particular embodiment said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, wherein the first, second and third antigen binding moiety are conventional Fab fragments and have identical light chains (VLCL), wherein the first antigen binding moiety capable of specific binding to CD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO: 36, a variable light chain comprising a sequence of SEQ ID NO: 31; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise a variable heavy chain comprising a sequence of SEQ ID NO: 15, a variable light chain comprising a sequence of SEQ ID NO: 31.
[0273] In a particular embodiment said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety and the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 65.
[0274] In a particular embodiment said T cell activating bispecific antigen binding molecule the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, and the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety and the first antigen binding moiety capable of specific binding to CD3 is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged, wherein the first antigen binding moiety capable of specific binding to CD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO: 36, a variable light chain comprising a sequence of SEQ ID NO: 31; and the second and the third antigen binding moiety capable of specific binding to FolR1 comprise a variable heavy chain comprising a sequence of SEQ ID NO: 55, a variable light chain comprising a sequence of SEQ ID NO: 65.
[0275] In one embodiment the T cell activating bispecific antigen binding molecule is monovalent for each antigen. In a particular embodiment the T cell activating bispecific antigen binding molecule can bind to human CD3 and human folate receptor alpha (FolR1) and was made without employing a hetero-dimerization approach, such as, e.g., knob-into-hole technology. For example, the molecule can be produced by employing a common light chain library and CrossMab technology. In a particular embodiment, The variable region of the CD3 binder is fused to the CH1 domain of a standard human IgG1 antibody to form the VLVH crossed molecule (fused to Fc) which is common for both specificities. To generate the crossed counterparts (VHCL), a CD3 specific variable heavy chain domain is fused to a constant human .lamda. light chain whereas a variable heavy chain domain specific for human FolR1 (e.g., isolated from a common light chain library) is fused to a constant human .kappa. light chain. The resulting desired molecule with correctly paired chains comprises both kappa and lambda light chains or fragments thereof. Consequently, this desired bispecific molecule species can be purified from mispaired or homodimeric species with sequential purification steps selecting for kappa and lambda light chain, in either sequence. In one particular embodiment, purification of the desired bispecific antibody employs subsequent purification steps with KappaSelect and LambdaFabSelect columns (GE Healthcare) to remove undesired homodimeric antibodies.
Fc Domain
[0276] The Fc domain of the T cell activating bispecific antigen binding molecule consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment the T cell activating bispecific antigen binding molecule of the invention comprises not more than one Fc domain.
[0277] In one embodiment according the invention the Fc domain of the T cell activating bispecific antigen binding molecule is an IgG Fc domain. In a particular embodiment the Fc domain is an IgG.sub.1 Fc domain. In another embodiment the Fc domain is an IgG.sub.4 Fc domain. In a more specific embodiment, the Fc domain is an IgG.sub.4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG.sub.4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is human. An exemplary sequence of a human IgG.sub.1 Fc region is given in SEQ ID NO:245.
[0278] Fc Domain Modifications Promoting Heterodimerization
[0279] T cell activating bispecific antigen binding molecules according to the invention comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of T cell activating bispecific antigen binding molecules in recombinant production, it will thus be advantageous to introduce in the Fc domain of the T cell activating bispecific antigen binding molecule a modification promoting the association of the desired polypeptides.
[0280] Accordingly, in particular embodiments the Fc domain of the T cell activating bispecific antigen binding molecule according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.
[0281] In a specific embodiment said modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other one of the two subunits of the Fc domain.
[0282] The knob-into-hole technology is described e.g. in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the T cell activating bispecific antigen binding molecule an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
[0283] The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
[0284] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
[0285] In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0286] In a particular embodiment the antigen binding moiety capable of binding to CD3 is fused (optionally via the antigen binding moiety capable of binding to FolR1 on a target cell antigen) to the first subunit of the Fc domain (comprising the "knob" modification). Without wishing to be bound by theory, fusion of the antigen binding moiety capable of binding to CD3 to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties capable of binding to CD3 (steric clash of two knob-containing polypeptides).
[0287] In an alternative embodiment a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
[0288] Fc Domain Modifications Abolishing Fc Receptor Binding and/or Effector Function
[0289] The Fc domain confers to the T cell activating bispecific antigen binding molecule favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the T cell activating bispecific antigen binding molecule to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the T cell activating properties and the long half-life of the antigen binding molecule, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the T cell activating bispecific antigen binding molecule due to the potential destruction of T cells e.g. by NK cells.
[0290] Accordingly, in particular embodiments the Fc domain of the T cell activating bispecific antigen binding molecules according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG.sub.1 Fc domain. In one such embodiment the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgG.sub.1 Fc domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG.sub.1 Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgG.sub.1 Fc domain domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG.sub.1 Fc domain). In one embodiment, the Fc domain domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fc.gamma. receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa, Fc.gamma.RI or Fc.gamma.RIIa, most specifically human Fc.gamma.RIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgG.sub.1 Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgG.sub.1 Fc domain (or the T cell activating bispecific antigen binding molecule comprising a native IgG.sub.1 Fc domain) to FcRn.
[0291] In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the T cell activating bispecific antigen binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fc.gamma. receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa, Fc.gamma.RI or Fc.gamma.RIIa, most specifically human Fc.gamma.RIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or T cell activating bispecific antigen binding molecules of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the T cell activating bispecific antigen binding molecule is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain).
[0292] In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329. In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329. In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331. In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fc.gamma. receptor binding of a human IgG.sub.1 Fc domain, as described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety. WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
[0293] IgG.sub.4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgG.sub.1 antibodies. Hence, in some embodiments the Fc domain of the T cell activating bispecific antigen binding molecules of the invention is an IgG.sub.4 Fc domain, particularly a human IgG.sub.4 Fc domain. In one embodiment the IgG.sub.4 Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P. To further reduce its binding affinity to an Fc receptor and/or its effector function, in one embodiment the IgG.sub.4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E. In another embodiment, the IgG.sub.4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G. In a particular embodiment, the IgG.sub.4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G. Such IgG.sub.4 Fc domain mutants and their Fc.gamma. receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
[0294] In a particular embodiment the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG.sub.1 Fc domain, is a human IgG.sub.1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG.sub.4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G.
[0295] In certain embodiments N-glycosylation of the Fc domain has been eliminated. In one such embodiment the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D).
[0296] In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
[0297] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
[0298] Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific antigen binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fc.gamma.IIIa receptor.
[0299] Effector function of an Fc domain, or a T cell activating bispecific antigen binding molecule comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96.RTM. non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
[0300] In some embodiments, binding of the Fc domain to a complement component, specifically to C1q, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. C1q binding assays may be carried out to determine whether the T cell activating bispecific antigen binding molecule is able to bind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
Biological Properties and Functional Characteristics of T Cell Activating Bispecific Antigen Binding Molecules
[0301] One of skill in the art can appreciate the advantageous efficiency of a molecule that selectively distinguishes between cancerous and non-cancerous, healthy cells. One way to accomplish this goal is by appropriate target selection. Markers expressed exclusively on tumor cells can be employed to selectively target effector molecules or cells to tumor cells while sparing normal cells that do not express such marker. However, in some instances, so called tumor cell markers are also expressed in normal tissue, albeit at lower levels. This expression in normal tissue raises the possibility of toxicity. Thus, there was a need in the art for molecules that can more selectively target tumor cells. The invention described herein provides for T cell activating bispecific antigen binding molecules that selectively target FolR1-positive tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all. In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least two, preferably two, FolR1 binding moieties of relatively low affinity that confer an avidity effect which allows for differentiation between high and low FolR1 expressing cells. Because tumor cells express FolR1 at high or intermediate levels, this embodiment of the invention selectively binds to, and/or induces killing of, tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all. In one embodiment, the T cell activating bispecific antigen binding molecule is in the 2+1 inverted format. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of FolR1-positive tumor cells and not non-tumor cells and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
[0302] In one specific embodiment, the T cell activating bispecific antigen binding molecule does not induce killing of a normal cells having less than about 1000 copies of FolR1 its surface.
[0303] In addition to the above advantageous characteristics, one embodiment of the invention does not require chemical cross linking or a hybrid approach to be produced. Accordingly, in one embodiment, the invention provides for T cell activating bispecific antigen binding molecule capable of production in CHO cells. In one embodiment, the T cell activating bispecific antigen binding molecule comprises humanized and human polypeptides. In one embodiment, the T cell activating bispecific antigen binding molecule does not cause FcgR crosslinking. In one such embodiment, the T cell activating bispecific antigen binding molecule is capable of production in CHO cells and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
[0304] As noted above, some embodiments contemplated herein include T cell activating bispecific antigen binding molecules having two binding moieties that confer specific binding to FolR1 and one binding moiety that confers specificity to the T cell activating antigen CD3, wherein each individual FolR1 binding moiety engages the antigen with low affinity. Because the molecule comprises two antigen binding moieties that confer binding to FolR1, the overall avidity of the molecule, nevertheless, provides effective binding to FolR1-expressing target cells and activation of T cells to induce T cell effector function. Considering that while FolR1 is expressed at various level on tumor cells, it is also expressed at very low levels (e.g., less than about 1000 copies on the cell surface) in certain normal cells, one of skill in the art can readily recognize the advantageous efficiency of such a molecule for use as a therapeutic agent. Such molecule selectively targets tumor cells over normal cells. Such molecule, thus, can be administered to an individual in need thereof with significantly less concern about toxicity resulting from FolR1 positive normal cells compared to molecules that bind to FolR1 with high affinity to induce effector function. In a preferred embodiment, the T cell activating bispecific antigen binding molecules have a monovalent binding affinity to huFolR1 in the micromolar range and an avidity to huFolR1 in the nanomolar range.
[0305] In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K.sub.D of about 10 nM to about 40 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K.sub.D of about 10 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K.sub.D of about 10 nM and about 30 nM, respectively. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of at least about 1000 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of about 1400 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of about 1400 nM and to cynomolgus FolR1 with a monovalent binding K.sub.D of about 5600 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K.sub.D of about 10 nM and with a monovalent binding K.sub.D of about 1400 nM.
[0306] In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K.sub.D of about 5.36 pM to about 4 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K.sub.D of about 4 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds murine FolR1 with an apparent K.sub.D of about 1.5 nM. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of at least about 1000 nM. In a specific embodiment, the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K.sub.D of about 4 nM, binds murine FolR1 with an apparent K.sub.D of about 1.5 nM, and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54. In one embodiment, the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K.sub.D of at least about 1000 nM and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
[0307] As described above, the T cell activating bispecific antigen binding molecules contemplated herein can induce T cell effector function, e.g., cell surface marker expression, cytokine production, T cell mediated killing. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing target cell, such as a human tumor cell, in vitro. In one embodiment, the T cell is a CD8 positive T cell. Examples of FolR1-expressing human tumor cells include but are not limited to Hela, Skov-3, HT-29, and HRCEpiC cells. Other FolR1 positive human cancer cells that can be used for in vitro testing are readily available to the skilled artisan. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing human tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours. Specifically contemplated are T cell activating bispecific antigen binding molecules that induce T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours. In one embodiment, the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours. The EC50 can be measure by methods known in the art, for example by methods disclosed herein by the examples.
[0308] In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments induces upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry. In one embodiment, the T cell is a CD4 positive T cell or a CD8 positive T cell.
[0309] In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to FolR1 expressed on a human tumor cell. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments binds to a conformational epitope on human FolR1. In one embodiment, the T cell activating bispecific antigen binding molecule of any of the above embodiments does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3). In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227). In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:227, to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:230 and to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 or 229. In one specific embodiment, the T cell activating bispecific antigen binding molecule comprises a FolR1 antigen binding moiety that binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 or 229, and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54.
[0310] In one embodiment of the T cell activating bispecific antigen binding molecule of any of the above embodiments, the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:227 and to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228, 229 or 230. In one specific embodiment, the T cell activating bispecific antigen binding molecule comprises a FolR1 antigen binding moiety that binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:227 and to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NO:231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228, 229 or 230, and comprises a CD3 antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties that each comprise the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 275, the heavy chain CDR3 of SEQ ID NO:315, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34.
[0311] With respect to the FolR1, the T cell activating bispecific antigen binding molecules contemplated herein can have agonist, antagonist or neutral effect. Examples of agonist effect include induction or enhancement of signaling through the FolR1 upon engagement by the FolR1 binding moiety with the FolR1 receptor on the target cell. Examples of antagonist activity include abrogation or reduction of signaling through the FolR1 upon engagement by the FolR1 binding moiety with the FolR1 receptor on the target cell. This can, for example, occur by blocking or reducing the interaction between folate with FolR1. Sequence variants of the embodiments disclosed herein having lower affinity while retaining the above described biological properties are specifically contemplated.
Immunoconjugates
[0312] The invention also pertains to immunoconjugates comprising a T cell activating bispecific antigen binding molecule conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Polynucleotides
[0313] The invention further provides isolated polynucleotides encoding a T cell activating bispecific antigen binding molecule as described herein or a fragment thereof.
[0314] Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in SEQ ID NOs:151-226 including functional fragments or variants thereof.
[0315] The polynucleotides encoding T cell activating bispecific antigen binding molecules of the invention may be expressed as a single polynucleotide that encodes the entire T cell activating bispecific antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional T cell activating bispecific antigen binding molecule. For example, the light chain portion of an antigen binding moiety may be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the heavy chain portion of the antigen binding moiety, an Fc domain subunit and optionally (part of) another antigen binding moiety. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding moiety. In another example, the portion of the T cell activating bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally (part of) one or more antigen binding moieties could be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the the other of the two Fc domain subunits and optionally (part of) an antigen binding moiety. When co-expressed, the Fc domain subunits will associate to form the Fc domain.
[0316] In some embodiments, the isolated polynucleotide encodes the entire T cell activating bispecific antigen binding molecule according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptides comprised in the T cell activating bispecific antigen binding molecule according to the invention as described herein.
[0317] In another embodiment, the present invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence as shown in SEQ ID NOs 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 and 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223. In another embodiment, the present invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide sequence as shown in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244.
[0318] In another embodiment, the invention is further directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence shown in SEQ ID NOs 97, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 12, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 246, 247. In another embodiment, the invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a nucleic acid sequence shown in SEQ ID NOs 97, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 12, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 246, 247.
[0319] In another embodiment, the invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 11, 13, 15, 19, 21, 12, 25, 27, 29, 31, 36, 41, 45, 49, 51, 55, 58, 62, 64, 66, 68, 70, 72, 74, 76, 78, 82, 113, 114, 115, 116, 117, 118, 119, 12, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135. In another embodiment, the invention is directed to an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide comprising one or more sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in SEQ ID NOs: 8, 9, 50, 37, 38, and 39. The invention encompasses an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence that encodes the variable region sequence of SEQ ID NOs 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 and 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223 with conservative amino acid substitutions. The invention also encompasses an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or fragment thereof, wherein the polynucleotide comprises a sequence that encodes the polypeptide sequence of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 and 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244 with conservative amino acid substitutions.
[0320] In certain embodiments the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded.
Recombinant Methods
[0321] T cell activating bispecific antigen binding molecules of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of a T cell activating bispecific antigen binding molecule (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) of the invention, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).
[0322] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of the T cell activating bispecific antigen binding molecule is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse .beta.-glucuronidase.
[0323] DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the T cell activating bispecific antigen binding molecule may be included within or at the ends of the T cell activating bispecific antigen binding molecule (fragment) encoding polynucleotide.
[0324] In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a T cell activating bispecific antigen binding molecule of the invention. As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the T cell activating bispecific antigen binding molecules of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of T cell activating bispecific antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the T cell activating bispecific antigen binding molecule for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.
[0325] Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES.TM. technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr.sup.- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).
[0326] Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an antibody, may be engineered so as to also express the other of the antibody chains such that the expressed product is an antibody that has both a heavy and a light chain.
[0327] In one embodiment, a method of producing a T cell activating bispecific antigen binding molecule according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the T cell activating bispecific antigen binding molecule, as provided herein, under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and recovering the T cell activating bispecific antigen binding molecule from the host cell (or host cell culture medium).
[0328] The components of the T cell activating bispecific antigen binding molecule are genetically fused to each other. T cell activating bispecific antigen binding molecule can be designed such that its components are fused directly to each other or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of T cell activating bispecific antigen binding molecules are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence.
[0329] In certain embodiments the one or more antigen binding moieties of the T cell activating bispecific antigen binding molecules comprise at least an antibody variable region capable of binding an antigenic determinant. Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Pat. No. 5,969,108, McCafferty).
[0330] Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used in the T cell activating bispecific antigen binding molecules of the invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate, or human origin. If the T cell activating bispecific antigen binding molecule is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the "guided selection" approach to FR shuffling). Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
[0331] In certain embodiments, the antigen binding moieties useful in the present invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of the T cell activating bispecific antigen binding molecule of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance technique (analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a particular antigen, e.g. an antibody that competes with the V9 antibody for binding to CD3. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In an exemplary competition assay, immobilized antigen (e.g. CD3) is incubated in a solution comprising a first labeled antibody that binds to the antigen (e.g. V9 antibody, described in U.S. Pat. No. 6,054,297) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0332] T cell activating bispecific antigen binding molecules prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the T cell activating bispecific antigen binding molecule binds. For example, for affinity chromatography purification of T cell activating bispecific antigen binding molecules of the invention, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate a T cell activating bispecific antigen binding molecule essentially as described in the Examples. The purity of the T cell activating bispecific antigen binding molecule can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the heavy chain fusion proteins expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing SDS-PAGE (see e.g. FIG. 2). Three bands were resolved at approximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to the predicted molecular weights of the T cell activating bispecific antigen binding molecule light chain, heavy chain and heavy chain/light chain fusion protein.
Assays
[0333] T cell activating bispecific antigen binding molecules provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
[0334] Affinity Assays
[0335] The affinity of the T cell activating bispecific antigen binding molecule for an Fc receptor or a target antigen can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression. Alternatively, binding of T cell activating bispecific antigen binding molecules for different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS). A specific illustrative and exemplary embodiment for measuring binding affinity is described in the following and in the Examples below. According to one embodiment, K.sub.D is measured by surface plasmon resonance using a BIACORE.RTM. T100 machine (GE Healthcare) at 25.degree. C.
[0336] To analyze the interaction between the Fc-portion and Fc receptors, His-tagged recombinant Fc-receptor is captured by an anti-Penta His antibody ("Penta His" disclosed as SEQ ID NO: 306) (Qiagen) immobilized on CM5 chips and the bispecific constructs are used as analytes. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to the supplier's instructions. Anti Penta-His antibody ("Penta-His" disclosed as SEQ ID NO: 306) is diluted with 10 mM sodium acetate, pH 5.0, to 40 .mu.g/ml before injection at a flow rate of 5 .mu.l/min to achieve approximately 6500 response units (RU) of coupled protein. Following the injection of the ligand, 1 M ethanolamine is injected to block unreacted groups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM. For kinetic measurements, four-fold serial dilutions of the bispecific construct (range between 500 nM and 4000 nM) are injected in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25.degree. C. at a flow rate of 30 .mu.l/min for 120 s.
[0337] To determine the affinity to the target antigen, bispecific constructs are captured by an anti human Fab specific antibody (GE Healthcare) that is immobilized on an activated CM5-sensor chip surface as described for the anti Penta-His antibody ("Penta-His" disclosed as SEQ ID NO: 306). The final amount of coupled protein is is approximately 12000 RU. The bispecific constructs are captured for 90 s at 300 nM. The target antigens are passed through the flow cells for 180 s at a concentration range from 250 to 1000 nM with a flowrate of 30 .mu.l/min. The dissociation is monitored for 180 s.
[0338] Bulk refractive index differences are corrected for by subtracting the response obtained on reference flow cell. The steady state response was used to derive the dissociation constant K.sub.D by non-linear curve fitting of the Langmuir binding isotherm. Association rates (k.sub.on) and dissociation rates (k.sub.off) are calculated using a simple one-to-one Langmuir binding model (BIACORE.RTM. T100 Evaluation Software version 1.1.1) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K.sub.D) is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).
[0339] Activity Assays
[0340] Biological activity of the T cell activating bispecific antigen binding molecules of the invention can be measured by various assays as described in the Examples. Biological activities may for example include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the induction of lysis of target cells such as tumor cells, and the induction of tumor regression and/or the improvement of survival.
Compositions, Formulations, and Routes of Administration
[0341] In a further aspect, the invention provides pharmaceutical compositions comprising any of the T cell activating bispecific antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
[0342] Further provided is a method of producing a T cell activating bispecific antigen binding molecule of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining a T cell activating bispecific antigen binding molecule according to the invention, and (b) formulating the T cell activating bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, whereby a preparation of T cell activating bispecific antigen binding molecule is formulated for administration in vivo.
[0343] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more T cell activating bispecific antigen binding molecule dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one T cell activating bispecific antigen binding molecule and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
[0344] The composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. T cell activating bispecific antigen binding molecules of the present invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the T cell activating bispecific antigen binding molecules of the invention.
[0345] Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the T cell activating bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the T cell activating bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the T cell activating bispecific antigen binding molecules of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
[0346] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof
[0347] In addition to the compositions described previously, the T cell activating bispecific antigen binding molecules may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the T cell activating bispecific antigen binding molecules may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0348] Pharmaceutical compositions comprising the T cell activating bispecific antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
[0349] The T cell activating bispecific antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
Therapeutic Methods and Compositions
[0350] Any of the T cell activating bispecific antigen binding molecules provided herein may be used in therapeutic methods. T cell activating bispecific antigen binding molecules of the invention can be used as immunotherapeutic agents, for example in the treatment of cancers.
[0351] For use in therapeutic methods, T cell activating bispecific antigen binding molecules of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
[0352] In one aspect, T cell activating bispecific antigen binding molecules of the invention for use as a medicament are provided. In further aspects, T cell activating bispecific antigen binding molecules of the invention for use in treating a disease are provided. In certain embodiments, T cell activating bispecific antigen binding molecules of the invention for use in a method of treatment are provided. In one embodiment, the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides a T cell activating bispecific antigen binding molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the T cell activating bispecific antigen binding molecule. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in inducing lysis of a target cell, particularly a tumor cell. In certain embodiments, the invention provides a T cell activating bispecific antigen binding molecule for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the T cell activating bispecific antigen binding molecule to induce lysis of a target cell. An "individual" according to any of the above embodiments is a mammal, preferably a human.
[0353] In a further aspect, the invention provides for the use of a T cell activating bispecific antigen binding molecule of the invention in the manufacture or preparation of a medicament. In one embodiment the medicament is for the treatment of a disease in an individual in need thereof. In a further embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for inducing lysis of a target cell, particularly a tumor cell. In still a further embodiment, the medicament is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the medicament to induce lysis of a target cell. An "individual" according to any of the above embodiments may be a mammal, preferably a human.
[0354] In a further aspect, the invention provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention. In one embodiment a composition is administered to said individual, comprising the T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. An "individual" according to any of the above embodiments may be a mammal, preferably a human.
[0355] In a further aspect, the invention provides a method for inducing lysis of a target cell, particularly a tumor cell. In one embodiment the method comprises contacting a target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, particularly a cytotoxic T cell. In a further aspect, a method for inducing lysis of a target cell, particularly a tumor cell, in an individual is provided. In one such embodiment, the method comprises administering to the individual an effective amount of a T cell activating bispecific antigen binding molecule to induce lysis of a target cell. In one embodiment, an "individual" is a human.
[0356] In certain embodiments the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using a T cell activating bispecific antigen binding molecule of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. A skilled artisan readily recognizes that in many cases the T cell activating bispecific antigen binding molecule may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of T cell activating bispecific antigen binding molecule that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.
[0357] In some embodiments, an effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to an individual for the treatment of disease.
[0358] For the prevention or treatment of disease, the appropriate dosage of a T cell activating bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of T cell activating bispecific antigen binding molecule, the severity and course of the disease, whether the T cell activating bispecific antigen binding molecule is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the T cell activating bispecific antigen binding molecule, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
[0359] The T cell activating bispecific antigen binding molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell activating bispecific antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the T cell activating bispecific antigen binding molecule would be in the range from about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligram/kg body weight, about 10 milligram/kg body weight, about 50 milligram/kg body weight, about 100 milligram/kg body weight, about 200 milligram/kg body weight, about 350 milligram/kg body weight, about 500 milligram/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 microgram/kg body weight to about 500 milligram/kg body weight, etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the T cell activating bispecific antigen binding molecule). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
[0360] The T cell activating bispecific antigen binding molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the T cell activating bispecific antigen binding molecules of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
[0361] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC.sub.50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
[0362] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
[0363] Dosage amount and interval may be adjusted individually to provide plasma levels of the T cell activating bispecific antigen binding molecules which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
[0364] In cases of local administration or selective uptake, the effective local concentration of the T cell activating bispecific antigen binding molecules may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
[0365] A therapeutically effective dose of the T cell activating bispecific antigen binding molecules described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of a T cell activating bispecific antigen binding molecule can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD.sub.50 (the dose lethal to 50% of a population) and the ED.sub.50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD.sub.50/ED.sub.50. T cell activating bispecific antigen binding molecules that exhibit large therapeutic indices are preferred. In one embodiment, the T cell activating bispecific antigen binding molecule according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety). The attending physician for patients treated with T cell activating bispecific antigen binding molecules of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
Other Agents and Treatments
[0366] The T cell activating bispecific antigen binding molecules of the invention may be administered in combination with one or more other agents in therapy. For instance, a T cell activating bispecific antigen binding molecule of the invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.
[0367] Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of T cell activating bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above. The T cell activating bispecific antigen binding molecules are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
[0368] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the T cell activating bispecific antigen binding molecule of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. T cell activating bispecific antigen binding molecules of the invention can also be used in combination with radiation therapy.
[0369] In another aspect, the invention provides for a bispecific antibody comprising a) a first antigen-binding site that comprises a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31; and b) a second antigen-binding site that comprises a variable heavy chain domain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ ID NO: 31 for use in combination with an antibody to PD-L1 or FAP-4-1BBL. In one embodiment, the bispecific antibody further comprises a third antigen-binding site that comprises a variable heavy chain domain (VH) of SEQ ID NO: 274 and a variable light chain domain of SEQ ID NO: 31.
Articles of Manufacture
[0370] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a T cell activating bispecific antigen binding molecule of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a T cell activating bispecific antigen binding molecule of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
EXAMPLES
[0371] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
[0372] General Methods
[0373] Recombinant DNA Techniques
[0374] Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturers' instructions. General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5.sup.th ed., NIH Publication No. 91-3242.
[0375] DNA Sequencing
[0376] DNA Sequences were Determined by Standard Double Strand Sequencing at Synergene (Schlieren).
[0377] Gene Synthesis
[0378] Desired gene segments where required were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. In cases where no exact gene sequence was available, oligonucleotide primers were designed based on sequences from closest homologues and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning/sequencing vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed with a 5'-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells.
[0379] Isolation of Primary Human Pan T Cells from PBMCs
[0380] Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors. Briefly, blood was diluted with sterile PBS and carefully layered over a Histopaque gradient (Sigma, H8889). After centrifugation for 30 minutes at 450.times.g at room temperature (brake switched off), part of the plasma above the PBMC containing interphase was discarded. The PBMCs were transferred into new 50 ml Falcon tubes and tubes were filled up with PBS to a total volume of 50 ml. The mixture was centrifuged at room temperature for 10 minutes at 400.times.g (brake switched on). The supernatant was discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps at 4.degree. C. for 10 minutes at 350.times.g). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree. C., 5% CO.sub.2 in the incubator until assay start.
[0381] T cell enrichment from PBMCs was performed using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130-091-156), according to the manufacturer's instructions. Briefly, the cell pellets were diluted in 40 .mu.l cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA, sterile filtered) and incubated with 10 .mu.l Biotin-Antibody Cocktail per 10 million cells for 10 min at 4.degree. C. 30 .mu.l cold buffer and 20 .mu.l Anti-Biotin magnetic beads per 10 million cells were added, and the mixture incubated for another 15 min at 4.degree. C. Cells were washed by adding 10-20.times. the current volume and a subsequent centrifugation step at 300.times.g for 10 min. Up to 100 million cells were resuspended in 500 .mu.l buffer. Magnetic separation of unlabeled human pan T cells was performed using LS columns (Miltenyi Biotec #130-042-401) according to the manufacturer's instructions. The resulting T cell population was counted automatically (ViCell) and stored in AIM-V medium at 37.degree. C., 5% CO.sub.2 in the incubator until assay start (not longer than 24 h).
[0382] Isolation of Primary Human Naive T Cells from PBMCs
[0383] Peripheral blood mononuclar cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors. T-cell enrichment from PBMCs was performed using the Naive CD8.sup.+ T cell isolation Kit from Miltenyi Biotec (#130-093-244), according to the manufacturer's instructions, but skipping the last isolation step of CD8.sup.+ T cells (also see description for the isolation of primary human pan T cells).
[0384] Isolation of Murine Pan T Cells from Splenocytes
[0385] Spleens were isolated from C57BL/6 mice, transferred into a GentleMACS C-tube (Miltenyi Biotech #130-093-237) containing MACS buffer (PBS+0.5% BSA+2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtain single-cell suspensions according to the manufacturer's instructions. The cell suspension was passed through a pre-separation filter to remove remaining undissociated tissue particles. After centrifugation at 400.times.g for 4 min at 4.degree. C., ACK Lysis Buffer was added to lyse red blood cells (incubation for 5 min at room temperature). The remaining cells were washed with MACS buffer twice, counted and used for the isolation of murine pan T cells. The negative (magnetic) selection was performed using the Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861), following the manufacturer's instructions. The resulting T cell population was automatically counted (ViCell) and immediately used for further assays.
[0386] Isolation of Primary Cynomolgus PBMCs from Heparinized Blood
[0387] Peripheral blood mononuclar cells (PBMCs) were prepared by density centrifugation from fresh blood from healthy cynomolgus donors, as follows: Heparinized blood was diluted 1:3 with sterile PBS, and Lymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterile PBS. Two volumes of the diluted blood were layered over one volume of the diluted density gradient and the PBMC fraction was separated by centrifugation for 30 min at 520.times.g, without brake, at room temperature. The PBMC band was transferred into a fresh 50 ml Falcon tube and washed with sterile PBS by centrifugation for 10 min at 400.times.g at 4.degree. C. One low-speed centrifugation was performed to remove the platelets (15 min at 150.times.g, 4.degree. C.), and the resulting PBMC population was automatically counted (ViCell) and immediately used for further assays.
Example 1
Purification of Biotinylated Folate Receptor-Fc Fusions
[0388] To generate new antibodies against human FolR1 the following antigens and screening tools were generated as monovalent Fc fusion proteins (the extracellular domain of the antigen linked to the hinge region of Fc-knob which is co-expressed with an Fc-hole molecule). The antigen genes were synthesized (Geneart, Regensburg, Germany) based on sequences obtained from GenBank or SwissProt and inserted into expression vectors to generate fusion proteins with Fc-knob with a C-terminal Avi-tag for in vivo or in vitro biotinylation. In vivo biotinylation was achieved by co-expression of the bacterial birA gene encoding a bacterial biotin ligase during production. Expression of all genes was under control of a chimeric MPSV promoter on a plasmid containing an oriP element for stable maintenance of the plasmids in EBNA containing cell lines.
[0389] For preparation of the biotinylated monomeric antigen/Fc fusion molecules, exponentially growing suspension HEK293 EBNA cells were co-transfected with three vectors encoding the two components of fusion protein (knob and hole chains) as well as BirA, an enzyme necessary for the biotinylation reaction. The corresponding vectors were used at a 9.5:9.5:1 ratio ("antigen ECD-Fc knob-avi tag": "Fc hole": "BirA").
[0390] For protein production in 500 ml shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 minutes at 210 g, and supernatant was replaced by pre-warmed CD CHO medium. Expression vectors were resuspended in 20 mL of CD CHO medium containing 200 .mu.g of vector DNA. After addition of 540 .mu.L of polyethylenimine (PEI), the solution was mixed for 15 seconds and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to a 500 mL shake flask and incubated for 3 hours at 37.degree. C. in an incubator with a 5% CO.sub.2 atmosphere. After the incubation, 160 mL of F17 medium was added and cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% Feed 1 (Lonza) were added to the culture. The production medium was also supplemented with 100 .mu.M biotin. After 7 days of culturing, the cell supernatant was collected by spinning down cells for 15 min at 210 g. The solution was sterile filtered (0.22 .mu.m filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and kept at 4.degree. C.
[0391] Secreted proteins were purified from cell culture supernatants by affinity chromatography using Protein A, followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. The bound protein was eluted using a linear pH-gradient created over 20 column volumes of 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. The column was then washed with 10 column volumes of 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0.
[0392] pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5 M sodium phosphate, pH 8.0. The protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0.
[0393] The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the FolR1-Fc-fusion was analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). The aggregate content of samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25.degree. C. Purified antigen-Fc-fusion proteins were analyzed by surface plasmon resonance assays using commercially available antibodies to confirm correct and natural conformation of the antigens (data not shown).
TABLE-US-00002 TABLE 1 Antigens produced for isolation, selection and counter selection of human FolR1 antibodies ECD Accession Seq ID Antigen (aa) number Sequence No human 25-234 P15328 RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWR 227 FolR1 KNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKR HFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLC KEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVG AACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRC IQMWFDPAQGNPNEEVARFYAAAM human 17-230 P14207 TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSP 228 FolR2 WKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPAC KRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVP LCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCP AGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSG RCIQMWFDSAQGNPNEEVARFYAAAMHVN human 24-243 P41439 SARARTDLLNVCMNAKHHKTQPSPEDELYGQCSPWKK 229 FolR3 NACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRH FIQDSCLYECSPNLGPWIRQVNQSWRKERILNVPLCK EDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAGA LCSTFESYFPTPAALCEGLWSHSFKVSNYSRGSGRCI QMWFDSAQGNPNEEVAKFYAAAMNAGAPSRGIIDS murine 25-232 P35846 TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTN 230 FolR1 SCCSTNTSQEAHKDISYLYRENWNHCGTMTSECKRHE IQDTCLYECSPNLGPWIQQVDQSWRKERILDVPLCKE DCQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGAS CHPFTFYFPTSAALCEEIWSHSYKLSNYSRGSGRCIQ MWFDPAQGNPNEEVARFYAEAMS cynomolgus 25-234 G7PR14 EAQTRTARARTELLNVCMNAKHHKEKPGPEDKLHEQC 231 FolR1 RPWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAP ACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLN VPLCKEDCERWWEDCRTSYCKSNWHKGWNWTSGFNKC PVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSRGS GRCIQMWFDPAQGNPNEEVARFYAAAMS
TABLE-US-00003 TABLE 2 Summary of the yield and final monomer content of the FolR-Fc-fusions. Monomer [%] Antigen (SEC) Yield huFolR1 100 30 mg/L cyFolR1 100 32 mg/L muFolR1 100 31 mg/L huFolR2 100 16 mg/L huFolR3 95 38 mg/L
Example 2
Generation of Common Light Chain with CD3.epsilon. Specificity
[0394] The T cell activating bispecific molecules described herein comprise at least one CD3 binding moiety. This moiety can be generated by immunizing laboratory animals, screening phage library or using known anti-CD3 antibodies. The common light chain with CD3.epsilon. specificity was generated by humanizing the light chain of a murine parental anti-CD3.epsilon. antibody (CH2527). For humanization of an antibody of non-human origin, the CDR residues from the non-human antibody (donor) have to be transplanted onto the framework of a human (acceptor) antibody. Generally, acceptor framework sequences are selected by aligning the sequence of the donor to a collection of potential acceptor sequences and choosing one that has either reasonable homology to the donor, or shows similar amino acids at some positions critical for structure and activity. In the present case, the search for the antibody acceptor framework was performed by aligning the mouse VL-domain sequence of the parental antibody to a collection of human germline sequences and choosing the human sequence that showed high sequence identity. Surprisingly, a good match in terms of framework sequence homology was found in a rather infrequent human light chain belonging to the V-domain family 7 of the lambda type, more precisely, hVL_7_46 (IMGT nomenclature, GenBank Acc No. Z73674). This infrequent human light chain was subsequently chosen as acceptor framework for humanization of the light chain of CH2527. The three complementarity determining regions (CDRs) of the mouse light chain variable domain were grafted onto this acceptor framework. Since the framework 4 region is not part of the variable region of the germline V-gene, the alignment for this region (J-element) was done individually. Hence the IGLJ3-02 sequence was chosen for humanization of this light chain.
[0395] Thirteen humanized variants were generated (CH2527-VL7_46-1 to VL7_46-10, VL7_46-12 to VL7_46-14). These differ in framework residues (and combinations thereof) that were back-mutated to the murine V-domain sequence or in CDR-residues (Kabat definition) that could be kept identical to the human germline sequence. The following framework residues outside the CDRs were back-mutated to the murine residues in the final humanized VL-domain variant VL7_46-13 (murine residues listed): V36, E38, F44, G46, G49, and G57, respectively. The human J-element IGLJ3-02 was 100% identical to the J-element of the murine parental antibody.
Example 3
SPR Assessment of Humanized Variants with CD3.epsilon. Specificity
[0396] Humanized VL variants were assessed as chimera in a 2+1 classical format (FIG. 1D), i.e. humanized light chain V-domains were paired with murine heavy chain V-domains. SPR assessment was carried out on a ProteOn XPR36 instrument (Bio-Rad). More precisely, the variants were captured directly from the culture supernatant on an anti-Fab derivatized GLM sensorchip (Goat Anti-Human IgG, F(ab')2 Fragment Specific, Jackson ImmunoResearch) in vertical orientation. The following analytes were subsequently injected horizontally as single concentrations to assess binding to human and cynomolgus CD3.epsilon.: 3 .mu.M hu CD3.epsilon.(-1-26)-Fc(knob)-avi (ID807) and 2.5 .mu.M cy CD3.epsilon.-(-1-26)-Fc(knob)-Avi-Fc(hole) (ID873), respectively. Binding responses were qualitatively compared to binding of the murine control construct and graded+(comparable binding observed), +/-(reduced binding observed) and -(no binding observed). The capture antibody was regenerated after each cycle of ligand capture and analyte binding and the murine construct was re-injected at the end of the study to confirm the activity of the capture surface. The results are summarized in Table 3.
TABLE-US-00004 TABLE 3 Qualitative binding assessment based on SPR for the humanized light chain variants combined with the murine heavy chain of CH2527. Only the humanized light chain variant that was finally chosen, CH2527-VL7_46-13, highlighted in bold letters, exhibited comparable binding to human and cynomolgus CD3.epsilon.. humanized VL variant binding to CD3.epsilon. murine_CH2527-VL + CH2527-VL7_46-1 - CH2527-VL7_46-2 - CH2527-VL7_46-3 - CH2527-VL7_46-4 - CH2527-VL7_46-5 - CH2527-VL7_46-6 - CH2527-VL7_46-7 - CH2527-VL7_46-8 - CH2527-VL7_46-9 - CH2527-VL7_46-10 - CH2527-VL7_46-12 +/- CH2527-VL7_46-13 + CH2527-VL7_46-14 -
Example 4
Properties of Humanized Common Light Chain with CD3.epsilon. Specificity
[0397] The light chain V-domain variant that was chosen for the humanized lead molecule is VL7_46-13. The degree of humanness, i.e. the sequence homology of the humanized V-domain to the human germline V-domain sequence was determined. For VL7_46-13, the overall sequence identity with the closest human germline homolog is 65% before humanization and 80% afterwards. Omitting the CDR regions, the sequence identity is 92% to the closest human germline homolog. As can be seen from Table 3, VL7_46-13 is the only humanized VL variant out of a panel of 13 variants that showed comparable binding to the parental murine antibody and also retained its cross-reactivity to cynomolgus CD3.epsilon.. This result indicates that it was not trivial to humanize the murine VL-domain without losing binding affinity to CD3.epsilon. which required several back-mutations to murine framework residues (in particular G46) while retaining G24 in CDR1. In addition, this result shows that the VL-domain plays a crucial role in target recognition. Importantly, the humanized VL-domain VL7_46-13 based on an infrequent human germline belonging to the V-domain family 7 of the lambda type and retaining affinity and specificity for CD3.epsilon., is also suitable to be used as a common light chain in phage-displayed antibody libraries of the Fab-format and enables successful selection for novel specificities which greatly facilitates the generation and production of bispecific molecules binding to CD3.epsilon. and e.g. a tumor target and sharing the same `common` light chain.
Example 5
Generation of a Phage Displayed Antibody Library Using a Human Germ-Line Common Light Chain Derived from HVK1-39
[0398] Several approaches to generate bispecific antibodies that resemble full length human IgG utilize modifications in the Fc region that induce heterodimerization of two distinct heavy chains. Such examples include knobs-into-holes (Merchant et al., Nat Biotechnol. 1998 July; 16(7):677-81) SEED (Davis et al., Protein Eng Des Sel. 2010 April; 23(4):195-202) and electrostatic steering technologies (Gunasekaran et al., J Biol Chem. 2010 Jun. 18; 285(25):19637-46). Although these approaches enable effective heterodimerization of two distinct heavy chains, appropriate pairing of cognate light and heavy chains remains a problem. Usage of a common light chain (LC) can solve this issue (Merchant, et al. Nat Biotech 16, 677-681 (1998)).
[0399] Here, we describe the generation of an antibody library for the display on a M13 phage. Essentially, we designed a multi framework library for the heavy chain with one constant (or "common") light chain. This library is designed for generating multispecific antibodies without the need to use sophisticated technologies to avoid light chain mispairing.
[0400] By using a common light chain the production of these molecules can be facilitated as no mispairing occurs any longer and the isolation of a highly pure bispecific antibody is facilitated. As compared to other formats the use of Fab fragments as building blocks as opposed to e.g. the use of scFv fragments results in higher thermal stability and the lack of scFv aggregation and intermolecular scFv formation.
[0401] Library Generation
[0402] In the following the generation of an antibody library for the display on M13 phage is described. Essentially, we designed a multi framework library for the heavy chain with one constant (or "common") light chain.
[0403] We used these heavy chains in the library (GenBank Accession Numbers in brackets):
[0404] IGHV1-46*01 (X92343) (SEQ ID NO:104),
[0405] IGHV1-69*06 (L22583), (SEQ ID NO:105)
[0406] IGHV3-15*01 (X92216), (SEQ ID NO:106)
[0407] IGHV3-23*01 (M99660), (SEQ ID NO:107)
[0408] IGHV4-59*01 (AB019438), (SEQ ID NO:108)
[0409] IGHV5-51*01 (M99686), (SEQ ID NO:109)
[0410] All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06 which uses IGHJ6 sequence.
[0411] The design of the randomization included the CDR-H1, CDR-H2, and CDR-H3. For CDR-H1 and CDR-H2 a "soft" randomization strategy was chosen, and the randomization oligonucleotides were such that the codon for the amino acid of the germ-line sequence was present at 50%. All other amino acids, except cysteine, were summing up for the remaining 50%. In CDR-H3, where no germ-line amino acid is present due to the presence of the genetic D-element, oligonucleotides were designed that allow for the usage of randomized inserts between the V-element and the J-element of 4 to 9 amino acids in length. Those oligonucleotides contained in their randomized part e.g. The three amino acids G/Y/S are present to 15% each, those amino acids A/D/T/R/P/L/V/N/W/F/I/E are present to 4.6% each.
[0412] Exemplary methods for generation of antibody libraries are described in Hoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-413; Lee et., al J. Mol. Biol. (2004) 340, 1073-1093.
[0413] The light chain is derived from the human sequence hVK1-39, and is used in an unmodified and non-randomized fashion. This will ensure that the same light chain can be used for other projects without additional modifications.
[0414] Exemplary Library Selection:
[0415] Selections with all affinity maturation libraries are carried out in solution according to the following procedure using a monomeric and biotinylated extracellular domain of a target antigen X. 1. 10 12 phagemid particles of each library are bound to 100 nM biotinylated soluble antigen for 0.5 h in a total volume of 1 ml. 2. Biotinylated antigen is captured and specifically bound phage particles are isolated by addition of .about.5.times.10 7 streptavidin-coated magnetic beads for 10 min. 3. Beads are washed using 5-10.times.1 ml PBS/Tween20 and 5-10.times.1 ml PBS. 4. Elution of phage particles is done by addition of 1 ml 100 mM TEA (triethylamine) for 10 min and neutralization by addition of 500 ul 1M Tris/HCl pH 7.4 and 5. Re-infection of exponentially growing E. coli TG1 bacteria, infection with helper phage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles is applied in subsequent selection rounds. Selections are carried out over 3-5 rounds using either constant or decreasing (from 10A-7M to 2.times.10 -9M) antigen concentrations. In round 2, capture of antigen/phage complexes is performed using neutravidin plates instead of streptavidin beads. All binding reactions are supplemented either with 100 nM bovine serum albumin, or with non-fat milk powder in order to compete for unwanted clones arising from mere sticky binding of the antibodies to the plastic support.
[0416] Selections are being carried out over three or four rounds using decreasing antigen concentrations of the antigen starting from 100 nM and going down to 5 nM in the final selection round. Specific binders are defined as signals ca. 5.times. higher than background and are identified by ELISA. Specific binders are identified by ELISA as follows: 100 .mu.l of 10 nM biotinylated antigen per well are coated on neutravidin plates. Fab-containing bacterial supernatants are added and binding Fabs are detected via their Flag-tags by using an anti-Flag/HRP secondary antibody. ELISA-positive clones are bacterially expressed as soluble Fab fragments in 96-well format and supernatants are subjected to a kinetic screening experiment by SPR-analysis using ProteOn XPR36 (BioRad). Clones expressing Fabs with the highest affinity constants are identified and the corresponding phagemids are sequenced. For further characterization, the Fab sequences are amplified via PCR from the phagemid and cloned via appropriate restriction sites into human IgG1 expression vectors for mammalian production.
[0417] Generation of a Phage Displayed Antibody Library Using a Humanized CD3.epsilon. Specific Common Light Chain
[0418] Here, the generation of an antibody library for the display on M13 phage is described. Essentially, we designed a multi framework library for the heavy chain with one constant (or "common") light chain. This library was designed for the generation of Fc-containing, but FcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PG isotype in which one or two Fab recognize a tumor surface antigen expressed on a tumor cell whereas the remaining Fab arm of the antibody recognizes CD3e on a T cell.
[0419] Library Generation
[0420] In the following the generation of an antibody library for the display on M13 phage is described. Essentially, we designed a multi framework library for the heavy chain with one constant (or "common") light chain. This library is designed solely for the generation of Fc-containing, but FcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PG isotype.
[0421] Diversity was introduced via randomization oligonucleotides only in the CDR3 of the different heavy chains. Methods for generation of antibody libraries are well known in the art and are described in (Hoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-413; or in: Lee et., al J. Mol. Biol. (2004) 340, 1073-1093).
[0422] We used these heavy chains in the library:
[0423] IGHV1-46*01 (X92343), (SEQ ID NO:104)
[0424] IGHV1-69*06 (L22583), (SEQ ID NO:105)
[0425] IGHV3-15*01 (X92216), (SEQ ID NO:106)
[0426] IGHV3-23*01 (M99660), (SEQ ID NO:107)
[0427] IGHV4-59*01 (AB019438), (SEQ ID NO:108)
[0428] IGHV5-51*01 (M99686), (SEQ ID NO:109)
[0429] We used the light chain derived from the humanized human and Cynomolgus CD3.epsilon. specific antibody CH2527 in the library: (VL7_46-13; SEQ ID NO:112). This light chain was not randomized and used without any further modifications in order to ensure compatibility with different bispecific binders.
[0430] All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06 which uses IGHJ6 sequence. The design of the randomization focused on the CDR-H3 only, and PCR oligonucleotides were designed that allow for the usage of randomized inserts between the V-element and the J-element of 4 to 9 amino acids in length.
Example 6
Selection of Antibody Fragments from Common Light Chain Libraries (Comprising Light Chain with CD3.epsilon. Specificity) to FolR1
[0431] The antibodies 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8, 19H3, 20G6, and 20H7 comprising the common light chain VL7_46-13 with CD3.epsilon. specificity were obtained by phage display selections against different species (human, cynomolgus and murine) of FolR1. Clones 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 21D1, 16F12, 19H3, 20G6, and 20H7 were selected from a sub-library in which the common light chain was paired with a heavy chain repertoire based on the human germline VH1_46. In this sub-library, CDR3 of VH1_46 has been randomized based on 6 different CDR3 lengths. Clones 16D5, 15E12, 21A5, and 21G8 were selected from a sub-library in which the common light chain was paired with a heavy chain repertoire based on the human germline VH3_15. In this sub-library, CDR3 of VH3_15 has been randomized based on 6 different CDR3 lengths. In order to obtain species cross-reactive (or murine FolR1-reactive) antibodies, the different species of FolR1 were alternated (or kept constant) in different ways over 3 rounds of biopanning: 16A3 and 15A1 (human-cynomolgus-human FolR1); 18D3 (cynomolgus-human-murine FolR1); 19E5 and 19A4 (3 rounds against murine FolR1); 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8 (human-cynomolgus-human FolR1); 19H3, 20G6, and 20H7 (3 rounds against murine FolR1).
[0432] Human, murine and cynomolgus FolR1 as antigens for the phage display selections as well as ELISA- and SPR-based screenings were transiently expressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and in vivo site-specifically biotinylated via co-expression of BirA biotin ligase at the avi-tag recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc knob chain). In order to assess the specificity to FolR1, two related receptors, human FolR2 and FolR3 were generated in the same way.
[0433] Selection rounds (biopanning) were performed in solution according to the following pattern:
[0434] 1. Pre-clearing of -10.sup.12 phagemid particles on maxisorp plates coated with 10 ug/ml of an unrelated human IgG to deplete the libraries of antibodies recognizing the Fc-portion of the antigen.
[0435] 2. Incubating the non-Fc-binding phagemid particles with 100 nM biotinylated human, cynomolgus, or murine FolR1 for 0.5 h in the presence of 100 nM unrelated non-biotinylated Fc knob-into-hole construct for further depletion of Fc-binders in a total volume of 1 ml.
[0436] 3. Capturing the biotinylated FolR1 and attached specifically binding phage by transfer to 4 wells of a neutravidin pre-coated microtiter plate for 10 min (in rounds 1 & 3).
[0437] 4. Washing the respective wells using 5.times.PBS/Tween20 and 5.times.PBS.
[0438] 5. Eluting the phage particles by addition of 250 ul 100 mM TEA (triethylamine) per well for 10 min and neutralization by addition of 500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.
[0439] 6. Post-clearing of neutralized eluates by incubation on neutravidin pre-coated microtiter plate with 100 nM biotin-captured FolR2 or FolR3 for final removal of Fc- and unspecific binders.
[0440] 7. Re-infection of log-phase E. coli TG1 cells with the supernatant of eluted phage particles, infection with helperphage VCSM13, incubation on a shaker at 30.degree. C. over night and subsequent PEG/NaCl precipitation of phagemid particles to be used in the next selection round.
[0441] Selections were carried out over 3 rounds using constant antigen concentrations of 100 nM. In round 2, in order to avoid enrichment of binders to neutravidin, capture of antigen:phage complexes was performed by addition of 5.4.times.10.sup.7 streptavidin-coated magnetic beads. Specific binders were identified by ELISA as follows: 100 ul of 25 nM biotinylated human, cynomolgus, or murine FolR1 and 10 ug/ml of human IgG were coated on neutravidin plates and maxisorp plates, respectively. Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags using an anti-Flag/HRP secondary antibody. Clones exhibiting signals on human FolR1 and being negative on human IgG were short-listed for further analyses and were also tested in a similar fashion against the remaining two species of FolR1. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.
[0442] Affinities (K.sub.D) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25.degree. C. with biotinylated human, cynomolgus, and murine FolR1 as well as human FolR2 and FolR3 (negative controls) immobilized on NLC chips by neutravidin capture. Immobilization of antigens (ligand): Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 .mu.g/ml, then injected at 30 .mu.l/minute in vertical orientation. Injection of analytes: For `one-shot kinetics` measurements, injection direction was changed to horizontal orientation, two-fold dilution series of purified Fab (varying concentration ranges) were injected simultaneously along separate channels 1-5, with association times of 200 s, and dissociation times of 600 s. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for referencing. Association rate constants (k.sub.on) and dissociation rate constants (k.sub.off) were calculated using a simple one-to-one Langmuir binding model in ProteOn Manager v3.1 software by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K.sub.D) was calculated as the ratio k.sub.off/k.sub.on. Table 4 lists the equilibrium dissociation constants (K.sub.D) of the selected clones specific for FolR1.
TABLE-US-00005 TABLE 4 Equilibrium dissociation constants (KD) for anti-FolR1 antibodies (Fab-format) selected by phage display from common light chain sub-libraries comprising VL7_46-13, a humanized light chain specific for CD3.epsilon.. KD in nM. huFolR1 muFolR1 huFolR2 huFolR3 Clone [nM] cyFolR1 [nM] [nM] [nM] [nM] 16A3 21.7 18 very weak no binding no binding 15A1 30.9 17.3 very weak no binding no binding 18D3 93.6 40.2 very weak no binding no binding 19E5 522 276 19.4 no binding no binding 19A4 2050 4250 43.1 no binding no binding 15H7 13.4 72.5 no binding no binding no binding 15B6 19.1 13.9 no binding no binding no binding 16D5 39.5 114 no binding no binding no binding 15E12 55.7 137 no binding no binding no binding 21D1 62.6 32.1 no binding no binding no binding 16F12 68 90.9 no binding no binding no binding 21A5 68.8 131 no binding no binding no binding 21G8 130 261 no binding no binding no binding 19H3 no binding no binding 89.7 no binding no binding 20G6 no binding no binding 78.5 no binding no binding
Example 7
Selection of Antibody Fragments from Generic Multi-Framework Libraries to FolR1
[0443] The antibodies 11F8, 36F2, 9D11, 5D9, 6B6, and 14E4 were obtained by phage display selections based on generic multi-framework sub-libraries against different species (human, cynomolgus and murine) of FolR1. In these multi-framework sub-libraries, different VL-domains with randomized CDR3 (3 different lengths) are paired with different VH-domains with randomized CDR3 (6 different lengths). The selected clones are of the following VL/VH pairings: 11F8 (Vk_1_5/VH_1_69), 36F2 (Vk_3_20/VH_1_46), 9D11 (Vk2D_28/VH1_46), 5D9 (Vk3_20/VH1_46), 6B6 (Vk3_20/VH1_46), and 14E4 (Vk3_20/VH3_23). In order to obtain species cross-reactive (or murine FolR1-reactive) antibodies, the different species of FolR1 were alternated (or kept constant) in different ways over 3 or 4 rounds of biopanning: 11F8 (cynomolgus-murine-human FolR1); 36F2 (human-murine-cynomolgus-murine FolR1); 9D11 (cynomolgus-human-cynomolgus FolR1); 5D9 (human-cynomolgus-human FolR1); 6B6 (human-cynomolgus-human FolR1) and 14E4 (3 rounds against murine FolR1).
[0444] Human, murine and cynomolgus FolR1 as antigens for the phage display selections as well as ELISA- and SPR-based screenings were transiently expressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and in vivo site-specifically biotinylated via co-expression of BirA biotin ligase at the avi-tag recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc knob chain). In order to assess the specificity to FolR1, two related receptors, human FolR2 and FolR3 were generated in the same way.
[0445] Selection rounds (biopanning) were performed in solution according to the following pattern:
[0446] 1. Pre-clearing of .about.10.sup.12 phagemid particles on maxisorp plates coated with 10 ug/ml of an unrelated human IgG to deplete the libraries of antibodies recognizing the Fc-portion of the antigen.
[0447] 2. Incubating the non-Fc-binding phagemid particles with 100 nM biotinylated human, cynomolgus, or murine FolR1 for 0.5 h in the presence of 100 nM unrelated non-biotinylated Fc knob-into-hole construct for further depletion of Fc-binders in a total volume of 1 ml.
[0448] 3. Capturing the biotinylated FolR1 and attached specifically binding phage by transfer to 4 wells of a neutravidin pre-coated microtiter plate for 10 min (in rounds 1 & 3).
[0449] 4. Washing the respective wells using 5.times.PBS/Tween20 and 5.times.PBS.
[0450] 5. Eluting the phage particles by addition of 250 ul 100 mM TEA (triethylamine) per well for 10 min and neutralization by addition of 500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.
[0451] 6. Post-clearing of neutralized eluates by incubation on neutravidin pre-coated microtiter plate with 100 nM biotin-captured FolR2 or FolR3 for final removal of Fc- and unspecific binders.
[0452] 7. Re-infection of log-phase E. coli TG1 cells with the supernatant of eluted phage particles, infection with helperphage VCSM13, incubation on a shaker at 30.degree. C. over night and subsequent PEG/NaCl precipitation of phagemid particles to be used in the next selection round.
[0453] Selections were carried out over 3 rounds using constant antigen concentrations of 100 nM. In round 2 and 4, in order to avoid enrichment of binders to neutravidin, capture of antigen:phage complexes was performed by addition of 5.4.times.10.sup.7 streptavidin-coated magnetic beads. Specific binders were identified by ELISA as follows: 100 ul of 25 nM biotinylated human, cynomolgus, or murine FolR1 and 10 ug/ml of human IgG were coated on neutravidin plates and maxisorp plates, respectively.
[0454] Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags using an anti-Flag/HRP secondary antibody. Clones exhibiting signals on human FolR1 and being negative on human IgG were short-listed for further analyses and were also tested in a similar fashion against the remaining two species of FolR1. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.
[0455] Affinities (K.sub.D) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25.degree. C. with biotinylated human, cynomolgus, and murine FolR1 as well as human FolR2 and FolR3 (negative controls) immobilized on NLC chips by neutravidin capture. Immobilization of antigens (ligand): Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 .mu.g/ml, then injected at 30 .mu.l/minute in vertical orientation. Injection of analytes: For `one-shot kinetics` measurements, injection direction was changed to horizontal orientation, two-fold dilution series of purified Fab (varying concentration ranges) were injected simultaneously along separate channels 1-5, with association times of 150 or 200 s, and dissociation times of 200 or 600 s, respectively. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for referencing. Association rate constants (k.sub.on) and dissociation rate constants (k.sub.off) were calculated using a simple one-to-one Langmuir binding model in ProteOn Manager v3.1 software by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K.sub.D) was calculated as the ratio k.sub.off/k.sub.on. Table 5 lists the equilibrium dissociation constants (K.sub.D) of the selected clones specific for FolR1.
TABLE-US-00006 TABLE 5 Equilibrium dissociation constants (K.sub.D) for anti-FolR1 antibodies (Fab-format) selected by phage display from generic multi-framework sub-libraries. K.sub.D in nM. K.sub.D (nM) Clone huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 11F8 632 794 1200 no binding no binding 36F2 1810 1640 737 no binding no binding 9D11 8.64 5.29 no binding no binding no binding 5D9 8.6 5.9 no binding no binding no binding 6B6 14.5 9.4 no binding no binding no binding 14E4 no binding no binding 6.09 no binding no binding
Example 8
Production and Purification of Novel FolR1 Binders in IgG and T-Cell Bispecific Formats
[0456] To identify FolR1 binders which are able to induce T-cell dependent killing of selected target cells the antibodies isolated from a common light chain- or Fab-library were converted into the corresponding human IgG1 format. In brief, the variable heavy and variable light chains of unique FolR1 binders from phage display were amplified by standard PCR reactions using the Fab clones as the template. The PCR products were purified and inserted (either by restriction endonuclease and ligase based cloning, or by `recombineering` using the InFusion kit from Invitrogen) into suitable expression vectors in which they are fused to the appropriate human constant heavy or human constant light chain. The expression cassettes in these vectors consist of a chimeric MPSV promoter and a synthetic polyadenylation site. In addition, the plasmids contain the oriP region from the Epstein Barr virus for the stable maintenance of the plasmids in HEK293 cells harboring the EBV nuclear antigen (EBNA). After PEI mediated transfection the antibodies were transiently produced in HEK293 EBNA cells and purified by standard ProteinA affinity chromatography followed by size exclusion chromatography as described:
[0457] Transient Transfection and Production
[0458] All (bispecific) antibodies (if not obtained from a commercial source) used herein were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells are centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 15 min at 210.times.g, the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4.degree. C. After production the supernatants were harvested and the antibody containing supernatants were filtered through 0.22 .mu.m sterile filters and stored at 4.degree. C. until purification.
[0459] Antibody Purification
[0460] All molecules were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA FF (GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 3, 100 mM NaCl, 100 mM glycine) over 12 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 0.5 M Na.sub.2HPO.sub.4 pH 8.0). Samples were concentrated to 2 ml using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a HiLoad.TM. 16/60 Superdex.TM. 200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. Fractions containing less than 2% oligomers were pooled and concentrated to final concentration of 1-1.5 mg/ml using ultra concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N.sub.2 and stored at -80.degree. C.
[0461] Based on in vitro characterization results selected binders were converted into a T-cell bispecific format. In these molecules the FolR1:CD3 binding moieties are arranged in a 2:1 order with the FolR1 Fabs being located at the N-terminus. For clones isolated from the standard Fab library the CD3 binding part was generated as a CrossFab (CH1C.kappa. crossing) while for the clones from the common light chain library no crossing was necessary. These bispecific molecules were produced and purified analogously to the IgGs.
TABLE-US-00007 TABLE 6 Yield and monomer content of novel FolR1 binders in IgG and TCB format, respectively. IgG TCB Yield Monomer Yield Monomer # Clone Library [mg/L] [%] [mg/L} [%] 1 11F8 Fab 8.03 96.26 -- -- 2 14E4 Fab 8.90 98.12 -- -- 3 15B6 CLC 7.72 100.00 -- -- 4 15E12 CLC 6.19 100.00 -- -- 5 15H7 CLC 8.94 100.00 -- -- 6 16A3 CLC 0.60 n.d. -- -- 7 16D5 CLC 36.50 96.96 4.36 97.19 8 16F12 CLC 5.73 97.17 -- -- 9 18D3 CLC 0.90 n.d. -- -- 10 19A4 CLC 38.32 100.00 37.50 100.00 11 19E5 CLC 46.09 100.00 -- -- 12 19H3 CLC 7.64 100.00 -- -- 13 20G6 CLC 24.00 100.00 -- -- 14 20H7 CLC 45.39 100.00 -- -- 15 21A5 CLC 1.38 98.56 47.31 95.08 16 21D1 CLC 5.47 100.00 -- -- 17 21G8 CLC 6.14 97.28 9.27 100.00 18 36F2 Fab 11.22 100.00 18.00 100.00 19 5D9 Fab 20.50 100.00 0.93 97.32 20 6B6 Fab 3.83 100.00 4.17 91.53 21 9D11 Fab 14.61 100.00 2.63 100.00 CLC: Common light chain
Example 9
2+1 and 1+1 T-Cell Bispecific Formats
[0462] Four different T-cell bispecific formats were prepared for one common light chain binder (16D5) and three formats for one binder from the Fab library (9D11) to compare their killing properties in vitro.
[0463] The standard format is the 2+1 inverted format as already described (FolR1:CD3 binding moieties arranged in a 2:1 order with the FolR1 Fabs located at the N-terminus). In the 2+1 classical format the FolR1:CD3 binding moieties are arranged in a 2:1 order with the CD3 Fab being located at the N-terminus. Two monovalent formats were also prepared. The 1+1 head-to-tail has the FolR1:CD3 binding moieties arranged in a 1:1 order on the same arm of the molecule with the FolR1 Fab located at the N-terminus. In the 1+1 classical format the FolR1:CD3 binding moieties are present once, each on one arm of the molecule. For the 9D11 clone isolated from the standard Fab library the CD3 binding part was generated as a CrossFab (CH1C.kappa. crossing) while for the 16D5 from the common light chain library no crossing was necessary. These bispecific molecules were produced and purified analogously to the standard inverted T-cell bispecific format.
TABLE-US-00008 TABLE 7 Summary of the yield and final monomer content of the different T-cell bispecific formats. Monomer [%] Construct (SEC) Yield 16D5 FolR1 TCB 2 + 1 (inverted) 96% 5.4 mg/L 16D5 FolR1 TCB 2 + 1 (classical) 90% 4.6 mg/L 16D5 FolR1 TCB 1 + 1 (head-to- 100% 5.4 mg/L tail) 16D5 FolR1 TCB 1 + 1 (classical) 100% 0.7 mg/L 9D11 FolR1 TCB 2 + 1 (inverted) 100% 2.6 mg/L 9D11 FolR1 TCB 1 + 1 (head-to- 100% 6.1 mg/L tail) 9D11 FolR1 TCB 1 + 1 (classical) 96% 1.3 mg/L Mov19 FolR1 TCB 2 + 1 (inverted) 98% 3 mg/L Mov19 FolR1 TCB 1 + 1 (head-to- 100% 5.2 mg/L tail)
Example 10
Biochemical Characterization of FolR1 Binders by Surface Plasmon Resonance
[0464] Binding of FolR1 binders as IgG or in the T-cell bispecific format to different recombinant folate receptors (human FolR1, 2 and 3, murine FolR1 and cynomolgus FolR1; all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).
[0465] Single Injections
[0466] First the anti-FolR1 IgGs were analyzed by single injections (Table 1) to characterize their crossreactivity (to human, murine and cyno FolR1) and specificity (to human FolR1, human FolR2, human FolR3). Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) or human Folate Receptor 2 and 3 (FolR2-Fc, FolR3-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The IgGs were injected for 60 seconds at a concentration of 500 nM. IgGs binding to huFolR2 and huFolR3 were rejected for lack of specificity. Most of the binders are only crossreactive between human and cyno FolR1, additional crossreactivity to murine FolR1 went most of the time hand in hand with loss of specificity.
TABLE-US-00009 TABLE 8 Crossreactivity and specificity of 25 new folate receptor 1 binders (as IgGs) as well as of two control IgGs (Mov19 and Farletuzumab). Binding to Binding to Binding to Binding to Binding to Clone name huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 Mov19 + + - - - Farletuzumab + + - - - 16A3 + + +/- - - 18D3 + + - - - 19E5 + + + + + 19A4 - - + + + 15H7 + + + - - 15B6 + + - - - 16D5 + + - - - 15E12 + + +/- + + 21D1 + + +/- - - 16F12 + + - - - 21A5 + + - - +/- 21G8 + + - + + 19H3 - - + - - 20G6 - - + - - 20H7 - - + - - 9D11 + + - - - 5D9 + + - + + 6B6 + + - + + 11F8 + + + + + 36F2 + + + - - 14E4 - - + - - + means binding, - means no binding, +/- means weak binding.
[0467] Avidity to Folate Receptor 1
[0468] The avidity of the interaction between the anti-FolR1 IgGs or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 9).
[0469] Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 2.1 to 500 nM with a flow of 30 .mu.L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 600 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion. For the analysis of the interaction of 19H3 IgG and murine folate receptor 1, folate (Sigma F7876) was added in the HBS-EP running buffer at a concentration of 2.3 .mu.M. The binding curves resulting from the bivalent binding of the IgGs or T cell bispecifics were approximated to a 1:1 Langmuir binding and fitted with that model (which is not correct, but gives an idea of the avidity). The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare).
TABLE-US-00010 TABLE 9 Bivalent binding (avidity with apparent KD) of selected FolR1 binders as IgGs or as T-cell bispecifics (TCB) on human and cyno FolR1. Apparent Analyte Ligand ka (1/Ms) kd (1/s) KD (M) 16D5 TCB huFolR1 8.31E+04 3.53E-04 4.24E-09 cyFolR1 1.07E+05 3.70E-04 3.45E-09 9D11 TCB huFolR1 1.83E+05 9.83E-05 5.36E-10 cyFolR1 2.90E+05 6.80E-05 2.35E-10 21A5 TCB huFolR1 2.43E+05 2.64E-04 1.09E-09 cyFolR1 2.96E+05 2.76E-04 9.32E-10 36F2 IgG huFolR1 2.62E+06 1.51E-02 5.74E-9 cyFolR1 3.02E+06 1.60E-02 5.31E-9 muFolR1 3.7E+05 6.03E-04 1.63E-9 Mov19 IgG huFolR1 8.61E+05 1.21E-04 1.4E-10 cyFolR1 1.29E+06 1.39E-04 1.08E-10 Farletuzumab huFolR1 1.23E+06 9E-04 7.3E-10 cyFolR1 1.33E+06 8.68E-04 6.5E-10 19H3 IgG muFolR1 7.1E+05 1.1E-03 1.55E-09
[0470] 1. Affinity to Folate Receptor 1
[0471] The affinity of the interaction between the anti-FolR1 IgGs or the T cell bispecifics and the recombinant folate receptors was determined as described below (Table 10).
[0472] For affinity measurement, direct coupling of around 6000-7000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cell bispecifics were captured at 20 nM with a flow rate of 10 .mu.l/min for 20 or 40 sec, the reference flow cell was left without capture. Dilution series (6.17 to 500 nM or 12.35 to 3000 nM) of human or cyno Folate Receptor 1 Fc fusion were passed on all flow cells at 30 .mu.l/min for 120 or 240 sec to record the association phase. The dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1 or pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
TABLE-US-00011 TABLE 10 Monovalent binding (affinity) of selected FolR1 binders as IgGs or as T-cell bispecifics (TCB) on human and cyno FolR1. Ligand Analyte ka (1/Ms) kd (1/s) KD (M) 16D5 TCB huFolR1 1.53E+04 6.88E-04 4.49E-08 cyFolR1 1.32E+04 1.59E-03 1.21E-07 9D11 TCB huFolR1 3.69E+04 3.00E-04 8.13E-09 cyFolR1 3.54E+04 2.06E-04 5.82E-09 21A5 TCB huFolR1 1.79E+04 1.1E-03 6.16E-08 cyFolR1 1.48E+04 2.06E-03 1.4E-07 Mov19 IgG huFolR1 2.89E+05 1.59E-04 5.5E-10 cyFolR1 2.97E+05 1.93E-04 6.5E-10 Farletuzumab huFolR1 4.17E+05 2.30E-02 5.53E-08 cyFolR1 5.53E+05 3.73E-02 6.73E-08
[0473] 2. Affinity to CD3
[0474] The affinity of the interaction between the anti-FolR1 T cell bispecifics and the recombinant human CD3.epsilon..delta.-Fc was determined as described below (Table 11).
[0475] For affinity measurement, direct coupling of around 9000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics were captured at 20 nM with a flow rate of 10 .mu.l/min for 40 sec, the reference flow cell was left without capture. Dilution series (6.17 to 500 nM) of human CD3.epsilon..delta.-Fc fusion were passed on all flow cells at 30 .mu.l/min for 240 sec to record the association phase. The dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
TABLE-US-00012 TABLE 11 Monovalent binding (affinity) of selected FolR1 T-cell bispecifics (TCB) on human CD3-Fc. Ligand Analyte ka (1/Ms) kd (1/s) KD (M) 16D5 TCB huCD3 4.25E+04 3.46E-03 8.14E-08 21A5 TCB huCD3 3.72E+04 3.29E-03 8.8E-08
[0476] The CD3 binding part is identical for all constructs and the affinity is similar for the measured T cell bispecifics (KD range between 60 and 90 nM).
Example 11
Simultaneous Binding T Cell Bispecifics on Folate Receptor 1 and CD3
[0477] Simultaneous binding of the anti-FolR1 T cell bispecifics on recombinant Folate Receptor 1 and recombinant human CD3.epsilon..delta.-Fc was determined by surface plasmon resonance as described below. Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The anti-FolR1 T cell bispecifics were injected for 60 s at 500 nM with a flow of 30 .mu.L/minutes through the flow cells, followed by an injection of hu CD.epsilon..delta.-Fc for 60 s at 500 nM. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion. The four T cell bispecifics tested (16D5 TCB, 21A5 TCB, 51C7 TCB and 45D2 TCB) were able to bind simultaneously to Folate Receptor 1 and human CD3 as expected.
Example 12
Epitope Binning
[0478] For epitope binning, the anti-FolR1 IgGs or T cell bispecifics were directly immobilized on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare), with a final response around 700 RU. 500 nM huFolR1-Fc was then captured for 60 s, followed by 500 nM of the different binders for 30 s. The surface was regenerated with two injections of 10 mM glycine pH 2 for 30 s each. It is assessed if the different binders can bind to huFolR1 captured on immobilized binders (Table 12).
TABLE-US-00013 TABLE 12 Epitope characterization of selected FolR1 binders as IgGs or as T-cell bispecifics (TCB) on human FolR1. Analytes in solution On 16D5 21A5 9D11 36F2 Mov19 huFolR1 TCB TCB TCB IgG IgG Farletuzumab Immobilized 16D5 TCB - - - + + + 21A5 TCB - - - + + + 9D11 TCB No additional binding on FolR1 possible once captured on 9D11 36F2 IgG Measure not possible, huFolR1 dissociates too rapidly Mov19 IgG + + +/- - - - + means binding, - means no binding, +/- means weak binding
[0479] Based on these results and additional data with simultaneous binding on immobilized huFolR1, the binders were separated in three groups. It is not clear if 9D11 has a separate epitope because it displaces all the other binders. 16D5 and 21A5 seem to be in the same group and Mov19, Farletuzumab (Coney et al., Cancer Res. 1991 Nov. 15; 51(22):6125-32; Kalli et al., Curr Opin Investig Drugs. 2007 December; 8(12):1067-73) and 36F2 in another (Table 13). However, 36F2 binds to a different epitope than Mov 19 and Farletuzumab as it binds to human, cynomous and murine FolR1.
TABLE-US-00014 TABLE 13 Epitope grouping of selected FolR1 binders as IgGs or as T-cell bispecifics (TCB) on human FolR1 Epitope 1 Epitope 2 Epitope 3 16D5 9D11 Mov19 21A5 Farletuzumab 36F2
Example 13
Selection of Binders
[0480] FolR1 binders in the IgG formats were screened by surface plasmon resonance (SPR) and by in vitro assay on cells to select the best candidates.
[0481] The anti-FolR1 IgGs were analyzed by SPR to characterize their crossreactivity (to human, murine and cynomolgus FolR1) and specificity (to human FolR1, human FolR2, human FolR3). Unspecific binding to human FolR2 and 3 was considered an exclusion factor. Binding and specificity to human FolR1 was confirmed on cells. Some binders did not bind on cells expressing FolR1 even though they recognized the recombinant human FolR1 in SPR. Aggregation temperature was determined but was not an exclusion factor because the selected binders were all stable. Selected binders were tested in a polyreactivity ELISA to check for unspecific binding, which led to the exclusion of four binders. This process resulted in an initial selection of three binders: 36F2 (Fab library), 9D11 (Fab library) and 16D5 (common light chain). 36F2 dissociated rapidly from huFolR1 and was, therefore, initially not favored.
Example 14
Specific Binding of Newly Generated FolR1 Binders to Human FolR1 Positive Tumor Cells
[0482] New FolR1 binders were generated via Phage Display using either a Fab library or a common light chain library using the CD3 light chain. The identified binders were converted into a human IgG1 format and binding to FolR1 high expressing HeLa cells was addressed. As reference molecule the human FolR1 binder Mov19 was included. Most of the binders tested in this assay showed intermediate to good binding to FolR1 with some clones binding equally well as Mov19 (see FIG. 2). The clones 16A3, 18D3, 15H7, 15B6, 21D1, 14E4 and 16F12 were excluded because binding to FolR1 on cells could not be confirmed by flow cytometry. In a next step the selected clones were tested for specificity to human FolR1 by excluding binding to the closely related human FolR2. HEK cells were transiently transfected with either human FolR1 or human FolR2 to address specificity. The clones 36F2 and 9D11 derived from the Fab library and the clones 16D5 and 21A5 derived from the CLC library bind specifically to human FolR1 and not to human FolR2 (see FIGS. 3A-B). All the other tested clones showed at least some binding to human FolR2 (see FIGS. 3A-B). Therefore these clones were excluded from further characterization. In parallel cross-reactivity of the FolR1 clones to cyno FolR1 was addressed by performing binding studies to HEK cells transiently transfected with cyno FolR1. All tested clones were able to bind cyno FolR1 and the four selected human FoLR1 specific clones 36F2, 9D11, 16D5 and 21A5 bind comparably well human and cyno FoLR1 (FIG. 4). Subsequently three human FolR1 specific cyno cross-reactive binders were converted into TCB format and tested for induction of T cell killing and T cell activation. These clones were 9D11 from the Fab library and 16D5 and 21A5 from the CLC library. As reference molecule Mov19 FolR1 TCB was included in all studies. These FolR1 TCBs were then used to compare induction of internalization after binding to FolR1 on HeLa cells. All three tested clones are internalized upon binding to FolR1 comparable to internalization upon binding of Mov19 FoLR1 TCB (FIG. 5). 21A5 FolR1 TCB was discontinued due to signs of polyreactivity.
Example 15
T Cell-Mediated Killing of FolR1-Expressing Tumor Target Cells Induced by FolR1 TCB Antibodies
[0483] The FolR1 TCBs were used to determine T cell mediated killing of tumor cells expressing FoLR1. A panel of potential target cell lines was used to determine FoLR1 binding sites by Qifikit analysis. The used panel of tumor cells contains FolR1 high, intermediate and low expressing tumor cells and a FolR1 negative cell line.
TABLE-US-00015 TABLE 14 FolR1 binding sites on tumor cells Cell line Origin FolR1 binding sites Hela Cervix adenocarcinoma 2'240'716 Skov3 Ovarian adenocarcinoma 91'510 OVCAR5 Ovarian adenocarcinoma 22'077 HT29 Colorectal adenocarcinoma 10'135 MKN45 Gastric adenocarcinoma 54
[0484] Binding of the three different FoLR1 TCBs (containing 9D11, 16D5 and Mov19 binders) to this panel of tumor cell lines was determined showing that the FoLR1 TCBs bind specifically to FolR1 expressing tumor cells and not to a FoLR1 negative tumor cell line. The amount of bound construct is proportional to the FolR1 expression level and there is still good binding of the constructs to the FolR1 low cell line HT-29 detectable. In addition there is no binding of the negative control DP47 TCB to any of the used cell lines (FIGS. 6A-E).
[0485] The intermediate expressing cell line SKOV3 and the low expressing cell line HT-29 were further on used to test T cell mediated killing and T cell activation using 16D5 TCB and 9D11 TCB; DP47 TCB was included as negative control. Both cell lines were killed in the presence of already very low levels of 16D5 TCB and 9D11 TCB and there was no difference in activity between both TCBs even though 9D11 TCB binds stronger to FolR1 than 16D5 TCB. Overall killing of SKOV3 cells was higher compared to HT-29 which reflects the higher expression levels of FolR1 on SKOV3 cells (FIGS. 7A-D). In line with this, a strong upregulation of the activation marker CD25 and CD69 on CD4.sup.+ T cells and CD8.sup.+ T cells was detected. Activation of T cells was very similar in the presence of SKOV3 cells and HT-29 cells. The negative control DP47 TCB does not induce any killing at the used concentrations and there was no significant upregulation of CD25 and CD69 on T cells.
TABLE-US-00016 TABLE 15 EC50 values of tumor cell killing and T cell activation with SKOV3 cells Killing Killing CD4+ CD4+ CD8+ CD8+ 24 h 48 h CD69+ CD25+ CD69+ CD25+ Construct (pM) (pM) (%) (%) (%) (%) 9D11 1.1 0.03 0.51 0.46 0.019 0.03 FolR1 TCB 16D5 0.7 0.04 0.34 0.33 0.025 0.031 FolR1 TCB
TABLE-US-00017 TABLE 16 EC50 values of tumor cell killing and T cell activation with HT-29 cells Killing Killing CD4+ CD4+ CD8+ CD8+ 24 h 48 h CD69+ CD25+ CD69+ CD25+ Construct (pM) (pM) (%) (%) (%) (%) 9D11 2.3 0.1 1.22 1.11 0.071 0.084 FolR1 TCB 16D5 2.8 0.1 0.69 0.62 0.021 0.028 FolR1 TCB
Example 16
Binding to Erythrocytes and T Cell Activation in Whole Blood
[0486] To prove that there is no spontaneous activation in the absence of FoLR1 expressing tumor cells we tested if there is binding of the FolR1 clones to erythrocytes which might potentially express FolR1. We could not observe any specific binding of 9D11 IgG, 16D5 IgG and Mov19 IgG to erythrocytes, as negative control DP47 IgG was included (FIG. 8).
[0487] To exclude any further unspecific binding to blood cells or unspecific activation via FoLR1 TCB, 9D11 TCB, 16D5 TCB and Mov19 TCB were added into whole blood and upregulation of CD25 and CD69 on CD4.sup.+ T cells and CD8.sup.+ T cells was analyzed by flow cytometry. DP47 TCB was included as negative control. No activation of T cells with any of the tested constructs could be observed by analyzing upregulation of CD25 and CD69 on CD4.sup.+ T cells and CD8.sup.+ T cells (FIG. 9).
Example 17
Removal of the N-Glycosylation Site in 9D11 Light Chain
[0488] During analysis of the different FolR1 binders to identify potential sequence hot spots, at the end of CDR L3 of the clone 9D11 a putative N-glycosylation site was identified. Usually the consensus motif for N-glycosylation is defined as N-X-S/T-X (where X is not P). The sequence of CDR L3 (MQASIMNRT) (SEQ ID NO: 61) perfectly matches this consensus motif having the sequence N-R-T. Since glycosylation might not be completely reproducible among different production batches this could have an impact on FolR1 binding, if the glycosylation in CDR L3 contributes to antigen binding. To evaluate if this N-glycosylation site is important for FolR1 binding, or could be replaced without impairing binding, different variants of the 9D11 light chain were generated in which the N-glycosylation site was exchanged by site specific mutagenesis.
[0489] 1. Transient Transfection and Production
[0490] The four T cell bispecifics were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells were centrifuged for 5 min by 210.times.g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210.times.g, the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4.degree. C. After production the supernatants were harvested and the antibody containing supernatants were filtered through 0.22 .mu.m sterile filters and stored at 4.degree. C. until purification.
[0491] 2. Antibody Purification
[0492] All molecules were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA HP (GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M NaCl, 0.01% Tween-20, pH 7.5). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 2.5, 0.5 M NaCl, 0.01% Tween-20) over 20 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 2 M Tris pH 8.0). Samples were concentrated to 1 ml using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a Superdex.TM. 200 10/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. Fractions containing less than 2% oligomers were pooled and concentrated to final concentration of 1-1.5 mg/ml using ultra concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N.sub.2 and stored at -80.degree. C.
[0493] 3. Aggregation Temperature
[0494] Stability of the four constructs was tested on an Optim 1000 (Avacta, PALL Corporation) by a gradient heating from 25.degree. to 80.degree. at 0.1.degree. C./min. The temperature at onset of aggregation is recorded.
TABLE-US-00018 TABLE 34 Yield, monomer content and aggregation temperature of four N-glycosylation site knock-out mutant of the 9D11 binder in the 2 + 1 inverted T-cell bispecific format. All four mutants behaved similarly to the wild-type 9D11 binder Yield Monomer Aggregation Clone Mutation [mg/L] [%] temperature 9D11 T102N 1.34 97 56.degree. 9D11 T102A 1.29 100 56.degree. 9D11 N100Q 2.5 100 56.degree. 9D11 N100S 2.05 100 56.degree. 9D11 -- 2.6 100 57.degree.
[0495] The following variants were generated: N100S (N95S); N100Q (N95Q), T102A (T97A) and T102N (T97N) (Kabat numbering indicated in parenthesis) and converted into the T-cell bispecific format. After transient production in HEK293 EBNA cells and purification the different variants were analyzed for target binding and cell killing activity in comparison to the original 9D11 clone.
TABLE-US-00019 TABLE 17 Primers used for removal of N-glycosylation site in CDR L3 of 9D11 (sequences see below) # Amino acid exchange Mutagenesis primer 1 N95S GAB-7735 2 N95Q GAB-7734 3 T97A GAB7736 4 T97N GAB-7737
Example 18
Binding and T Cell Mediated Killing with 9D11 a-Glyco Variants
[0496] Due to a glycosylation site in the CDRs four different 9D11 variants were produced with a mutation removing the glycosylation site (Example 17). These four variants were tested in comparison to the original 9D11 for binding to FolR1 on HeLa cells (FIG. 10) and induction of tumor cell killing on SKOV3 and HT-29 (FIG. 11A-B, E-F). None of the variants showed differences in binding or induction of tumor cell killing. In parallel unspecific killing of the FolR1 negative cell lines MKN-45 was addressed (FIGS. 11C-D). Also, no differences between the variants and the original binder could be observed. None of the constructs induced unspecific killing on FoLR1 negative tumor cells.
Example 19
FolR1 Expression on Primary Epithelial Cells
[0497] FolR1 is expressed at low levels on primary epithelial cells. Here we wanted to test if these levels are sufficient to induce T cell mediated killing in the presence of the FolR1 TCBs. To test this we used primary human bronchial epithelial cells, primary human choroid plexus epithelial cell, primary human renal cortical epithelial cells and primary human retinal pigment epithelial cells. As positive control either FolR1 positive SKOV3 cells or HT-29 cells were included. First we verified FolR1 expression on the used primary cells and determined the amount of FolR1 binding sites on these cells. Bronchial epithelial cells, renal cortical epithelial cells and retinal pigment epithelial cells express very low but significant levels of FolR1 compared to the levels expressed on tumor cells. The choroid plexus epithelial cells do not express significant levels of FolR1.
TABLE-US-00020 TABLE 18 FolR1 binding sites on primary epithelial cells Cell line Binding sites Bronchial epithelium 492 Choroid plexus epithelium 104 Renal cortical epithelium 312 Retinal pigment epithelium 822 Skov3 69'890
[0498] The primary epithelial cells that demonstrated FolR1 expression on the surface were used to address the question if these cells can be killed by T cells in the presence of FoLR1 TCBs. No significant levels of killing could be measured but induction of T cell activation in the presence of retinal pigment epithelial cells, bronchial epithelial cells and renal cortical cells resulting in upregulation of CD25 and CD69 was detected. The strongest activation is seen with retinal pigment epithelial cells resulting in upregulation of CD25 and CD69 both on CD4.sup.+ T cells and CD8.sup.+ T cells. In the presence of bronchial epithelial cells lower activation of T cells is induced with upregulation of CD69 on CD4.sup.+ T cells and CD8.sup.+ T cells but very low upregulation of CD25 only on CD4.sup.+ T cells but not on CD8.sup.+ T cells. The lowest activation of T cells is obtained in the presence of renal epithelial cells with no upregulation of CD25 on CD4 T.sup.+ cells and CD8.sup.+ T cells and CD69 been only upregulated on CD8.sup.+ T cells (FIGS. 12A-X).
Example 20
Comparison of Different TCB Formats Containing Either 16D5 or 9D11 Binder
[0499] To determine if the TCB 2+1 inverted format is the most active format with the selected FolR1 binder, different formats containing either 16D5 or 9D11 were produced and compared in target cell binding, T cell mediated killing and T cell activation. The 16D5 binder was tested in the TCB 2+1 inverted (FIG. 1A), TCB 2+1 classical (FIG. 1D), TCB 1+1 classical (FIG. 1C) and TCB 1+1 head-to-tail (FIG. 1B) format; the 9D11 binder was tested in the TCB 2+1 inverted (FIG. 1A), TCB 1+1 classical (FIG. 1C) and TCB 1+1 head-to-tail (FIG. 1B) format.
[0500] All constructs were tested for binding to FolR1 on HeLa cells. The molecules bivalent for binding to FolR1 bind stronger compared to the monovalent constructs due to avidity. The difference between the bivalent vs. monovalent constructs is more pronounced for 16D5. The reason might be that due to the lower affinity of 16D5 the avidity effect for this binder is stronger. Between the two 1+1 TCBs there is no significant difference in binding but there is a difference between the two 2+1 constructs. The inverted 2+1 construct binds stronger to FolR1 than the classical 2+1 construct. This indicates that in the classical 2+1 construct the binding to FoLR1 is influenced by the presence of the CD3 Fab whereas in the inverted construct binding is less influenced.
[0501] By testing T cell mediated killing with these constructs we could show that stronger binding of the 2+1 inverted TCB in converted into stronger tumor cell killing and T cell activation compared to the 2+1 classical TCB. The 16D5 FoLR1 TCB 2+1 classical is only a little bit more active than the respective 1+1 head-to-tail construct. The 1+1 head-to-tail construct is significantly more active than the 1+1 classical construct. This does not reflect the situation seen in binding and might be due to better crosslinking with the head-to-tail construct. Overall tumor cell killing and T cell activation is comparable with all tested constructs, the differences in potency seen with the differences are only in terms of EC50 values. In general it can be concluded that the FolR1 TCB 2+1 inverted independent of the used binder is the preferred format to induce T cell mediated tumor cell killing and T cell activation (see FIG. 13A-C and FIG. 14A-C).
TABLE-US-00021 TABLE 19 EC50 values of target cell binding and T cell mediated killing with different TCB formats Killing 24 h Killing 48 h Construct Binding EC50 (nM) (pM) (pM) 16D5 FolR1 TCB 11.03 1.43 0.18 2 + 1 inverted 16D5 FolR1 TCB 17.07 5.60 2.18 2 + 1 classical 16D5 FolR1 TCB 107.3 n.d. n.d. 1 + 1 classical 16D5 FoLR1 TCB 102.6 26.24 6.06 1 + 1 head-to-tail 9D11 FoLR1 TCB 17.52 0.74 0.14 2 + 1 inverted 9D11 FoLR1 TCB 38.57 20.92 n.d. 1 + 1 classical 9D11 FoLR1 TCB 44.20 4.73 n.d. 1 + 1 head-to-tail
TABLE-US-00022 TABLE 20 EC50 values of T cell activation in the presence of SKOV3 cells with different TCB formats CD4+CD25+ CD4+CD69+ CD8+CD25+ CD8+CD69+ Construct (%) (%) (%) (%) 16D5 1.96 0.33 2.10 n.d. FolR1 TCB 2 + 1 inverted 16D5 13.83 3.67 12.88 4.47 FolR1 TCB 2 + 1 classical 16D5 38.54 n.d. n.d. n.d. FolR1 TCB 1 + 1 classical 16D5 FoLR1 17.14 7.47 25.15 n.d. TCB 1 + 1 head-to-tail 9D11 1.41 0.27 1.24 0.35 FoLR1 TCB 2 + 1 inverted 9D11 34.01 n.d. 34.39 7.40 FoLR1 TCB 1 + 1 classical 9D11 FoLR1 3.73 2.47 4.98 2.89 TCB 1 + 1 head-to-tail
Example 21
Tumor Cell Lines and Primary Cells
[0502] HeLa cells (CCL-2) were obtained from ATCC and cultured in DMEM with 10% FCS and 2 mM Glutamine, SKOV3 (HTB-77) were obtained from ATCC and cultured in RPMI with 10% FCS and 2 mM Glutamine, OVCAR5 were obtained from NCI and cultured in RPMI with 10% FCS and 2 mM Glutamine, HT-29 (ACC-299) were obtained from DSMZ and cultured in McCoy's 5A medium with 10% FCS and 2 mM Glutamine, MKN-45 (ACC-409) were obtained from DSMZ and cultured in RPMI with 10% FCS and 2 mM Glutamine.
[0503] All tested primary epithelial cells were obtained from ScienCell Research Laboratories. Human Bronchial Epithelium Cells (HBEpiC, Catalog Number 3210 were cultured in Bronchial Epithelial Cell Medium (BEpiCM, Cat. No. 3211, ScienCell). Human Colonic Epithelial Cells (HCoEpiC), Catalog Number 2950 were cultured in Colonic Epithelial Cell Medium (CoEpiCM, Cat. No. 2951, ScienCell). Human Retinal Pigment Epithelial Cells (HRPEpiC), Catalog Number 6540 were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell). Human Renal Cortical Epithelial Cells (HRCEpiC), Catalog Number 4110, were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell). Human Choroid Plexus Epithelial Cells (HCPEpiC), Catalog Number 1310 were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell).
Example 22
Target Binding by Flow Cytometry
[0504] Target cells as indicated were harvested with Cell Dissociation Buffer, washed with PBS and resuspended in FACS buffer. The antibody staining was performed in a 96well round bottom plate. Therefore 200'000 cells were seeded per well. The plate was centrifuged for 4 min at 400 g and the supernatant was removed. The test antibodies were diluted in FACS buffer and 20 .mu.l of the antibody solution were added to the cells for 30 min at 4.degree. C. To remove unbound antibody the cells were washed twice with FACS buffer before addition of the diluted secondary antibody (FITC conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fcg Fragment, Jackson ImmunoResearch #109-096-098 or PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg Fragment Specific, Jackson ImmunoResearch #109-116-170. After 30 min incubation on 4.degree. C. unbound secondary antibody was washed away. Before measurement the cells were resuspended in 200 .mu.l FACS buffer and analyzed by flow cytometry using BD Canto II or BD Fortessa.
Example 23
Internalization
[0505] The cells were harvested and the viability was determined. The cells were re-suspended in fresh cold medium at 2 Mio cells per ml and the cell suspension was transferred in a 15 ml falcon tube for each antibody. The antibodies that should be tested for internalization were added with a final concentration of 20 .mu.g per ml to the cells. The tubes were incubated for 45 min in the cold room on a shaker. After incubation the cells were washed three times with cold PBS to remove unbound antibodies. 0.2 Mio cells per well were transfer to the FACS plate as time point zero. The labeled cells were re-suspended in warm medium and incubated at 37.degree. C. At the indicated time-points 0.2 Mio cells per well were transferred in cold PBS, washed in plated on the FACS plate. To detect the constructs that remain on the surface the cells were stained with PE-labeled anti-human Fc secondary antibody. Therefore 20 .mu.l of the diluted antibody were added per well and the plate was incubated for 30 min at 4.degree. C. Then the cells were washed twice to remove unbound antibodies and then fixed with 1% PFA to prevent any further internalization. The fluorescence was measured using BD FACS CantoII.
Example 24
QIFIKIT.RTM. Analysis
[0506] QIFIKIT.RTM. contains a series of beads, 10 .mu.m in diameter and coated with different, but well-defined quantities of mouse Mab molecules (high-affinity anti-human CD5, Clone CRIS-1, isotype IgG2a). The beads mimic cells with different antigen densities which have been labeled with a primary mouse Mab, isotype IgG. Briefly, cells were labeled with primary mouse monoclonal antibody directed against the antigen of interest. In a separate test well, cells were labeled with irrelevant mouse monoclonal antibody (isotype control). Then, cells, Set-Up Beads and Calibration Beads were labeled with a fluorescein-conjugated anti-mouse secondary antibody included in the kit. The primary antibody used for labeling of the cells has to be used at saturating concentration. The primary antibody may be of any mouse IgG isotype. Under these conditions, the number of bound primary antibody molecules corresponds to the number of antigenic sites present on the cell surface. The secondary antibody is also used at saturating concentration. Consequently, the fluorescence is correlated with the number of bound primary antibody molecules on the cells and on the beads.
Example 25
T Cell Mediated Tumor Cell Killing and T Cell Activation
[0507] Target cells were harvested with Trypsin/EDTA, counted and viability was checked. The cells were resuspended in their respective medium with a final concentration of 300'000 cells per ml. Then 100 .mu.l of the target cell suspension was transferred into each well of a 96-flat bottom plate. The plate was incubated overnight at 37.degree. C. in the incubator to allow adherence of the cells to the plate. On the next day PBMCs were isolated from whole blood from healthy donors. The blood was diluted 2:1 with PBS and overlayed on 15 ml Histopaque-1077 (#10771, Sigma-Aldrich) in Leucosep tubes and centrifuged for 30 min at 450 g without break. After centrifugation the band containing the cells was collected with a 10 ml pipette and transferred into 50 ml tubes. The tubes were filled up with PBS until 50 ml and centrifuged (400 g, 10 min, room temperature). The supernatant was removed and the pellet resuspended in PBS. After centrifugation (300 g, 10 min, room temperature), supernatants were discarded, 2 tubes were pooled and the washing step was repeated (this time centrifugation 350.times.g, 10 min, room temperature). Afterwards the cells were resuspended and the pellets pooled in 50 ml PBS for cell counting. After counting cells were centrifuged (350 g, 10 min, room temperature) and resuspended at 6 Mio cells per ml in RPMI with 2% FCS and 2 nM Glutamine. Medium was removed from plated target cells and the test antibodies diluted in RPMI with 2% FCS and 2 nM Glutamine were added as well as. 300'000 cells of the effector cell solution were transferred to each well resulting in a E:T ratio of 10:1. To determine the maximal release target cells were lysed with Triton X-100. LDH release was determined after 24 h and 48 h using Cytotoxicity Detection Kit (#1644793, Roche Applied Science). Activation marker upregulation on T cells after tumor cell killing was measured by flow cytometry. Briefly PBMCs were harvested, transferred into a 96 well round bottom plate and stained with CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) antibodies diluted in FACS buffer. After 30 min incubation at 4.degree. C. the cells were washed twice with FACS buffer. Before measuring the fluorescence using BD Canto II the cells were resuspended in 200 .mu.l FACS buffer.
Example 26
T Cell Activation in Whole Blood
[0508] 280 .mu.l of fresh blood were added into a 96 well conical deep well plate. Then 20 .mu.l of the diluted TCBs were added to the blood and mixed well by shaking the plate. After 24 h incubation at 37.degree. C. in an incubator the blood was mixed and 35 .mu.l were transferred to a 96well round bottom plate. Then 20 .mu.l of the antibody staining mix were added consisting of CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) and CD45 V500 (#560777, BD Horizon) and incubated for 15 min in the dark at room temperature. Before measuring 200 .mu.l of the freshly prepared BD FACS lysing solution (#349202, BD FCAS) was added to the blood. After 15 min incubation at room temperature the cells were measured with BD Fortessa.
Example 27
SDPK (Single Dose Pharmacokinetics) Study of Humanized FOLR1 TCB (Clone 16D5) in Immunodeficient NOD/Shi-Scid/IL-2R.gamma. null (NOG) Mice
[0509] Female NOD/Shi-scid/IL-2R.gamma. null (NOG) mice, age 6-7 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2011/128). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
[0510] Mice were injected i.v. with 10/1/0.1 .mu.g/mouse of the FOLR1 TCB whereas 3 mice were bled per group and time point. All mice were injected with a total volume of 200 .mu.l of the appropriate solution. To obtain the proper amount of the FOLR1 TCB per 200 the stock solutions were diluted with PBS when necessary. Serum samples were collected 5 min, 1 h, 3 h, 8 h, 24 h, 48 h, 72 h, 96 h and 168 h after therapy injection.
[0511] FIG. 15 shows that the 16D5 FOLR1 TCB shows typical and dose proportional IgG-like PK properties in NOG mice with slow clearance.
TABLE-US-00023 TABLE 21 Experimental conditions. Formulation Concentration Compound Dose buffer (mg/mL) FOLR1 TCB 10 .mu.g 20 mM Histidine, 5.43 (16D5) (corresponding 140 mM NaCl, (=stock solution) to ca. 0.5 mg/kg) pH 6.0 FOLR1 TCB 1 .mu.g 20 mM Histidine, 5.43 (16D5) (corresponding 140 mM NaCl, (=stock solution) to ca. 0.05 mg/kg) pH 6.0 FOLR1 TCB 0.1 .mu.g 20 mM Histidine, 5.43 (16D5) (corresponding 140 mM NaCl, (=stock solution) to ca. 0.005 mg/kg) pH 6.0
Example 28
[0512] In Vivo Efficacy of FOLR1 TCB (Clone 16D5) after Human PBMC Transfer in Skov3-Bearing NOG Mice
[0513] The FOLR1 TCB was tested in the human ovarian carcinoma cell line Skov3, injected s.c. into PBMC engrafted NOG mice.
[0514] The Skov3 ovarian carcinoma cells were obtained from ATCC (HTB-77). The tumor cell line was cultured in RPMI containing 10% FCS (Gibco) at 37.degree. C. in a water-saturated atmosphere at 5% CO.sub.2. Passage 35 was used for transplantation, at a viability >95%. 5.times.10.sup.6 cells per animal were injected s.c. into the right flank of the animals in a total of 100 .mu.l of RPMI cell culture medium (Gibco).
[0515] Female NOD/Shi-scid/IL-2R.gamma. null (NOG) mice, age 6-7 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2011/128). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
[0516] According to the protocol (FIG. 16), mice were injected s.c. on study day 0 with 5.times.10.sup.6 of the Skov3. At study day 21, human PBMC of a healthy donor were isolated via the Ficoll method and 10.times.10.sup.6 cells were injected i.p. into the tumor-bearing mice. Two days after, mice were randomized and equally distributed in five treatment groups (n=12) followed by i.v. injection with either 10/1/0.1 .mu.g/mouse of the FOLR1 TCB or 10 .mu.g/mouse of the DP47 control TCB once weekly for three weeks. All mice were injected i.v. with 200 .mu.l of the appropriate solution. The mice in the vehicle group were injected with PBS. To obtain the proper amount of TCB per 200 the stock solutions were diluted with PBS when necessary. Tumor growth was measured once weekly using a caliper (FIG. 17) and tumor volume was calculated as followed:
T.sub.v: (W.sup.2/2).times.L (W:Width,L:Length)
[0517] The once weekly injection of the FOLR1 TCB resulted in a dose-dependent anti-tumoral effect. Whereas a dose of 10 .mu.g/mouse and 1 .mu.g/mouse induced tumor shrinkage and 0.1 .mu.g/mouse a tumor stasis (FIG. 17, Table 22). Maximal tumor shrinkage was achieved at a dose of 10 .mu.g/mouse as compared to a non-targeted control DP47 TCB.
TABLE-US-00024 TABLE 22 In vivo efficacy. Tumor growth Compound Dose inhibition DP47 TCB 10 .mu.g 7% control TCB (corresponding to ca. 0.5 mg/kg) FOLR1 TCB 10 .mu.g 90% (16D5) (corresponding to ca. 0.5 mg/kg) FOLR1 TCB 1 .mu.g 74% (16D5) (corresponding to ca. 0.05 mg/kg) FOLR1 TCB 0.1 .mu.g 56% (16D5) (corresponding to ca. 0.005 mg/kg)
[0518] For PD read-outs, three mice per treatment group were sacrificed at study day 32, tumors were removed and single cell suspensions were prepared through an enzymatic digestion with Collagenase V, Dispase II and DNAse for subsequent FACS-analysis (FIGS. 19 and 20). Single cells where either used directly for staining of extracellular antigens and activation markers or were re-stimulated using 5 ng/ml PMA and 500 ng/ml Ionomycin in the presence of a protein transport inhibitor Monensin for 5 h in normal culture medium. After re-stimulation, cells were stained for surface antigens, followed by a fixation and permeabilization step. Fix samples were then stained intracellulary for TNF-.alpha., IFN-.gamma., IL-10 and IL-2 and analyzed by flow cytometry. Same procedure was used for the degranulation of cells, but an anti-CD107a antibody was added during the restimulation period and fixed samples were staining for intracellular perforin and granzyme-B contents. The FACS analysis revealed statistically higher number of infiltrating CD4.sup.+ and CD8.sup.+ T-cells in the tumor tissue upon treatment with FOLR1 TCB compared to vehicle and untargeted control TCB. Furthermore, higher numbers of TNF-.alpha., IFN-.gamma. and IL-2 producing as well as perforin.sup.+/granzym-B.sup.+ CD4.sup.+ and CD8.sup.+ T-cells were detected in FOLR1 TCB treated tumors. Tumor infiltrating T-cells treated with FOLR1 TCB also showed higher degranulation rates compared to control groups.
[0519] At study termination day 38, all animals were sacrificed; tumors were removed and weight (FIG. 18). The weight of the tumors treated with 10 and 1 .mu.g/mouse of the FOLR1 TCB showed a statistically significant difference compared to the control groups.
TABLE-US-00025 TABLE 23 Experimental conditions. Concentration Compound Dose Formulation buffer (mg/mL) PBS FOLR1 TCB 10 .mu.g 20 mM Histidine, 3.88 (16D5) 140 mM NaCl, (=stock solution) pH 6.0 FOLR1 TCB 1 .mu.g 20 mM Histidine, 3.88 (16D5) 140 mM NaCl, (=stock solution) pH 6.0 FOLR1 TCB 0.1 .mu.g 20 mM Histidine, 3.88 (16D5) 140 mM NaCl, (=stock solution) pH 6.0 DP47 TCB 10 .mu.g 20 mM Histidine, 4.35 140 mM NaCl, (=stock solution) pH 6.0
Example 29
Generation of a Bispecific FolR1/CD3-Kappa-Lambda Antibody
[0520] To generate a bispecific antibody (monovalent for each antigen) that simultaneously can bind to human CD3 and human folate receptor alpha (FolR1) without using any hetero-dimerization approach (e.g. knob-into-hole technology), a combination of a common light chain library with the so-called CrossMab technology was applied: The variable region of the humanized CD3 binder (CH2527_VL7_46/13) was fused to the CH1 domain of a standard human IgG1 antibody to form the VLVH crossed molecule (fused to Fc) which is common for both specificities. To generate the crossed counterparts (VHCL), a CD3 specific variable heavy chain domain (CH2527_VH_23/12) was fused to a constant human .lamda. light chain whereas a variable heavy chain domain specific for human FolR1 (clone 16D5, isolated from common light chain library) was fused to a constant human .kappa. light chain. This enables the purification of the desired bispecific antibody by applying subsequent purification steps with KappaSelect and LambdaFab Select columns (GE Healthcare) to remove undesired homodimeric antibodies.
[0521] All antibody expression vectors were generated using standard recombinant DNA technology as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Molecular biological reagents were used according the manufacturer's recommendations. Genes or gene fragments were either amplified by polymerase chain reaction (PCR) or generated from synthetic oligonucleotides at Geneart AG (Regensburg, Germany) by automated gene synthesis. PCR-amplified or subcloned DNA fragments were confirmed by DNA sequencing (Synergene GmbH, Switzerland). Plasmid DNA was transformed into and amplified in suitable E. coli host strains for preparation of transfection-grade plasmid DNA using standard Maxiprep kits (Qiagen). For production of the bispecific molecules HEK293 EBNA cells were transfected with plasmids encoding the respective genes using a standard polyethlenimine (PEI) based method. The used plasmid ratio of the three expression vectors was 1:1:1. Transfected cells were cultivated for 7 days before supernatants were harvested for purification. The bispecific FolR1/CD3-kappa-lambda antibodies were produced and purified as follows.
[0522] 1. Transient Transfection and Production
[0523] The kappa-lambda bispecific antibody was transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells were centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210.times.g, the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4.degree. C.
[0524] 2. Purification
[0525] The kappa-lambda bispecific antibody was purified in three steps, using an affinity step specific for kappa light chains, followed by an affinity step specific for lambda light chains and finally by a size exclusion chromatography step for removal of aggregates. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to Capture Select kappa affinity matrix, or HiTrap KappaSelect, GE Healthcare, column volume (cv)=1 ml, equilibrated with 5 column volumes (cv) buffer A (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 8.0). After washing with 15 cv of buffer A, the protein was eluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0) over 25 cv. Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0). The neutralized pooled fractions were applied to Capture Select lambda affinity matrix (now: HiTrap LambdaFab Select, GE Healthcare, column volume (cv)=1 ml) equilibrated with 5 column volumes (cv) buffer A (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 8.0). After washing with 15 cv of buffer A, the protein was eluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0) over 25 cv. Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0). This solution was concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a Superdex.TM. 200 10/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The pooled fractions after size exclusion were again concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius).
[0526] The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Only small amounts of protein could be purified with a final yield of 0.17 mg/L.
Example 30
T Cell Mediated Killing with Bispecific FolR1/CD3-Kappa-Lambda Antibody
[0527] Activity of kappa lambda FolR1 TCB was tested on SKOV3 cells in the presence of freshly isolated PBMCs. As negative control DP47 TCB was included. T cell mediated killing of SKOV3 cells was determined after 24 h and 48 h by LDH release. After 48 h the T cells were harvested and CD69 and CD25 upregulation on CD4 T cells and CD8 T cells was measured by flow cytometry.
[0528] The kappa lambda FolR1 construct induces killing of SKOV3 cells in a concentration dependent manner which is accompanied by CD69 and CD25 upregulation both on CD4 T cells and on CD8 T cells.
[0529] SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 24 h and 48 h killing of tumor cells was determined by measuring LDH release (FIG. 21). SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells and CD8 T cells was measured by flow cytometry (FIG. 22).
Example 31
Biochemical Characterization of 16D5 and 36F2 FolR1 Binders by Surface Plasmon Resonance
[0530] Binding of anti-FolR1 16D5 in different monovalent or bivalent T-cell bispecific formats and of anti-FolR1 36F2 as IgG or as T-cell bispecific to recombinant human, cynomolgus and murine folate receptor 1 (all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, GE Healthcare).
[0531] 1. Molecules Tested
[0532] The molecules used for affinity and avidity determination are described in Table 24.
TABLE-US-00026 TABLE 24 Name and description of the 6 constructs used in SPR analysis Name Description 16D5 TCB 2 + 1 T-cell bispecific, inverted format (common light chain) 16D5 TCB classical 2 + 1 T-cell bispecific, classical format (common light chain) 16D5 TCB 1 + 1 1 + 1 T-cell bispecific (common light chain) 16D5 TCB 1 + 1 HT 1 + 1 T-cell bispecific head-to- tail (common light chain) 36F2 IgG Human IgG1 with P329G LALA 36F2 TCB 2 + 1 T-cell bispecific, inverted format, crossfab
[0533] 2. Avidity to Folate Receptor 1
[0534] The avidity of the interaction between the anti-FolR1 IgG or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 25).
[0535] Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, GE Healthcare). The immobilization level was about 300-400 RU. The anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 3.7 to 900 nM with a flow of 30 .mu.L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 240 or 600 seconds. The chip surface was regenerated after every cycle using a double injection of 30 sec 10 mM Glycine-HCl pH 2. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine CD134 Fc fusion. The binding curves resulting from the bivalent binding of the IgG or T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent KD representing the avidity of the bivalent binding. The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare). For the 1+1 T cell bispecifics format the interaction is a real 1:1 and the KD represents affinity since there is only one FolR1 binder in this construct.
TABLE-US-00027 TABLE 25 Bivalent binding (avidity with apparent KD) of anti-FolR1 16D5 and 36F2 as IgG or as T-cell bispecifics (TCB) on human, cyno and murine FolR1. Apparent Analyte Ligand ka (1/Ms) kd (1/s) KD 36F2 IgG huFolR1 2.07E+06 1.3E-02 6 nM cyFolR1 2.78E+06 1.75E-02 6 nM muFolR1 4.28E+05 8.23E-04 2 nM 36F2 TCB huFolR1 2.45E+06 9.120E-03 4 nM cyFolR1 4.31E+06 1.45E-02 3 nM muFolR1 6.97E+05 9.51E-04 1 nM 16D5 TCB huFolR1 1.57E+05 3.92E-04 3 nM cyFolR1 2.01E+05 3.81E-04 2 nM 16D5 TCB classical huFolR1 2.04E+05 1.84E-04 0.9 nM cyFolR1 2.50E+05 3.05E-04 1 nM 16D5 TCB 1 + 1 HT huFolR1 5.00E+04 2.25E-03 45 nM cyFolR1 5.75E+04 4.10E-03 70 nM 16D5 TCB 1 + 1 huFolR1 3.65E+04 2.04E-03 56 nM cyFolR1 4.09E+04 3.60E-03 90 nM
[0536] 3. Affinity to Folate Receptor 1
[0537] The affinity of the interaction between the anti-FolR1 IgG or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 26).
[0538] For affinity measurement, direct coupling of around 12000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 IgG or T cell bispecifics were captured at 20 nM with a flow rate of 10 .mu.l/min for 40 sec, the reference flow cell was left without capture. Dilution series (12.3 to 3000 nM) of human, cyno or murine Folate Receptor 1 Fc fusion were passed on all flow cells at 30 .mu.l/min for 240 sec to record the association phase. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
TABLE-US-00028 TABLE 26 Monovalent binding (affinity) of anti-FolR1 16D5 and 36F2 as IgG or as T-cell bispecifics (TCB) on human, cyno and murine FolR1. Analyte Ligand ka (1/Ms) kd (1/s) KD 36F2 IgG huFolR1 9.10E+04 6.65E-02 730 nM cyFolR1 1.02E+05 5.78E-02 570 nM muFolR1 8.32E+04 1.78E-02 210 nM 36F2 TCB huFolR1 5.94E+04 6.13E-02 1000 nM cyFolR1 6.29E+04 5.42E-02 860 nM muFolR1 5.68E+04 1.75E-02 300 nM 16D5 TCB huFolR1 2.23E+04 7.33E-04 33 nM cyFolR1 1.57E+04 1.60E-03 100 nM 16D5 TCB classical huFolR1 1.03E+04 7.59E-04 74 nM cyFolR1 9.18E+03 1.61E-03 175 nM 16D5 TCB 1 + 1 HT huFolR1 2.05E+04 7.08E-04 35 nM cyFolR1 1.67E+04 1.53E-03 92 nM 16D5 TCB 1 + 1 huFolR1 1.43E+04 9.91E-04 69 nM cyFolR1 1.20E+04 1.80E-03 150 nM
[0539] The affinity (monovalent binding) to human and cyno FolR1-Fc of 36F2 TCB is similar and around 1000 nM for both, whereas the affinity to murine FolR1-Fc is slightly better and around 300 nM. The 36F2 can be used in murine and primate models, there is no need for a surrogate.
[0540] The avidity (apparent KD) of 36F2 TCB to human FolR1 is around 30 times lower than the affinity of the 16D5 TCB to human FolR1. In the bivalent format, 36F2 TCB is in the low nanomolar range, whereas 16D5 TCB is in the low picomolar range (1000 fold difference).
[0541] FolR1 is expressed on tumor cells overexpressed, at intermittent and high levels, on the surface of cancer cells in a spectrum of epithelial malignancies, including ovarian, breast, renal, colorectal, lung and other solid cancers and is also expressed on the apical surface of a limited subset of polarized epithelial cells in normal tissue. These non-tumorous, normal cells express FolR1 only at low levels, and include, e.g., bronchiolal epithelial cells on alveolar surface, renal cortical luminal border of tubular cells, retinal pigment epithelium (basolateral membrane) and choroid plexus. 16D5 TCB binds to normal tissues cells expressing low amounts of FolR1 which results in their T cell mediated killing. This might, at least in part, account for limited tolerance observed at 10 .mu.g/kg in cynomolgus monkeys. The inventors wanted to determine if lowering the affinity of the T cell bispecific molecule could increase the differentiation between high and low target density tissues and, thereby, lower toxicity by making use of bivalent binding and avidity. Low affinity binders are ordinarily not selected as suitable candidates for further analysis because low affinity is often associated with low potency and efficacy. Nevertheless, the low affinity FolR1 binder 36F2 was developed in several formats and characterized for its biological properties. For the 36F2 used in the bivalent T cell bispecific format the avidity effect (difference between monovalent and bivalent binding) is around 250 fold (1000 nM versus 4 nM). At low target density the affinity defined the interaction and with 1000 nM led to a low potency of the TCB. However, at high target density the molecule's avidity comes into play and with 4 nM led to a high activity of the TCB (see Example 32).
[0542] In an alternatively approach, the inventors generated monovalent formats of 16D5 and low affinity variant of 16D5 (affinity about 10-40 nM) in a bivalent format. The 16D5 binder used in a monovalent format (1+1) has an affinity of about 50 nM. The differentiation between high and low target density tissues can be better achieved by taking advantage of the avidity effect.
Example 32
T-Cell Killing of SKov-3 Cells Induced by 36F2 TCB, Mov19 TCB and 21A5 TCB
[0543] T-cell killing mediated by 36F2 TCB, Mov19 TCB and 21A5 TCB was assessed on SKov-3 cells (medium FolR1). Human PBMCs were used as effectors and the killing was detected at 24 h and 48 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350.times.g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree. C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.005 pM-5 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h and 48 h of incubation at 37.degree. C., 5% CO.sub.2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct.
[0544] The results show that the killing induced by 36F2 is strongly reduced in comparison to Mov19 TCB and 21A5 TCB (FIGS. 23A-B). The EC50 values related to killing assays, calculated using GraphPadPrism6 are summarized in Table 27.
TABLE-US-00029 TABLE 27 EC50 values (pM) for T-cell mediated killing of FolR1-expressing SKov-3 cells induced by 36F2 TCB, Mov19 TCB and 21A5 TCB. EC50 [pM] Antibody 24 h 48 h 36F2 TCB 1406.07* 134.5 Mov19 TCB 0.75 0.05 21A5 TCB 2.83 0.10 *curve did not reach saturation, value is hypothetical
Example 33
T-Cell Killing Induced by 36F2 TCB and 16D5 TCB in Different Monovalent and Bivalent T-Cell Bispecific Formats
[0545] T-cell killing mediated by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, 16D5 TCB 1+1 and 16D5 TCB HT antibodies of Hela (high FolR1, about 2 million copies, Table 14, FIG. 27), Skov-3 (medium FolR1, about 70000-90000 copies, Table 14, FIG. 27) and HT-29 (low FolR1, about 10000, Table 14, FIG. 27) human tumor cells was assessed. DP47 TCB antibody was included as negative control. Human PBMCs were used as effectors and the killing was detected at 24 h of incubation with the bispecific antibody. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350.times.g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree. C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-100 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h of incubation at 37.degree. C., 5% CO.sub.2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct.
[0546] The results show that target-specific killing of all three FolR1+ target cell lines induced by 36F2 TCB is much weaker compared to the killing induced by 16D5 TCB (FIGS. 24A-C, Table 29). Target-specific killing induced by the monovalent 16D5 TCBs (16D5 HT and 16D5 1+1) is worse compared to the bivalent 16D5 TCBs (16D5 TCB and 16D5 TCB classical). The EC50 values related to killing assays, calculated using GraphPadPrism6, are summarized in Table 28. Importantly, this data shows that using the 36F2 FolR1 binder in the bivalent 2+1 TCB format widens the therapeutic window compared to the 16D5 FOLR1 TCB (FIG. 24A-C). Whereas the potency reduction between 16D5 and 36F2 FOLR1 TCB is approximately 45-fold for Hela cells (high FOLR1 expression, see Table 28: 16D5 TCB=0.8 versus 36F2 TCB 36.0) and approximately 297-fold for Skov3 cells (medium FOLR1 expression, see Table 28: 16D5 TCB=0.6 versus 36F2 TCB 178.4), this reduction is almost 7000-fold for HT29 with low FOLR1 expression (see Table 28: 16D5 TCB=5.7 versus 36F2 TCB 39573). Thus, the 36F2 FOLR1 TCB differentiates between high and low expressing cells which is of special importance to reduce toxicity as the cells of some normal, non-tumorous tissues express very low levels of FolR1 (approximately less than 1000 copies per cell). Consistent with this observation, the results discussed in Example 35 below show that 36F2 TCB does not induce T-cell killing of primary cells (FIGS. 26A-D) whereas for 16D5 TCB some killing can be observed on HRCEpiC and HRPEpiC cells after 48 h of incubation (FIGS. 26B and C). This important characteristic of 36F2 TCB allows for dosing for the treatment of FolR1-positive tumors so that it mediates potent killing of tumor tissues with high or medium FOLR1 expression, but not of normal tissues with low (partially polarized) expression. Notably, this characteristic appears to be mediated by the avidity of 36F2 TCB in the bivalent 2+1 inverted format, as it was not observed when using the 1+1 monovalent formats carrying the same low affinity 36F2 binder.
[0547] Stated another way, 36F2 TCB in the bivalent 2+1 format comprises FolR1 binding moieties of relatively low affinity but it possesses an avidity effect which allows for differentiation between high and low FolR1 expressing cells. Because tumor cells express FolR1 at high or intermediate levels, this TCB selectively binds to tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all.
[0548] In addition to the above advantageous characteristics, the 36F2 TCB in the bivalent 2+1 inverted format also has the advantage that it does not require chemical cross linking or other hybrid approach. This makes it suitable for manufacture of a medicament to treat patients, for example patients having FolR1-positive cancerous tumors. The 36F2 TCB in the bivalent 2+1 inverted format can be produced using standard CHO processes with low aggregates. Further, the 36F2 TCB in the bivalent 2+1 comprises human and humanized sequences making it superior to molecules that employ rat and murine polypeptides that are highly immunogenic when administered to humans. Furthermore, the 36F2 TCB in the bivalent 2+1 format was engineered to abolish FcgR binding and, as such, does not cause FcgR crosslinking and infusion reactions, further enhancing its safety when administered to patients.
[0549] As demonstrated by the results described above, its head-to-tail geometry make the 36F2 TCB in the bivalent 2+1 inverted format a highly potent molecule that induces absolute target cell killing. Its bivalency enhance avidity and potency, but also allow for differentiation between high and low expressing cells. Its preference for high or medium target expressing cells due to its avidity affect reduce toxicity resulting from T cell mediated killing of normal cells that express FolR1 at low levels.
[0550] A further advantage of the 36F2 TCB in the bivalent 2+1 format and other embodiments disclosed herein is that their clinical development does not require the use of surrogate molecules as they bind to human, cynomous and murine FolR1. As such, the molecules disclosed herein recognize a different epitope than antibodies to FolR1 previously described that do not recognize FolR1 from all three species.
TABLE-US-00030 TABLE 28 EC50 values (pM) for T-cell mediated killing of FolR1-expressing tumor cells induced by 36F2 TCB and 16D5 TCB in different monovalent and bivalent T-cell bispecific formats after 24 h of incubation. Skov-3 Hela (FolR1 HT-29 Antibody (FolR1 high) medium) (FolR1 low) 16D5 TCB 0.8 0.6 5.7 16D5 TCB 4.6 2.0 13.0 classical 16D5 TCB 11.6 12.3 15.1 HT 16D5 TCB 23.8 48.9 883.8* 1 + 1 36F2 TCB 36.0 178.4 39573.0* *curve did not reach saturation, only hypothetical value
[0551] Table 29 shows a comparison of EC50 values of 16D5 TCB and 36F2 TCB on the different cell lines tested. Out of the obtained EC50 values the delta (EC50 of 16D5 TCB minus EC50 of 36F2 TCB) and the x-fold difference (EC50 of 16D5 TCB divided by the EC50 of 36F2 TCB) was calculated.
TABLE-US-00031 TABLE 29 Comparison of EC50 values of 16D5 TCB and 36F2 TCB. Hela Skov-3 HT-29 Antibody (FolR1 high) (FolR1 medium) (FolR1 low) 16D5 TCB 0.82 0.63 5.73 36F2 TCB 35.99 178.40 39573.00* .DELTA. 35.17 177.77 39567.27 x-fold 43.83 284.61 6906.58 *curve did not reach saturation, only hypothetical value
[0552] The calculated EC50 values clearly show that the difference between 36F2 TCB and 16D5 TCB gets larger the lower the FolR1 expression on the target cells is.
[0553] The same calculations as done for the comparison of the EC50 values of 16D5 TCB and 36F2 TCB were done for 16D5 TCB and the two monovalent 16D5 TCBs (16D5 TCB HT and 16D5 1+1). Tables 30 and 31 show the comparisons of the EC50 values of 16D5 TCB vs 16D5 TCB HT (Table 30) and 16D5 TCB vs 16D5 TCB 1+1 (Table 31) as well as the corresponding deltas (EC50 of 16D5 TCB minus EC50 of 16D5 TCB HT/1+1) and the x-fold differences (EC50 of 16D5 TCB divided by the EC50 of 16D5 TCB HT/1+1).
TABLE-US-00032 TABLE 30 Comparison of EC50 values of 16D5 TCB (2 + 1 inverted) and 16D5 TCB HT. Hela Skov-3 HT-29 Antibody (FolR1 high) (FolR1 medium) (FolR1 low) 16D5 TCB 0.82 0.63 5.73 16D5 TCB HT 11.61 12.27 15.11 .DELTA. 10.79 11.65 9.38 x-fold 14.14 19.58 2.64
TABLE-US-00033 TABLE 31 Comparison of EC50 values of 16D5 TCB and 16D5 TCB 1 + 1. Hela Skov-3 HT-29 Antibody (FolR1 high) (FolR1 medium) (FolR1 low) 16D5 TCB 0.82 0.63 5.73 16D5 TCB 1 + 1 23.84 48.86 883.78* .DELTA. 23.02 48.24 878.05 x-fold 29.03 77.95 154.24 *curve did not reach saturation, only hypothetical value
[0554] The comparison of the EC50 values of 16D5 TCB and 36F2 TCB (Table 29) shows that the difference in the EC50 values gets larger the lower the FolR1 expression on the target cells is. This effect cannot be seen in the comparison of 16D5 TCB and the monovalent 16D5 TCBs (Table 29 and Table 30). For 16D5 TCB 1+1 (Table 31) there is also a slight increase in the difference between the EC50 of 16D5 TCB and 16D5 TCB 1+1 with decreasing FolR1 expression but by far not as pronounced as can be seen in the comparison of 16D5 TCB vs 36F2 TCB.
Example 34
CD25 and CD69 Upregulation on CD8+ and CD4+Effector Cells after T Cell-Killing of FolR1-Expressing Tumor Cells Induced by 36F2 TCB and 16D5 TCB Antibody
[0555] Activation of CD8.sup.+ and CD4.sup.+ T cells after T-cell killing of FolR1-expressing Hela, SKov-3 and HT-29 tumor cells mediated by 36F2 TCB and 16D5 TCB was assessed by FACS analysis using antibodies recognizing the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). DP47 TCB was included as non-binding control. The antibody and the killing assay conditions were essentially as described above (Example 32) using the same antibody concentration range (0.01 pM-100 nM in triplicates), E:T ratio 10:1 and an incubation time of 48 h.
[0556] After the incubation, PBMCs were transferred to a round-bottom 96-well plate, centrifuged at 400.times.g for 4 min and washed twice with PBS containing 0.1% BSA. Surface staining for CD8 (PE anti-human CD8, BD #555635), CD4 (Brilliant Violet 421.TM. anti-human CD4, Biolegend #300532), CD69 (FITC anti-human CD69, BD #555530) and CD25 (APC anti-human CD25 BD #555434) was performed according to the manufacturer's instructions. Cells were washed twice with 150 .mu.l/well PBS containing 0.1% BSA. After centrifugation, the samples were resuspended in 200 .mu.l/well PBS 0.1% for the FACS measurement. Samples were analyzed at BD FACS Canto II.
[0557] 36F2 TCB induced a target-specific up-regulation of activation markers (CD25, CD69) on CD8+ and CD4+ T cells after killing of Hela (FIG. 25A) and SKov-3 (FIG. 25B) cells. In comparison to 16D5 TCB the up-regulation of CD25 and CD69 on CD8+ and CD4+ T cells induced by 36F2 is much weaker.
[0558] On HT-29 (low FolR1) an up-regulation of activation markers can only be seen at the highest concentration of 36F2 TCB. In contrast, with 16D5 TCB up-regulation of CD25 and CD69 can be seen already at much lower antibody concentrations (FIG. 25C).
[0559] As seen as well in the tumor lysis experiment, the analysis of activation markers (CD25 and CD69) on T cells (CD4+ and CD8+) after killing clearly shows that the difference between 16D5 TCB and 36F2 TCB becomes larger the lower the FolR1 expression level on the target cells is.
Example 35
T-Cell Killing of Primary Cells Induced by 36F2 TCB and 16D5 TCB
[0560] T-cell killing mediated by 36F2 TCB and 16D5 TCB was assessed on primary cells (Human Renal Cortical Epithelial Cells (HRCEpiC) (ScienCell Research Laboratories; Cat No 4110) and Human Retinal Pigment Epithelial Cells (HRPEpiC) (ScienCell Research Laboratories; Cat No 6540)). HT-29 cells (low FolR1) were included as control cell line. DP47 TCB served as non-binding control. Human PBMCs were used as effectors and the killing was detected at 24 h and 48 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350.times.g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree. C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h and 48 h of incubation at 37.degree. C., 5% CO.sub.2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct.
[0561] The results show that 36F2 TCB does not induce T-cell killing of primary cells (FIG. 26A-D) whereas for 16D5 TCB some killing can be observed on HRCEpiC and HRPEpiC cells after 48 h of incubation (FIGS. 26B and D). As described above, a strong difference in T-cell killing between of HT-29 cells was observed between 16D5 TCB and 36F2 TCB (FIG. 26E, F).
Example 36
Preparation of DP47 GS TCB
(2+1 Crossfab-IgG P329G LALA Inverted="Untargeted TCB")
[0562] The "untargeted TCB" was used as a control in the above experiments. The bispecific antibody engages CD3e but does not bind to any other antigen and therefore cannot crosslink T cells to any target cells (and subsequently cannot induce any killing). It was therefore used as negative control in the assays to monitor any unspecific T cell activation. This untargeted TCB was prepared as described in WO2014/131712. In brief, the variable region of heavy and light chain DNA sequences have been subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression was driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3' end of the CDS. In addition each vector contains an EBV OriP sequence.
[0563] The molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:2:1:1 ratio ("vector heavy chain Fc(hole)": "vector light chain": "vector light chain Crossfab": "vector heavy chain Fc(knob)-FabCrossfab").
[0564] For transfection HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 g DNA. After addition of 540 .mu.l PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210.times.g, the solution was sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4.degree. C.
[0565] The secreted protein was purified from cell culture supernatants by affinity chromatography using ProteinA. Supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volume 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Target protein was eluted during a gradient over 20 column volume from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium chloride solution of pH 6.0.
[0566] The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.
[0567] Purity and molecular weight of molecules were analyzed by CE-SDS analyses in the presence and absence of a reducing agent. The Caliper LabChip GXII system (Caliper lifescience) was used according to the manufacturer's instruction. 2 ug sample is used for analyses.
[0568] The aggregate content of antibody samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25.degree. C.
TABLE-US-00034 TABLE 32 Summary production and purification of DP47 GS TCB. Aggregate after 1.sup.st purification Mono- Titer Yield step HMW LMW mer Construct [mg/l] [mg/l] [%] [%] [%] [%] DP47 GS 103.7 8.04 8 2.3 6.9 91.8 TCB
TABLE-US-00035 TABLE 33 CE-SDS analyses of DP47 GS TCB. Peak kDa Corresponding Chain DP47 GS TCB non reduced 1 165.22 Molecule with 2 missing (A) light chains 2 181.35 Molecule with 1 missing light chain 3 190.58 Correct molecule without N-linked glycosylation 4 198.98 Correct molecule DP47 GS TCB reduced (B) 1 27.86 Light chain DP47 GS 2 35.74 Light chain huCH2527 3 63.57 Fc(hole) 4 93.02 Fc(knob)
Example 37
Binding of 16D5 TCB and 9D11 TCB and their Corresponding CD3 Deamidation Variants N100A and S100aA to CD3-Expressing Jurkat Cells
[0569] The binding of 16D5 TCB and the corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the demidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 was assessed on a CD3-expressing immortalized T lymphocyte line (Jurkat).
[0570] Briefly, cells were harvested, counted, checked for viability and resuspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of cell suspension (containing 0.2.times.10.sup.6 cells) was incubated in round-bottom 96-well plates for 30 min at 4.degree. C. with different concentrations of the bispecific antibodies (686 pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4.degree. C. with a PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-116-170). After washing the samples twice with cold PBS 0.1% BSA they were immediately analyzed by FACS using a FACS CantoII (Software FACS Diva). Binding curves were obtained using GraphPadPrism6 (FIG. 28A-B).
[0571] The results show reduced binding of the deamidation variants N100A and S100aA to CD3 compared to the parental antibodies 16D5 TCB (FIG. 28A) and 9D11 TCB (FIG. 28B).
Example 38
T-Cell Killing of SKov-3 and HT-29 Cells Induced by 16D5 TCB and 9D11 TCB and their CD3 Deamidation Variants N100A and S100aA
[0572] T-cell killing mediated by 16D5 TCB and the corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the demidation variants 9D11 TCB N100A and 9D11 TCB S100aA was assessed on SKov-3 (medium FolR1) and HT-29 (low FolR1) cells. Human PBMCs were used as effectors and the killing was detected at 24 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at a density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350.times.g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree. C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h of incubation at 37.degree. C., 5% CO.sub.2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct.
[0573] The results show that on SKov-3 cells the killing induced by the CD3 deamidation variants 16D5 TCB N100A and 16D5 S100aA is comparable to the one induced by 16D5 TCB (FIG. 29A). The same is true for 9D11 TCB and its variants 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 29B). On FolR1 low expressing HT-29 cells the S100aA variant shows an impaired killing efficiency which is the case for 16D5 TCB (FIG. 30A) as well as for 9D11 TCB (FIG. 30B). The EC50 values related to killing assays, calculated using GraphPadPrism6 are given in Table 35.
TABLE-US-00036 TABLE 35 EC50 values (pM) for T-cell mediated killing of FolR1-expressing SKov-3 and HT-29 cells induced by 16D5 TCB and 9D11 TCB and their deamidation variants N100A and A100aA. EC50 [pM] Antibody SKov-3 HT-29 16D5 TCB 1.283 56.67 16D5 TCB 1.886 91.95 N100A 16D5 TCB 1.939 165.6 S100aA 9D11 TCB 1.283 2.827 9D11 TCB 1.886 37.72 N100A 9D11 TCB 1.939 n.d.* S100aA *not determined
Example 39
Biochemical Characterization by Surface Plasmon Resonance as TCBs of Two CD3 Binder Variants (N100A and S100aA) to Remove a Deamidation Site
[0574] Binding of two 16D5 TCBs with CD3 binder variants (N100A or S100aA) to human recombinant CD3 (CD3epsilon-CD3delta heterodimer as Fc fusion) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).
[0575] Affinity to CD3ed-Fc
[0576] The affinity of the interaction between the anti-FolR1 T cell bispecifics and the recombinant CD3 epsilon-delta heterodimer was determined as described below (Table 36).
[0577] For affinity measurement, direct coupling of around 6000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics were captured at 200 nM with a flow rate of 20 .mu.l/min for 60 sec, the reference flow cell was left without capture. Dilution series (4.1 to 3000 nM) of human and cyno Folate Receptor 1 Fc fusion were passed on all flow cells at 30 .mu.l/min for 240 sec to record the association phase. The dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
TABLE-US-00037 TABLE 36 Monovalent binding (affinity) of two 16D5 CD3 deamidation variants as TCBs on human CD3ed-Fc. Ligand Analyte ka (1/Ms) kd (1/s) KD 16D5 TCB N100A huCD3 1.23E+04 4.67E-03 380 nM 16D5 TCB S100aA huCD3 1.21E+04 5.49E-03 460 nM 16D5 TCB huCD3 2.03E+04 4.41E-03 220 nM
[0578] The two CD3 deamidation variants have a slightly reduced affinity compared to the wild-type CD3 binder (CH2527), but the difference is not grave.
Example 40
Production and Purification of Two Variants of the 16D5 T-Cell Bispecific with Mutations to Remove the Deamidation Site in the CD3 Binder: 16D5 TCB N100A, 16D5 TCB S100aA
[0579] Transient Transfection and Production
[0580] The two deamidation variants 16D5 TCBs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells are centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 15 min at 210.times.g, the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4.degree. C. After production the supernatant was harvested, filtered through 0.22 .mu.m sterile filters and stored at 4.degree. C. until purification.
[0581] Purification
[0582] The two deamidation variants 16D5 TCBs were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to Mab Select SuRe (GE Healthcare, column volume (cv)=2 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate pH 7.5, 20 mM sodium citrate). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 3.0, 100 mM NaCl, 100 mM glycine) over 20 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 0.5 M Na.sub.2HPO.sub.4 pH 8.0). Samples were concentrated to 1 ml using ultra-concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore) and subsequently applied to a HiLoad.TM. 16/60 Superdex.TM. 200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. Fractions containing less than 2% oligomers were pooled. The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N.sub.2 and stored at -80.degree. C.
TABLE-US-00038 TABLE 36 Yield, monomer content and purity by CE-SDS of the two 16D5 deamidation variants in the T cell bispecific format. Mutation in the Monomer Purity by CE- Name CD3 binder Yield [mg/L] [%] SDS [%] 16D5 TCB N100A 9 100 89 16D5 TCB S100aA 23 100 83 16D5 TCB Wild-type 9 100 93
[0583] Both TCBs were produced in good quality, similar to the construct with the wild-type CD3 binder.
Example 41
Production and Purification of Two 16D5 Binder Variants (D52dE and D52dQ) as IgGs to Remove a Hotspot in the CDR
[0584] Transient Transfection and Production
[0585] The two IgGs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells are centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 15 min at 210.times.g, the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4.degree. C. After production the supernatants were harvested and the antibody containing supernatants were filtered through 0.22 .mu.m sterile filters and stored at 4.degree. C. until purification.
[0586] Antibody Purification
[0587] The two IgGs were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to POROS MabCapture A (Applied Biosystems, column volume (cv)=1 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5). After washing with 10 cv of buffer A, the protein was eluted using a pH step to buffer B (20 mM sodium citrate pH 3.0, 100 mM NaCl, 100 mM glycine) over 5 cv. The 5 ml containing the protein of interest are stored in a loop on the Akta Explorer and subsequently applied to a HiLoad 16/60 Superdex.TM. 200 (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl (no TWEEn was used). Fractions containing the IgGs were pooled and concentrated using ultra concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore). The aggregate content of the final pool was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l were applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were stored at 4.degree. C.
TABLE-US-00039 TABLE 37 Yield, monomer content and purity by CE-SDS of two 16D5 IgG hotspot variants. Mutation Monomer Purity by CE- Clone HC/LC Yield [mg/L} [%] SDS [%] 16D5 D52dE 9 100 96 16D5 D52dQ 20 100 96
[0588] Both IgGs produced well and in good quality.
Example 42
Biochemical Characterization by Surface Plasmon Resonance of Two 16D5 Binder Variants (D52dE and D52dQ) as IgGs to Remove a Hotspot in the CDR
[0589] Binding of two 16D5 binder variants (D52dE and D52dQ) as IgGs to human and cyno recombinant folate receptor 1 (both as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).
[0590] 1. Avidity to Folate Receptor 1
[0591] The avidity of the interaction between the anti-FolR1 IgGs or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 38).
[0592] Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 160. The anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 3.7 to 900 nM with a flow of 30 .mu.L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 600 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine IL2 receptor Fc fusion. The binding curves resulting from the bivalent binding of the IgG or T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent KD representing the avidity of the bivalent binding. The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare).
TABLE-US-00040 TABLE 38 Bivalent binding (avidity with apparent KD) of two 16D5 hot spot variants as IgGs on human, murine and cyno FolR1 (no binding on muFolR1 as expected). Analyte Ligand ka (1/Ms) kd (1/s) Apparent KD 16D5 D52dE IgG huFolR1 1.62E+05 5.45E-04 3.4 nM cyFolR1 2.98E+06 7.47E-03 2.5 nM 16D5 D52dQ IgG huFolR1 8.40E+04 7.75E-04 9.2 nM cyFolR1 4.12E+05 2.04E-03 5 nM 16D5 TCB huFolR1 2.25E+05 5.00E-04 2.2 nM cyFolR1 2.71E+05 6.63E-04 2.5 nM
[0593] 2. Affinity to Folate Receptor 1
[0594] The affinity of the interaction between the anti-FolR1 IgGs or the T cell bispecifics and the recombinant folate receptors was determined as described below (Table 39).
[0595] For affinity measurement, direct coupling of around 10000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cell bispecifics were captured at 20 nM with a flow rate of 10 .mu.l/min for 40 sec, the reference flow cell was left without capture. Dilution series (12.35 to 3000 nM) of human and cyno Folate Receptor 1 Fc fusion were passed on all flow cells at 30 .mu.l/min for 240 sec to record the association phase. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
TABLE-US-00041 TABLE 39 Monovalent binding (affinity) of two 16D5 hot spot variants as IgGs on human and cyno FolR1. Ligand Analyte ka (1/Ms) kd (1/s) KD 16D5 D52dE IgG huFolR1 2.40E+04 2.27E-03 95 nM cyFolR1 2.25E+04 1.20E-02 530 nM 16D5 D52dQ IgG huFolR1 6.97E+03 1.62E-03 230 nM cyFolR1 8.20E+03 3.32E-03 410 nM 16D5 TCB huFolR1 2.05E+04 7.05E-04 35 nM cyFolR1 1.72E+04 1.62E-03 90 nM
[0596] The two 16D5 hot spot variants have similar avidity (bivalent binding) than the wild-type 16D5 binder. The avidity is slightly decreased for the D52dQ variant and this difference is even more visible in affinity (monovalent binding).
Example 43
Production and Purification as IgGs of Twelve Variants of the 16D5 Binder with Mutations in the Heavy and Light Chain to Reduce Affinity to FolR1
[0597] Transient Transfection and Production
[0598] The twelve IgGs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells are centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After addition of 540 .mu.l PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 15 min at 210.times.g, the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4.degree. C. After production the supernatants were harvested and the antibody containing supernatants were filtered through 0.22 .mu.m sterile filters and stored at 4.degree. C. until purification.
[0599] Antibody Purification
[0600] All molecules were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to POROS MabCapture A (Applied Biosystems, column volume (cv)=1 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5). After washing with 10 cv of buffer A, the protein was eluted using a pH step to buffer B (20 mM sodium citrate pH 3.0, 100 mM NaCl, 100 mM glycine) over 5 cv. The 5 ml containing the protein of interest are stored in a loop on the Akta Explorer and subsequently applied to a HiLoad 16/60 Superdex.TM. 200 (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. Fractions containing the IgGs were pooled and concentrated using ultra concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore). The aggregate content of the final pool was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l were applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were stored at 4.degree. C.
TABLE-US-00042 TABLE 40 Yield, monomer content and purity by CE-SDS of twelve 16D5 IgG variants Mutation Monomer Purity by CE- Clone HC/LC Yield [mg/L} [%] SDS [%] 16D5 W98Y/wt 32 100 100 16D5 W98Y/K53A 24 100 100 16D5 S35H/wt 21 100 100 16D5 S35H/K53A 18 100 100 16D5 S35H/S93A 18 100 100 16D5 W96Y/wt 40 100 100 16D5 W96Y/K53A 21 100 100 16D5 W96Y/S93A 25 98 100 16D5 R50S/K53A 10 98 100 16D5 R50S/S93A 7 100 100 16D5 G49S/K53A 42 100 100 16D5 G49S/S93A 45 100 100
[0601] All twelve IgGs produced well and in good quality.
Example 44
Biochemical Characterization of 16D5 Heavy and Light Chain Combination Variants as IgG by Surface Plasmon Resonance
[0602] Binding of FolR1 16D5 heavy and light chain combination variants binders as IgG to different recombinant folate receptors (human, murine and cynomolgus FolR1; all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).
[0603] Avidity to Folate Receptor 1
[0604] The avidity of the interaction between the anti-FolR1 IgGs or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 41).
[0605] Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300. The anti-FolR1 IgGs or T cell bispecifics were passed at a concentration range from 11.1 to 900 nM with a flow of 30 .mu.L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 240 or 600 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine IL2 receptor Fc fusion. The binding curves resulting from the bivalent binding of the IgG or T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent KD representing the avidity of the bivalent binding. The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare). For low affinity kinetics with association and dissociation phases too fast to be fitted by the 1:1 Langmuir binding model, the steady state analysis model was applied using the Bia Evaluation software (GE Healthcare). The steady state analysis gives the KD of the binding reaction at equilibrium.
TABLE-US-00043 TABLE 41 Bivalent binding (avidity with apparent KD) of twelve 16D5 variants binders as IgGs on human, murine and cyno FolR1. Analyte HC variant/LC Apparent variant Ligand ka (1/Ms) kd (1/s) KD W98Y/K53A huFolR1 Weak binding cyFolR1 Weak binding S35H/K53A huFolR1 2.10E+04 2.91E-02 1400 nM cyFolR1 3.47E+04 4.04E-02 1100 nM W96Y/K53A huFolR1 580 nM (steady state) cyFolR1 660 nM (steady state) W98Y/wt huFolR1 1.36E+05 3.28E-02 240 nM cyFolR1 1.71E+05 3.61E-02 200 nM S35H/S93A huFolR1 2.43E+05 2.20E-02 90 nM cyFolR1 6.12E+05 6.77E-02 110 nM G49S/K53A huFolR1 1.90E+05 1.15E-02 60 nM cyFolR1 3.93E+05 3.28E-02 80 nM R50S/K53A huFolR1 3.28E+05 1.97E-02 60 nM cyFolR1 5.50E+05 4.55E-02 80 nM S35H/wt huFolR1 1.32E+05 5.68E-03 40 nM cyFolR1 2.23E+05 1.24E-02 55 nM R50S/S93A huFolR1 1.25E+05 3.23E-03 30 nM cyFolR1 4.39E+05 7.80E-03 20 nM W96Y/S93A huFolR1 6.55E+05 1.89E-02 30 nM cyFolR1 6.25E+05 1.74E-02 30 nM G49S/S93A huFolR1 1.52E+05 3.06E-03 20 nM cyFolR1 3.58E+05 6.22E-03 20 nM W96Y/wt huFolR1 1.29E+05 2.13E-03 20 nM cyFolR1 1.73E+05 2.11E-03 10 nM 36F2 TCB huFolR1 2.44E+06 1.37E-02 6 nM cyFolR1 4.12E+06 2.15E-02 5 nM muFolR1 4.86E+05 1.20E-03 2.5 nM 16D5 TCB huFolR1 1.41E+05 4.25E-04 3 nM cyFolR1 1.78E+05 6.39E-04 3.5 nM
[0606] Affinity to Folate Receptor 1
[0607] The affinity of the interaction between the anti-FolR1 IgGs or the T cell bispecifics and the recombinant folate receptors was determined as described below (Table 42).
[0608] For affinity measurement, direct coupling of around 10000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cell bispecifics were captured at 200 nM with a flow rate of 10 ul/min for 40 sec, the reference flow cell was left without capture. Dilution series (12.35 to 3000 nM) of human Folate Receptor 1 Fc fusion were passed on all flow cells at 30 .mu.l/min for 240 sec to record the association phase. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).
TABLE-US-00044 TABLE 42 Monovalent binding (affinity) of twelve 16D5 variants FolR1 binders as IgGs on human, cyno and murine FolR1. Ligand HC variant/LC variant Analyte ka (1/Ms) kd (1/s) KD W98Y/K53A huFolR1 No binding S35H/K53A huFolR1 Weak binding W96Y/K53A huFolR1 Weak binding W98Y/wt huFolR1 5400 nM (steady state) G49S/K53A huFolR1 9.19E+03 1.74E-02 1900 nM R50S/K53A huFolR1 1.35E+04 2.45E-02 1800 nM 36F2 TCB huFolR1 5.00E+04 8.57E-02 1700 nM S35H/S93A huFolR1 8.43E+03 1.12E-02 1300 nM S35H/wt huFolR1 8.96E+03 1.13E-02 1200 nM R50S/S93A huFolR1 1.57E+04 1.23E-02 780 nM G49S/S93A huFolR1 1.05E+04 7.99E-03 760 nM W96Y/wt huFolR1 9.95E+03 5.44E-03 550 nM W96Y/S93A huFolR1 4.05E+04 1.72E-02 420 nM 16D5 TCB huFolR1 1.18E+04 7.22E-04 60 nM
[0609] Twelve "affinity reduced" variants of the 16D5 FolR1 binder were analyzed by surface plasmon resonance in comparison to the 16D5 wild-type binder and the 36F2 binder. The goal was to find a 16D5 variant with an affinity and an avidity comparable to 36F2. When measuring monovalent binding (affinity) there were variants with a higher and variants with a lower affinity than 36F2. However in the bivalent binding (avidity) all the variants have a higher apparent KD value than 36F2. This is mainly due to the fast association rate (ka) of 36F2 that results in a small apparent KD for 36F2. The big avidity effect when 36F2 binds bivalently seems to be unique to this binder. As noted above, 36F2 was the only human, murine and cyno crossreactive binder that could be identified.
Example 45
Binding of 16D5 HC/LC Variants to Human FolR1 Expressed on Hela Cells
[0610] The binding of 36F2 TCB, 16D5 TCB and various HC/LC variants of 16D5 to human FolR1 was assessed on Hela cells. Briefly, cells were harvested, counted, checked for viability and resuspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of cell suspension (containing 0.2.times.10.sup.6 cells) was incubated in round-bottom 96-well plates for 30 min at 4.degree. C. with different concentrations of the bispecific antibodies (229 pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4.degree. C. with a PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-116-170). After washing the samples twice with cold PBS 0.1% BSA they were fixed with 1% PFA overnight. Afterwards samples were centrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using a FACS CantoII (Software FACS Diva). Binding curves were obtained using GraphPadPrism6 (FIG. 32A-E). The 36F2 TCB bound FolR2, was not well tolerated in mice, and did not demonstrate the desired efficacy.
Example 46
Production and Purification of Four Variants of the 16D5 T-Cell Bispecific with Mutations to Reduce the Affinity to Human and Cynomolgus FolR1: 16D5 TCB G49S/S93A, G49S/K53A, W96Y, W96Y/D52E
[0611] Transient Transfection and Production
[0612] Four additional variants of 16D5 TCBs having reduced affinity to FolR1 were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. For transfection HEK293 EBNA cells are cultivated in suspension serum free in Excell culture medium containing 6 mM L-Glutamine and 250 mg/l G418 culture medium. For the production in 600 ml tubespin flask (max. working volume 400 mL) 600 million HEK293 EBNA cells are seeded 24 hours before transfection. For transfection cells are centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 400 .mu.g DNA. After addition of 1080 .mu.l PEI solution (2.7 .mu.g/ml) is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 600 ml tubespin flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 360 ml Excell+6 mM L-Glutamine+5 g/L Pepsoy+1.0 mM VPA medium is added and cells are cultivated for 24 hours. One day after transfection 7% Feed 7 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 20-30 min at 3600.times.g (Sigma 8K centrifuge), the solution is sterile filtered (0.22 mm filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4.degree. C.
[0613] Purification
[0614] The reduced affinity variants 16D5 TCBs were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap Protein A (GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate pH 7.5, 20 mM sodium citrate). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 3.0, 100 mM NaCl, 100 mM glycine) over 20 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 0.5 M Na.sub.2HPO.sub.4 pH 8.0). Samples were concentrated to 1 ml using ultra-concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore) and subsequently applied to a HiLoad.TM. 16/60 Superdex.TM. 200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween 20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. Fractions containing less than 2% oligomers were pooled. The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N.sub.2 and stored at -80.degree. C.
TABLE-US-00045 TABLE 43 Yield, monomer content and purity by CE-SDS of the reduced affinity 16D5 variants in the T cell bispecific format. Mutations to Yield Purity by CE- Name reduce affinity [mg/L] Monomer [%] SDS [%] 16D5 TCB G49S S93A 10.3 100 88 16D5 TCB G49S K53A 22.3 98.5 96 16D5 TCB W96Y 15.2 98.7 92.5 16D5 TCB W96Y D52E 9.9 99.3 92.9 16D5 TCB Wild-type 5.4 96 91.6
[0615] All variants with reduced affinity could be produced in good quality.
Example 47
Binding of 36F2 TCB, 16D5 TCB and the Two 16D5 Affinity Reduced Variants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to Human FolR1 Expressed on Hela Cells
[0616] The binding of 36F2 TCB, 16D5 TCB and the two 16D5 affinity reduced variants 16D5 W96Y/D52E TCB and 16D5 G49S/S93A TCB to human FolR1 was assessed on Hela cells. Briefly, cells were harvested, counted, checked for viability and resuspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of cell suspension (containing 0.2.times.10.sup.6 cells) was incubated in round-bottom 96-well plates for 30 min at 4.degree. C. with different concentrations of the bispecific antibodies (30 pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4.degree. C. with a FITC-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-096-098). After washing the samples twice with cold PBS 0.1% BSA samples were centrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using a FACS CantoII (Software FACS Diva). Binding curves were obtained using GraphPadPrism6 (FIG. 33).
Example 48
Production and Purification of Three T-Cell Bispecifics with Intermediate Affinity to Human and Cynomolgus FolR1: 14B1, 6E10, 2C7
[0617] Transient Transfection and Production
[0618] The intermediate affinity TCBs were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. For transfection HEK293 EBNA cells are cultivated in suspension serum free in Excell culture medium containing 6 mM L-Glutamine and 250 mg/l G418 culture medium. For the production in 600 ml tubespin flask (max. working volume 400 mL) 600 million HEK293 EBNA cells are seeded 24 hours before transfection. For transfection cells are centrifuged for 5 min by 210.times.g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 400 .mu.g DNA. After addition of 1080 .mu.l PEI solution (2.7 .mu.g/ml) is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 600 ml tubespin flask and incubated for 3 hours by 37.degree. C. in an incubator with a 5% CO2 atmosphere. After incubation time 360 ml Excell+6 mM L-Glutamine+5 g/L Pepsoy+1.0 mM VPA medium is added and cells are cultivated for 24 hours. One day after transfection 7% Feed 7 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 20-30 min at 3600.times.g (Sigma 8K centrifuge), the solution is sterile filtered (0.22 .mu.m filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4.degree. C.
[0619] Purification
[0620] The intermediate affinity TCBs were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap Protein A (GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate pH 7.5, 20 mM sodium citrate). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 3.0, 100 mM NaCl, 100 mM glycine) over 20 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 0.5 M Na.sub.2HPO.sub.4 pH 8.0). Samples were concentrated to 1 ml using ultra-concentrators (Amicon Ultra-15, 30.000 MWCO, Millipore) and subsequently applied to a HiLoad.TM. 16/60 Superdex.TM. 200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween 20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 .mu.l of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Toso h) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C. Fractions containing less than 2% oligomers were pooled. The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis (CE-SDS) in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N.sub.2 and stored at -80.degree. C.
TABLE-US-00046 TABLE 44 Yield, monomer content and purity by CE-SDS of the intermediate affinity TCBs. Purity by CE- Name Yield [mg/L] Monomer [%] SDS [%] 6E10 TCB 2.3 93 95 14B1 TCB 1.8 94 70 9C7 TCB 3.4 98 99
[0621] All intermediate affinity T cell bispecifics could be produced. The yields are not high. The quality is good for 9C7 and acceptable for 14B1 and 6E10.
Example 49
Binding of 16D5 HC/LC Variants to Human FolR1 Expressed on HT-29 Cells
[0622] The binding of 36F2 TCB, 16D5 TCB and various HC/LC variants (FIG. 34A-E) of 16D5 to human FolR1 was assessed on HT-29 cells. Briefly, cells were harvested, counted, checked for viability and resuspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of cell suspension (containing 0.2.times.10.sup.6 cells) was incubated in round-bottom 96-well plates for 30 min at 4.degree. C. with different concentrations of the bispecific antibodies (229 pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4.degree. C. with a PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-116-170). After washing the samples twice with cold PBS 0.1% BSA they were fixed with 1% PFA overnight. Afterwards samples were centrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using a FACS CantoII (Software FACS Diva). Binding curves were obtained using GraphPadPrism6 (FIG. 34A-E).
Example 50
Binding of Intermediate FolR1 Binders to Human and Mouse FolR1 and FolR2
[0623] Cross-reactivity of the intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB), as well as 16D5 TCB and 36F2 TCB to human and mouse FolR1 and FolR2 was assessed in a FACS binding assay on transfected HEK293T cells.
[0624] Briefly, cells were harvested, counted, checked for viability and resuspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of cell suspension (containing 0.2.times.10.sup.6 cells) was incubated in round-bottom 96-well plates for 30 min at 4.degree. C. with 100 nM of the bispecific antibodies. After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4.degree. C. with a Fluorescein (FITC) AffiniPure F(ab').sub.2 Fragment Goat Anti-Human IgG, Fc.gamma. Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-096-098). After washing the samples twice with cold PBS 0.1% BSA they were fixed with 1% PFA overnight. Afterwards samples were centrifuged, resuspended in PBS 0.1% BSA and analyzed by FACS using a FACS CantoII (Software FACS Diva). Graphs were obtained using GraphPadPrism6 (FIG. 35A-D).
[0625] The results show that 36F2 TCB and 14B1 TCB are cross-reactive to mouse FolR1 and human and mouse FolR2. For 6E10 TCB a weak binding to human FolR2 can be observed. 16D5 TCB and 9C7 TCB are specific for human FolR1 and show no cross-reactivity to mouse FolR1 or human and mouse FolR2.
Example 51
Biochemical Characterization by Surface Plasmon Resonance of 16D5 Reduced Affinity Variants and Additional Intermediate Affinity Binders in the T-Cell Bispecific Format
[0626] Binding of anti-FolR1 16D5 reduced affinity variants and additional intermediate affinity binders in the bivalent T-cell bispecific format to recombinant human, cynomolgus and murine folate receptor 1 (all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, GE Healthcare). The molecules used for affinity and avidity determination are described in Table 45.
TABLE-US-00047 TABLE 45 Name, description and figure reference of the nine constructs used in SPR analysis. Figure Name Description reference 16D5 reduced affinity 2 + 1 T-cell bispecific, inverted FIG. 1A variants format (common light chain) 16D5 TCB 16D5 G49S/S93A TCB 16D5 G49S/K53A TCB 16D5 W96Y TCB 16D5 W96Y/D52E TCB Intermediate affinity 2 + 1 T-cell bispecific, inverted FIG. 1F binders format, crossfab 36F2 TCB 6E10 TCB 14B1 TCB 9C7 TCB
[0627] Single Injections
[0628] First the anti-FolR1 TCBs were analyzed by single injections (Table 46) to characterize their crossreactivity (to human, murine and cyno FolR1) and specificity (to human FolR1, human FolR2, human FolR3). Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) or human Folate Receptor 2 and 3 (FolR2-Fc, FolR3-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The TCBs were injected for 60 seconds at a concentration of 500 nM.
TABLE-US-00048 TABLE 46 Crossreactivity and specificity of 7 folate receptor 1 T cell bispecifics. + means binding, - means no binding, +/- means weak binding. Binding to Binding to Binding to Binding to Binding to Clone name huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 16D5 TCB + + - - - 16D5 + + - - - G49S/S93A TCB 16D5 + + - - - W96Y/D52E TCB 36F2 TCB + + + +/- - 6E10 TCB + + - - - 14B1 TCB + + + +/- - 9C7 TCB + + - - -
[0629] Avidity to Folate Receptor 1
[0630] The avidity of the interaction between the anti-FolR1 T cell bispecifics and the recombinant folate receptors was determined as described below (Table 47).
[0631] Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, GE Healthcare). The immobilization level was about 200-300 RU. The anti-FolR1 T cell bispecifics were passed at a concentration range from 11.1 to 900 nM (for the 16D5 reduced affinity variants) or 0.2 to 500 nM (for the additional intermediate affinity binders and 36F2) with a flow of 30 .mu.L/minutes through the flow cells over 180 seconds. The dissociation was monitored for 240 or 600 seconds. The chip surface was regenerated after every cycle using a double injection of 30 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine IL2R Fc fusion (unrelated Fc fused receptor). The binding curves resulting from the bivalent binding of the T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent KD representing the avidity of the bivalent binding. The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare).
TABLE-US-00049 TABLE 47 Bivalent binding (avidity with apparent KD) of anti-FolR1 T-cell bispecifics (TCB) on human, cyno and murine FolR1. Apparent Analyte Ligand ka (1/Ms) kd (1/s) KD 16D5 TCB huFolR1 1.68E+05 4.33E-04 3 nM cyFolR1 2.08E+05 6.95E-04 3 nM 16D5 G49S/S93A huFolR1 1.49E+05 2.09E-03 10 nM TCB cyFolR1 4.54E+05 7.84E-03 20 nM 16D5 G49S/K53A huFolR1 1.32E+05 5.86E-03 40 nM TCB cyFolR1 3.73E+05 2.56E-02 70 nM 16D5 W96Y TCB huFolR1 1.15E+05 1.44E-03 10 nM cyFolR1 1.37E+05 1.68E-03 10 nM 16D5 W96Y/D52E huFolR1 1.24E+05 1.40E-03 10 nM TCB cyFolR1 5.17E+05 1.41E-02 30 nM 36F2 TCB huFolR1 1.12E+06 7.90E-03 7 nM cyFolR1 1.97E+06 1.10E-02 6 nM muFolR1 5.54E+05 1.47E-03 3 nM 6E10 TCB huFolR1 7.93E+06 8.74E-03 1 nM cyFolR1 5.56E+06 5.72E-03 1 nM 14B1 TCB huFolR1 1.12E+06 1.40E-03 1 nM cyFolR1 1.02E+06 1.66E-03 2 nM muFolR1 8.03E+06 8.20E-04 0.1 nM 9C7 TCB huFolR1 1.18E+06 1.42E-03 1 nM cyFolR1 4.98E+06 4.82E-03 1 nM
[0632] 3. Affinity to Folate Receptor 1
[0633] The affinity of the interaction between the anti-FolR1 T cell bispecifics and the recombinant folate receptors was determined as described below (Table 48).
[0634] For affinity measurement, direct coupling of around 12000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics were captured at 20 nM with a flow rate of 10 .mu.l/min for 40 sec, the reference flow cell was left without capture. Dilution series (12.3 to 3000 nM) of human, cyno or murine Folate Receptor 1 Fc fusion were passed on all flow cells at 30 .mu.l/min for 240 sec to record the association phase. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare). For low affinity kinetics with association and dissociation phases too fast to be fitted by the 1:1 Langmuir binding model, the steady state analysis model was applied using the Bia Evaluation software (GE Healthcare). The steady state analysis gives the KD of the binding reaction at equilibrium.
TABLE-US-00050 TABLE 48 Monovalent binding (affinity) of anti-FolR1 T-cell bispecifics (TCB) on human, cyno and murine FolR1. Analyte Ligand ka (1/Ms) kd (1/s) KD 16D5 TCB huFolR1 1.22E+04 7.02E-04 57 nM cyFolR1 1.29E+04 1.71E-03 130 nM 16D5 G49S/ huFolR1 1.01E+04 8.37E-03 830 nM S93A TCB cyFolR1 2.05E+04 8.60E-03 420 nM 16D5 G49S/ huFolR1 9.17E+03 1.59E-02 1700 nM K53A TCB cyFolR1 1900 nM (steady state analysis) 16D5 W96Y huFolR1 1.11E+04 4.05E-03 370 nM TCB cyFolR1 1.17E+04 5.16E-03 440 nM 16D5 huFolR1 1400 nM W96Y/ D52E TCB (steady state analysis) cyFolR1 5600 nM (steady state analysis) 36F2 TCB huFolR1 1400 nM (steady state analysis) cyFolR1 1500 nM (steady state analysis) muFolR1 3.50E+04 1.73E-02 490 nM 6E10 TCB huFolR1 1200 nM (steady state analysis) cyFolR1 1500 nM (steady state analysis) 14B1 TCB huFolR1 6.16E+04 3.03E-02 490 nM cyFolR1 1200 nM (steady state analysis) muFolR1 7.03E+04 2.28E-03 30 nM 9C7 TCB huFolR1 840 nM (steady state analysis) cyFolR1 1400 nM (steady state analysis)
[0635] The mutations introduced into the 16D5 binders reduce its affinity to human and cynomolgus FolR1 as determined by surface plasmon resonance. The ranking with decreasing affinity is 16D5 WT (57 nM)>W96Y (6.5 fold lower)>G49S/S93A (14.5 fold lower)>W96Y/D52E (24.5 fold lower)>G49S/K53A (30 fold lower). The same ranking is visible in the avidity values, however the fold differences are smaller 16D5 WT (3 nM)>W96Y, G49S/S93A, W96Y/D52E (3 fold lower)>G49S/K53A (13 fold lower).
[0636] The intermediate affinity binders have following ranking in affinity 16D5 (57 nM)>14B1 (8.5 fold lower)>9C7 (15 fold lower)>6E10 (21 fold lower)>36F2 (24.5 fold lower). These differences however disappear in the avidity measurement 14B1, 9C7, 6E10 (1 nM)>16D5 (3 nM)>36F2 (7 nM).
[0637] 16D5 W96Y/D52E TCB addresses the problems observed with previous candidates. 16D5 W96Y/D52E TCB is based on the common light chain 16D5 binder and has two point mutations on the heavy chain with respect to the parental 16D5 binder. The W96Y mutation reduces the affinity of the binder to FolR1 compared to the parental binder and the D52E mutation removes a deamidation site and also contributes to the reduction in affinity. 16D5 W96Y/D52E TCB binds to human and cynomolgus FolR1, but not to murine FolR1. It is specific for FolR1 and does not bind to recombinant human FolR2 or human FolR3. The affinity (monovalent binding) of 16D5 W96Y/D52E is around 1.4 .mu.M for human FolR1 (24.5 fold lower than the parental 16D5 binder) and the avidity (bivalent binding) is around 10 nM (3 fold lower than the parental 16D5 binder).
Example 52
T-Cell Killing of Hela, SKov-3 and HT-29 Cells Induced by Intermediate FolR1 TCBs
[0638] T-cell killing mediated by intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB), was assessed on Hela (high FolR1), SKov-3 (medium FolR1) and HT-29 (low FolR1) cells. 16D5 TCB and 36F2 TCB were included as benchmarks. Human PBMCs were used as effectors and the killing was detected at 24 h and 48 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at a density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350.times.g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree. C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h and 48 h of incubation at 37.degree. C., 5% CO.sub.2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct.
[0639] The results show that tumor lysis induced by the intermediate FolR1 binders (6E10 TCB, 14B1 TCB and 9C7 TCB) ranges between the one obtained for the high affinity 16D5 TCB and the low affinity 36F2 TCB (FIG. 36A-F). Among the intermediate FolR1 binders, 14B1 TCB shows the strongest killing as can be seen after 48 h of incubation (FIG. 36D-F). The EC50 values related to killing assays after 24 h and 48 h of incubation, calculated using GraphPadPrism6, are given in Table 49 and Table 50.
TABLE-US-00051 TABLE 49 EC50 values (pM) for T-cell mediated killing of Hela, SKov-3 and HT-29 cells induced by intermediate FolR1 TCBs after 24 h of incubation. EC50 [pM] Antibody Hela SKov-3 HT-29 6E10 TCB 6.5 n.d. *n.d. 14B1 TCB 8.5 30.1 *n.d. 9C7 TCB 2.8 741.4 *n.d. 16D5 TCB 2.2 1.5 *n.d. 36F2 TCB 31.1 *n.d. *n.d. *not determined
TABLE-US-00052 TABLE 50 EC50 values (pM) for T-cell mediated killing of Hela, SKov-3 and HT-29 cells induced by intermediate FolR1 TCBs after 48 h of incubation. EC50 [pM] Antibody Hela SKov-3 HT-29 6E10 TCB 2.1 2164.0 *n.d. 14B1 TCB 5.5 4.7 397.7 9C7 TCB 4.3 519.6 *n.d. 16D5 TCB 2.3 *n.d. 4.9 36F2 TCB 10.5 *n.d. n.d. *not determined
Example 53
T-Cell Killing of Hela, SKov-3 and HT-29 Cells Induced by Affinity Reduced 16D5 Variants
[0640] T-cell killing mediated by affinity reduced 16D5 variants (16D5-G49S/S93A TCB, 16D5-G49S/K53A TCB, 16D5 W96Y TCB, 16D5 W96Y/D52E TCB) was assessed on Hela (high FolR1), SKov-3 (medium FolR1) and HT-29 (low FolR1) cells. 16D5 TCB and 36F2 TCB were included as benchmarks. The assay was performed as described above (Example 52).
[0641] The results show that tumor lysis induced by affinity reduced 16D5 variants (16D5-G49S/S93A TCB, 16D5-G49S/K53A TCB, 16D5 W96Y TCB, 16D5 W96Y/D52E TCB), ranges between the one obtained for the high affinity 16D5 TCB and the low affinity 36F2 TCB. The EC50 values related to killing assays after 24 h and 48 h of incubation, calculated using GraphPadPrism6, are given in Table 51 and Table 52 (FIG. 37A-F).
TABLE-US-00053 TABLE 51 EC50 values (pM) for T-cell mediated killing of FolR1-expressing Hela, SKov-3 and HT-29 cells induced by 16D5 TCB and its affinity reduced variants after 24 h of incubation. EC50 [pM] Antibody Hela SKov-3 HT-29 16D5 TCB 2.2 1.5 *n.d. 16D5- 2.3 430.4 *n.d. G49S/S93A TCB 16D5- 4.4 1701.9 *n.d. G49S/K53A TCB 16D5 W96Y 3.0 164.5 *n.d. TCB 16D5 1.3 235.4 *n.d. W96Y/D52E TCB 36F2 TCB 31.1 *n.d. *n.d. *not determined
TABLE-US-00054 TABLE 52 EC50 values (pM) for T-cell mediated killing of FolR1-expressing Hela, SKov-3 and HT-29 cells induced by 16D5 TCB and its affinity reduced variants after 48 h of incubation. EC50 [pM] Antibody Hela SKov-3 HT-29 16D5 TCB 2.3 0.1 4.9 16D5- 0.9 95.9 99.3 G49S/S93A TCB 16D5- 0.5 950.4 1790.7 G49S/K53A TCB 16D5 W96Y 1.8 24.7 99.3 TCB 16D5 0.9 93.0 399.4 W96Y/D52E TCB 36F2 TCB 10.5 968.5 *n.d. *not determined
[0642] Thus, as with 36F2 FOLR1 TCB described above, the 16D5 W96Y/D52E TCB differentiates between high and low expressing cells which is of special importance to reduce toxicity as the cells of some normal, non-tumorous tissues express very low levels of FolR1 (approximately less than 1000 copies per cell). Consistent with this observation, the results discussed in Example 54 below show that 16D5 W96Y/D52E TCB induces much lower levels of T-cell-mediated killing of primary cells (FIG. 38A-F) compared to the parental 16D5 TCB. As such, 16D5 W96Y/D52E TCB mediates potent killing of tumor tissues with high or medium FOLR1 expression, but not of normal tissues with low expression. 16D5 W96Y/D52E TCB in the bivalent 2+1 format comprises FolR1 binding moieties of relatively low affinity but it possesses an avidity effect which allows for differentiation between high and low FolR1 expressing cells. Because tumor cells express FolR1 at high or intermediate levels, this TCB selectively binds to tumor cells and not normal, non-cancerous cells that express FolR1 at low levels or not at all. As an additional advantage over the 36F2 FOLR1 TCB described above, the 16D5 W96Y/D52E TCB binds specifically to FolR1 and not to FolR2 or FolR3, further enhancing its safety for in vivo treatment.
[0643] In addition to the above advantageous characteristics, the 16D5 W96Y/D52E TCB in the bivalent 2+1 inverted format also has the advantage that it does not require chemical cross linking or other hybrid approach. This makes it suitable for manufacture of a medicament to treat patients, for example patients having FolR1-positive cancerous tumors. The 16D5 W96Y/D52E TCB in the bivalent 2+1 inverted format can be produced using standard CHO processes with low aggregates. Further, the 16D5 W96Y/D52E TCB in the bivalent 2+1 comprises human and humanized sequences making it superior to molecules that employ rat and murine polypeptides that are highly immunogenic when administered to humans. Furthermore, the 16D5 W96Y/D52E TCB in the bivalent 2+1 format was engineered to abolish FcgR binding and, as such, does not cause FcgR crosslinking and infusion reactions, further enhancing its safety when administered to patients.
[0644] As demonstrated by the results described above, its head-to-tail geometry make the 16D5 W96Y/D52E TCB in the bivalent 2+1 inverted format a highly potent molecule that induces absolute target cell killing. Its bivalency enhance avidity and potency, but also allow for differentiation between high and low expressing cells. Its preference for high or medium target expressing cells due to its avidity affect reduce toxicity resulting from T cell mediated killing of normal cells that express FolR1 at low levels.
[0645] A further advantage of the 16D5 W96Y/D52E TCB in the bivalent 2+1 format and other embodiments disclosed herein is that their clinical development does not require the use of surrogate molecules as they bind to human and cynomous FolR1. As such, the molecules disclosed herein recognize a different epitope than antibodies to FolR1 previously described that do not recognize FolR1 from both species (see also FIG. 41).
Example 54
T-Cell Killing of Primary Cells Induced by Affinity Reduced 16D5 Variants and Intermediate FolR1 TCBs
[0646] T-cell killing mediated by affinity reduced 16D5 variants (16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB) and the intermediate FolR1 binder 14B1 TCB was assessed on primary cells (Human Renal Cortical Epithelial Cells (HRCEpiC) (ScienCell Research Laboratories; Cat No 4110) and Human Retinal Pigment Epithelial Cells (HRPEpiC) (ScienCell Research Laboratories; Cat No 6540)). HT-29 cells (low FolR1) were included as control cell line. 16D5 TCB and 36F2 TCB were included as benchmarks and DP47 TCB served as non-binding control.
[0647] The assay was performed as described in Example 52, with a concentration range of the antibodies of 0.1 pM-100 nM (in triplicates).
[0648] When human primary cells are used as targets, the overall lysis is much lower due to a lower expression rate of FolR1 on these cells (FIG. 38A-F). For the high affinity FolR1 binder 16D5 TCB a T-cell mediated lysis can be observed on both primary cell types used. As observed previously when tumor cell lines were used as targets, lysis induced by the intermediate FolR1 binder 14B1 TCB and the affinity reduced 16D5 variants (16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB), ranges between the one obtained for the high affinity 16D5 TCB and the low affinity 36F2 TCB. The significantly reduced lysis of cells that express FolR1 at low levels is consistent with low off target activity and the affinity reduced 16D5 variants 16D5-G49S/S93A TCB and 16D5 W96Y/D52E TCB are, thus, expected to be well tolerated in vivo.
Example 55
Single Dose PK of FOLR1 TCB Constructs in Female NOG Mice
[0649] Female NOD/Shi-scid/IL-2R.gamma. null (NOG) mice at an average age of 8-10 weeks at start of experiment (purchased from Taconic, SOPF facility) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by local government (ZH193/2014). After arrival animals were maintained for one week to get accustomed to new environment and for observation. Continuous health monitoring was carried out on regular basis.
[0650] A single dose pharmacokinetic study (SDPK) was performed to evaluate exposure of FOLR1 TCB constructs (36F2, 16D5, 16D5 G49S/S93A and 16D5 W96Y/D52E). An i.v. bolus administration of 0.5 mg/kg was administered to NOG mice and blood samples were taken at selected time points for pharmacokinetic evaluation. Mouse serum samples were analyzed by ELISA. Biotinylated a-huCD3-CDR (mAb<ID-mAb<CD3>>M-4.25.93-IgG-Bi), test samples, Digoxygenin labelled a-huFc antibody (mAb<H-FC pan>M-R10Z8E9-IgG-Dig) and anti-Digoxygenin detection antibody (POD) were added stepwise to a 96-well streptavidin-coated microtiter plate and incubated after every step for 1 h at roomtemperature. The plate is washed three times after each step to remove unbound substances. Finally, the peroxidase-bound complex is visualized by adding ABTS substrate solution to form a colored reaction product. The reaction product intensity, which is photometrically determined at 405 nm (with reference wavelength at 490 nm), is proportional to the analyte concentration in the serum sample. The calibration range of the standard curve for the constructs is was 0.078 to 5 ng/ml, where 1.5 ng/ml is the lower limit of quantification (LLOQ).
[0651] The SDPK study revealed an IgG-like PK-profile for the 16D5, 16D5 W96Y/D52E and 16D5 G49S/S93A constructs (FIG. 39A-B). Because of that, a once per weeks scheduling was chosen for the efficacy study (FIG. 40B). The half-life for 36F2 is lower as compared to the other clones. 36F2 is the only out of the four molecules tested that is cross-reactive to mouse FOLR1, which might explain the lower half-life for this molecule and indicates a TMDD (Target Mediated Drug Disposition).
Example 56
In Vivo Efficacy of FOLR1 TCB Constructs (16D5, 16D5 G49S/S93A and 16D5 W96Y/D52E) after Human PBMC Transfer in Hela-Bearing NOG Mice
[0652] The FOLR1 TCB constructs were tested in the FOLR1-expressing human cervical cancer cell line Hela, injected s.c. into PBMC engrafted NOG mice.
[0653] Hela cells were originally obtained from ATCC (CCL2) and after expansion deposited in the Roche-Glycart internal cell bank. The tumor cell line was routinely cultured in RPMI containing 10% FCS (Gibco) at 37.degree. C. in a water-saturated atmosphere at 5% CO2. Passage 13 was used for transplantation, at a viability >95%. 1.times.106 cells per animal were injected s.c. into the right flank of the animals in a total of 100 .mu.l of RPMI cell culture medium (Gibco).
[0654] 60 female NOG mice, age 8-10 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (ZH193/2014). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
[0655] According to the study protocol (FIG. 40B), mice were injected s.c. on study day 0 with 1.times.106 Hela cells. At study day 30, when tumor reached a size of app. 150 mm3, human PBMC of a healthy donor were isolated via the Ficoll method and 10.times.106 cells were injected i.v. into the tumor-bearing mice. Two days after (day 32), mice were randomized and equally distributed in six treatment groups (n=10) followed by i.v. injection with either 16D5 (0.5 mg/kg), 16D5 G49S/S93A (2.5 or 0.5 mg/kg) and 16D5 W96Y/D52E (2.5 or 0.5 mg/kg). All treatments group were injected once weekly for three weeks in total. Mice were injected i.v. with 200 .mu.l of the appropriate solution. The mice in the vehicle group were injected with PBS. To obtain the proper amount of TCB per 200 the stock solutions were diluted with PBS when necessary. Tumor growth was measured once weekly using a caliper (FIG. 40C-E) and tumor volume was calculated as followed:
Tv: (W2/2).times.L (W: Width,L: Length)
[0656] The once weekly injection of the FOLR1 TCB constructs resulted in significant tumor regression (FIG. 40C-E). The efficacy of 16D5 (0.5 mg/kg) and 16D5 W96Y/D52E16D5 (0.5 mg/kg) was comparable, whereas 16D5 G49S/S93A (0.5 mg/kg) showed slight less potency. The higher doses of 2.5 mg/kg of 16D5 W96Y/D52E16D5 and 16D5 G49S/S93A didn't show increased efficacy compared to 0.5 mg/kg doses. For PD read-outs, mice were sacrificed at study day 52, tumors were removed, weighted and single cell suspensions were prepared through an enzymatic digestion with Collagenase V, Dispase II and DNAse for subsequent FACS-analysis. Explanted tumors of all treatment groups showed significant lower tumor weight at study termination as compared to vehicle control tumors (FIG. 40F). Single cell suspensions from tumors where stained for huCD45 and huCD3 and DAPI for dead cell exclusion and were analyzed at the BD Fortessa. The FACS analysis revealed statistically higher numbers of infiltrated CD3-positive human T-cells in the tumor tissue upon treatment with 16D5 as well as 16D5 W96Y/D52E16D5 compared to vehicle control tumors (FIG. 40C).
Example 57
Toxicity Study in Cynomolgus Monkey
[0657] A pharmacokinetic (PK), pharmacodynamic (PD) and tolerability study is performed to investigate the tolerability, PK and PD effects of a single intravenous dose of affinity reduced 16D5 variant TCBs (e.g., 16D5-G49S/S93A TCB, 16D5 W96Y/D52E TCB) in cynomolgus monkeys. In this study, naive cynomolgus monkeys, (1 male and 1 female monkey/group), receive a single intravenous dose of affinity reduced 16D5 variant TCBs, including 16D5 W96Y/D52E TCB, following a dose escalating protocol. Exemplary dose levels include 0.003, 0.03, and 0.09 mg/kg. Standard toxicity parameters (clinical signs, body weights, hematology & clinical chemistry) and the kinetics of T cell numbers and activation status in blood and the kinetics of cytokine release are assessed. Blood samples are also taken for PK for a period of 28 days for the measurement of affinity reduced 16D5 variant TCBs, including 16D5 W96Y/D52E TCB, and of anti-drug antibodies.
[0658] Amino Acid Sequences of Exemplary Embodiments
[0659] 1) FolR Binders Useful in Common Light Chain Format, Variable Heavy Chain
TABLE-US-00055 Description Sequence Seq ID No 16A3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 1 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNYYAGVTPFDYWGQGTLVTVSS 18D3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 2 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNYYTGGSSAFDYWGQGTLVTVS 15H7 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 3 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNYYLFSTSFDYWGQGTLVTVSS 15B6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 4 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNYYIGIVPFDYWGQGTLVTVSS 21D1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 5 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNYYVGVSPFDYWGQGTLVTVSS 16F12 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 6 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNFTVLRVPFDYWGQGTLVTVSS 15A1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 7 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARNYYIGVVTFDYWGQGTLVTVSS 15A1 CDR1 SYYMH 8 15A1 CDR2 IINPSGGSTSYAQKFQG 9 15A1 CDR3 NYYIGVVTFDY 10 19E5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 11 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARGEWRRYTSFDYWGQGTLVTVSS 19E5_CDR1 SYYMH 8 19E5_CDR2 IINPSGGSTSYAQKFQG 9 19E5_CDR3 GEWRRYTSFDY 12 19A4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 13 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARGGWIRWEHFDYWGQGTLVTVSS 19A4_CDR1 SYYMH 8 19A4_CDR2 IINPSGGSTSYAQKFQG 9 19A4_CDR3 GGWIRWEHFDY 14 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 15 WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED TAVYYCTTPWEWSWYDYWGQGTLVTVSS 16D5_CDR1 NAWMS 16 16D5_CDR2 RIKSKTDGGTTDYAAPVKG 17 16D5_CD3R PWEWSWYDY 18 15E12 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 19 WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED TAVYYCTTPWEWSYFDYWGQGTLVTVSS 15E12_CDR1 NAWMS 16 15E12_CDR2 RIKSKTDGGTTDYAAPVKG 17 15E12_CDR3 PWEWSYFDY 20 21A5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 21 WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED TAVYYCTTPWEWAWFDYWGQGTLVTVSS 21A5_CDR1 NAWMS 16 21A5_CDR2 RIKSKTDGGTTDYAAPVKG 17 21A5_CDR3 PWEWAWFDY 22 21G8 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 23 WVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED TAVYYCTTPWEWAYFDYWGQGTLVTVSS 21G8_CDR1 NAWMS 16 21G8_CDR2 RIKSKTDGGTTDYAAPVKG 17 21G8_CDR3 PWEWAYFDY 24 19H3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 25 WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARTGWSRWGYMDYWGQGTLVTVSS 19H3_CDR1 SYYMH 8 19H3_CDR2 IINPSGGSTSYAQKFQG 9 19H3_CDR3 TGWSRWGYMDY 26 20G6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 27 WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARGEWIRYYHFDYWGQGTLVTVSS 20G6_CDR1 SYYMH 8 20G6_CDR2 IINPSGGSTSYAQKFQG 9 20G6_CDR3 GEWIRYYHFDY 28 20H7 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 29 WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARVGWYRWGYMDYWGQGTLVTVSS 20H7_CDR1 SYYMH 8 20H7_CDR2 IINPSGGSTSYAQKFQG 9 20H7_CDR3 VGWYRWGYMDY 30
[0660] 2) CD3 Binder Common Light Chain (CLC)
TABLE-US-00056 Description Sequence Seq ID No common QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 31 CD3 light GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED chain (VL) EAEYYCALWYSNLWVFGGGTKLTVL common GSSTGAVTTSNYAN 32 CD3 light chain_CDR1 common GTNKRAP 33 CD3 light chain_CDR2 common ALWYSNLWV 34 CD3 light chain_CDR3 common QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 35 CD3 light GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED chain EAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSE (VLCL) ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS
[0661] 3) CD3 Binder, Heavy Chain
TABLE-US-00057 Seq ID Description Sequence No CD3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG 36 variable LEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSL heavy chain RAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (VH) CD3 heavy TYAMN 37 chain (VH)_CDR1 CD3 heavy RIRSKYNNYATYYADSVKG 38 chain (VH)_CDR2 CD3 heavy HGNFGNSYVSWFAY 39 chain (VH)_CDR3 CD3 full EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG 40 heavy chain LEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSL (VHCH1)_ RAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSC CD3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG 84 constant ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK heavy chain PSNTKVDKKVEPKSC CH1
[0662] 4) FolR Binders Useful for Crossfab Format
TABLE-US-00058 Seq ID Description Sequence No 11F8_VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE 41 WMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTA VYYCARAVFYRAWYSFDYWGQGTTVTVSS 11F8_VH_CDR1 SYAIS 42 11F8_VH_CDR2 GIIPIFGTANYAQKFQG 43 11F8_VH_CDR3 AVFYRAWYSFDY 44 11F8_VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKL 45 LIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYT SPPPTFGQGTKVEIK 11F8_VL_CDR1 RASQSISSWLA 46 11F8_VL_CDR2 DASSLES 47 11F8_VL_CDR3 QQYTSPPPT 48 36F2_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 49 WMGIINPSGGSTSYAQKFQGRVIMTHDTSTSTVYMELSSLRSEDTA VYYCARSFFTGFHLDYWGQGTLVTVSS 36F2_VH_CDR1 SYYMH 8 36F2_VH_CDR2 IINPSGGSTSYAQKFQG 9 36F2_VH_CDR3 SFFTGFHLDY 50 36F2_VL EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 51 LLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQY TNEHYYTFGQGTKVEIK 36F2_VL_CDR1 RASQSVSSSYLA 52 36F2_VL_CDR2 GASSRAT 53 36F2_VL_CDR3 QQYTNEHYYT 54 9D11_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 55 WMGIINPSGGPTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARGDFAWLDYWGQGTLVTVSS 9D11_VH_CDR1 SYYMH 8 9D11_VH_CDR2 IINPSGGPTSYAQKFQG 56 9D11_VH_CDR3 GDFAWLDY 57 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 58 QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRTFGQGTKVEIK 9D11_VL_CDR1 RSSQSLLHSNGYNYLD 59 9D11_VL_CDR2 LGSNRAS 60 9D11_VL_CDR3 MQASIMNRT 61 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 62 N95S QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMSRTFGQGTKVEIK 9D11_VL MQASIMSRT 63 N95S_CDR3 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 64 N95Q QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMQRTFGQGTKVEIK 9D11_VL MQASIMQRT 65 N95Q_CDR3 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 66 T97A QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRAFGQGTKVEIK 9D11_VL MQASIMNRA 67 T97A 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 68 T97N QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRNFGQGTKVEIK 9D11_VL MQASIMNRN 69 T97N_CDR3 5D9_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 70 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARSYIDMDYWGQGTLVTVSS 5D9_VH_CDR1 SYYMH 8 5D9_VH_CDR2 IINPSGGSTSYAQKFQG 9 5D9_VH_CDR3 SYIDMDY 71 5D9_VL EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 72 LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQD NWSPTFGQGTKVEIK 5D9_VL_CDR1 RASQSVSSSYLA 52 5D9_VL_CDR2 GASSRAT 53 5D9_VL_CDR3 QQDNWSPT 73 6B6_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 74 WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA VYYCARSYVDMDYWGQGTLVTVSS 6B6_VH_CDR1 SYYMH 8 6B6_VH_CDR2 IINPSGGSTSYAQKFQG 9 6B6_VH_CDR3 SYVDMDY 75 6B6_VL EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 76 LLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQD IWSPTFGQGTKVEIK 6B6_VL_CDR1 RASQSVSSSYLA 52 6B6_VL_CDR2 GASSRAT 53 6B6_VL_CDR3 QQDIWSPT 77 14E4_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 78 WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKDSSYVEWYAFDYWGQGTLVTVSS 14E4_VH_CDR1 SYAMS 79 14E4_VH_CDR2 AISGSGGSTYYADSVKG 80 14E4_VH_CDR3 DSSYVEWYAFDY 81 14E4_VL EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 82 LLIYGASSRATGIPDRFSGSGSGTDSTLTISRLEPEDFAVYYCQQP TSSPITFG QGTKVEIK 14E4_VL_CDR1 RASQSVSSSYLA 52 14E4_VL_CDR2 GASSRAT 53 14E4_VL_CDR3 QQPTSSPIT 83
[0663] 5) CD3 Binder Useful in Crossfab Format
TABLE-US-00059 Description Sequence Seq ID No CD3 heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG 36 chain (VH) KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS CD3 heavy TYAMN 37 chain (VH)_CDR1 CD3 heavy RIRSKYNNYATYYADSVKG 38 chain (VH)_CDR2 CD3 heavy HGNFGNSYVSWFAY 39 chain (VH)_CDR3 CD3 light QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 31 chain (VL) GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED EAEYYCALWYSNLWVFGGGTKLTVL CD3 light GSSTGAVTTSNYAN 32 chain_CDR1 CD3 light GTNKRAP 33 chain_CDR2 CD3 light ALWYSNLWV 34 chain_CDR3 pETR12940: QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 86 crossed GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED common EAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSS CD3 light KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL chain QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (VLCH1) PKSC Crossed EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG 87 CD3 heavy KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ chain MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSA (VHC.kappa.); SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV e.g. in DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY pCON1057 ACEVTHQGLSSPVTKSFNRGEC CD3- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN 85 CH1 SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSC CD3- VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD 88 ckappa NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC
[0664] 6)--Exemplary Amino Acid Sequences of CD3-FolR Bispecific Antibodies 2+1 Inverted Crossmab Format
TABLE-US-00060 Description Sequence Seq ID No VHCH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 94 [9D11]_VHCL WMGIINPSGGPTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA [CD3]_ VYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG Fcknob_ TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS PGLALA VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGG pCON1057 SEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGL EWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAE DTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVICVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_Fchole_ QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 95 PGLALA_ WMGIINPSGGPTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTA HYRF VYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCA VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_LC DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 96 pCON1063 QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VLCH1 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAF 86 [CD3] RGLIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCAL pETR12940 WYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSC CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL 307 TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCD VHCH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 308 [36F2]_VHCL WMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTA [CD3]_ VYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS Fcknob_ GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL PGLALA SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGG pCON1056 GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK GLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLR AEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVIC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPP CRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 36F2-Fc QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 309 hole WMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTA PGLALA VYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS pCON1050 GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLS CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36F2 LC EIVLIQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 310 pCON1062 LLIYGASSRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQY TNEHYYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHXGLSSPVTKSFNRGEC CD3 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAF 86 VLCH1 RGLIGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCAL pETR12940 WYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSC
[0665] 7) Exemplary Amino Acid Sequences of CD3-FolR Bispecific Antibodies with Common Light Chain
TABLE-US-00061 VHCH1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 89 [16D5]_ RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC VHCH1 TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC [CD3]_ LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL Fcknob GTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGL pCON999 VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYAT YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYV SWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK VHCH1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 90 [16D5]_ RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC Fchole TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC pCON983 LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN NYKITPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQ KSLSLSPGK CD3_common QAVVTQEPSLIVSPGGIVTLICGSSTGAVITSNYANWVQEKPGQAFRGL 35 light IGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW chain VFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV pETR13197 TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS VHCH1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS 91 [CD3]_ RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC VHCH1 VRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT [16D5]_ AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV Fcknob_ PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVE PGLALA SGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKT pETR13932 DGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW SWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK CD3_Fcknob_ EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS 92 PGLALA RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC pETR13917 VRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK Fc_hole_ DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE 93 PGLALA_ DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE HYRF YKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSC pETR10755 AVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNRFTQKSLSLSPGK VHCL[CD3]_ EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS 98 Fcknob_ RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC PGLALA VRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGT pETR13378 ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 99 inverted RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC 2 + 1 with TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC N100A in LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL CDR H3 GTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGL pETR14096 VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYAT YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGASYV SWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 100 inverted RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC 2 + 1 with TTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC S100aA in LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL CDR H3 GTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGL pETR14097 VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYAT YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYV SWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK CD3 light QAVVTQEPSLIVSPGGIVTLICGSSTGAVITSNYANWVQEKPGQAFRGL 101 chain fused IGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW to CH1; VFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP Fc_PGLALA; VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV pETR13862 NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16D5 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 102 fused to RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC constant TTPWEWSWYDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVC kappa chain; LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK pETR13859 ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CD3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS 103 fused to RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC constant VRHGNFGNSYVSWFAYWGQGTLVTVSSASPKAAPSVTLFPPSSEELQAN lambda KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSY chain; LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS pETR13860 IGHV1- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG 104 46*01 IINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCAR (X92343), GGSGGSFDYWGQGTLVTVSS plus JH4 element IGHV1- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG 105 69*06 GIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR (L22583), GGSGGSMDAWGQGTTVTVSS plus JH6 element IGHV3- EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG 106 15*01 RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC (X92216), TTGGSGGSFDYWGQGTLVTVSS plus JH4 element IGHV3- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS 107 23*01 AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (M99660), GGSGGSFDYWGQGTLVTVSS plus JH4 element IGHV4- QVQLQESGPGLVKPSETLSLICTVSGGSISSYYWSWIRQPPGKGLEWIG 108 59*01 YIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG (AB019438), GSGGSFDYWGQGTLVTVSS plus JH4 element IGHV5- EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMG 109 51*01 IIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR (M99686), GGSGGSFDYWGQGTLVTVSS plus JH4 element CD3 specific QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGL 110 antibody IGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW based on VFGGGTKLTVL humanized CH2527 light chain hVK1-39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY 111 (JK4 J- AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF element) GGGTKVEIK VL7_46-13 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGL 112 (humanized IGGINKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW anti-CD3 VFGGGTKLTVL antibody light chain)
[0666] 8) Exemplary 16D5 Variants with Reduced Affinity
[0667] a. Exemplary Light Chain Variants with Reduced Affinity
TABLE-US-00062 Seq ID Name Sequence No K53A QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTT 113 aa SNYANWVQQKPGQAPRGLIGGTNARAPGTPA RFSGSLLGGKAALTLSGVQPEDEAEYYCALW YSNLWVFGGGTKLTVL K53A_VL_ GSSTGAVTTSNYAN 32 CDR1 K53A_VL_ GTNARAP 311 CDR2 K53A_VL_ ALWYSNLWV 34 CDR3 S93A QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTT 114 aa SNYANWVQQKPGQAPRGLIGGTNKRAPGTPA RFSGSLLGGKAALTLSGVQPEDEAEYYCALW YANLWVFGGGTKLTVL S93A_VL_ GSSTGAVTTSNYAN 32 CDR1 S93A_VL_ GTNKRAP 33 CDR2 S93A_VL_ ALWYANLWV 312 CDR3
[0668] b. Exemplary Heavy Chain Variants with Reduced Affinity
TABLE-US-00063 Seq Name Sequence ID No S35H EVQLVESGGGLVKPGGSLRLSCAASGETFS 115 aa NAWMHWVRQAPGKGLEWVGRIKSKTDGGIT DYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEWSWYDYWGQGTLVTVSS AS S35H_VH_ NAWMH 313 CDR1 S35H_VH_ RIKSKTDGGTTDYAAPVKG 17 CDR2 S35H_VH_ PWEWSWYDY 18 CDR3 G49S EVQLVESGGGLVKPGGSLRLSCAASGETFS 116 aa NAWMSWVRQAPGKGLEWVSRIKSKTDGGTT DYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEWSWYDYWGQGTLVTVSS AS G49S_VH_ NAWMS 16 CDR1 G49S_VH_ RIKSKTDGGTTDYAAPVKG 17 CDR2 G49S_VH_ PWEWSWYDY 18 CDR3 R50S EVQLVESGGGLVKPGGSLRLSCAASGETFS 117 aa NAWMSWVRQAPGKGLEWVGSIKSKTDGGTT DYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEWSWYDYWGQGTLVTVSS AS R50S_VH_ NAWMS 16 CDR1 R50S_VH_ SIKSKTDGGTTDYAAPVKG 314 CDR2 R50S_VH_ PWEWSWYDY 18 CDR3 W96Y EVQLVESGGGLVKPGGSLRLSCAASGFTFS 118 aa NAWMSWVRQAPGKGLEWVGRIKSKTDGGTT DYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPYEWSWYDYWGQGTLVTVSS AS W96Y_VH_ NAWMS 16 CDR1 W96Y_VH_ RIKSKTDGGTTDYAAPVKG 17 CDR2 W96Y_VH_ PYEWSWYDY 315 CDR3 W98Y EVQLVESGGGLVKPGGSLRLSCAASGFTFS 119 aa NAWMSWVRQAPGKGLEWVGRIKSKTDGGTT DYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPWEYSWYDYWGQGTLVTVSS AS W98Y_VH_ NAWMS 16 CDR1 W98Y_VH_ RIKSKTDGGTTDYAAPVKG 17 CDR2 W98Y_VH_ PWEYSWYDY 232 CDR3
[0669] 9) Additional Exemplary Embodiments Generated from a Phage Display Library (CDRs Underlined)
TABLE-US-00064 Name Sequence Seq ID No 90D7 QVQLVQSGAEVKKPGASVKVSCKASGYTF 120 aa TSYYMHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARNYTIVVSPFDYWGQGTLV TVSSAS 90D7_VH_ SYYMH 8 CDR1 90D7_VH_ IINPSGGSTSYAQKFQG 9 CDR2 90D7_VH_ NYTIVVSPFDY 233 CDR3 90C1 QVQLVQSGAEVKKPGASVKVSCKASGYTF 121 aa TSYYMHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARNYFIGSVAMDYWGQGTLV TVSSAS 90C1_VH_ SYYMH 8 CDR1 90C1_VH_ IINPSGGSTSYAQKFQG 9 CDR2 90C1_VH_ NYFIGSVAMDY 234 CDR3 5E8 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 122 aa TSYYMHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGLTYSMDYWGQGTLVTVS SAS 5E8_VH_ SYYMH 8 CDR1 5E8_VH_ IINPSGGSTSYAQKFQG 9 CDR2 5E8_VH_ GLTYSMDY 235 CDR3 5E8 VL DIVMTQSPLSLPVTPGEPASISCRSSQSL 123 aa LHSNGYNYLDWYLQKPGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQALQIPNTFGQGTKVEIKRT 5E8_VL_ RSSQSLLHSNGYNYLD 59 CDR1 5E8_VL_ LGSNRAS 60 CDR2 5E8_VL_ MQALQIPNT 236 CDR3 12A4 VH EVQLLESGGGLVQPGGSLRLSCAASGETF 124 aa SSYAMSWVRQAPGKGLEWVSAISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKYAYALDYWGQGTLVTVSS AS 12A4_VH_ SYAMS 79 CDR1 12A4_VH_ AISGSGGSTYYADSVKG 80 CDR2 12A4_VH_ YAYALDY 237 CDR3 12A4 VL EIVLTQSPGTLSLSPGERATLSCRASQSV 125 aa SSSYLAWYQQKPGQAPRLLIYGASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQHGSSSTFGQGTKVEIKRT 12A4_VL_ RASQSVSSSYLA 52 CDR1 12A4_VL_ GASSRAT 53 CDR2 12A4_VL_ QQHGSSST 238 CDR3 7A3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 126 aa TSYYMHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGDFSAGRLMDYWGQGTLV TVSSAS 7A3_VH_ SYYMH 8 CDR1 7A3_VH_ IINPSGGSTSYAQKFQG 9 CDR2 7A3_VH_ GDFSAGRLMDY 239 CDR3 7A3 VL DIVMTQSPLSLPVTPGEPASISCRSSQSL 127 aa LHSNGYNYLDWYLQKPGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQALQTPPITFGQGTKVEIKRT 7A3_VL_ RSSQSLLHSNGYNYLD 59 CDR1 7A3_VL_ LGSNRAS 60 CDR2 7A3_VL_ MQALQTPPIT 240 CDR3 6E10 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 128 aa TSYYMHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGDYNAFDYWGHGTLVTVS SAS 6E10 VH_ SYYMH 8 CDR1 6E10 VH_ IINPSGGSTSYAQKFQG 9 CDR2 6E10 VH_ GDYNAFDY 241 CDR3 6E10 VL DIVMTQSPLSLPVTPGEPASISCRSSQSL 129 aa LHSNGYNYLDWYLQKPGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQAWHSPTFGQGTKVEIKRT 6E10 VL RSSQSLLHSNGYNYLD 59 CDR1 6E10 VL LGSNRAS 60 CDR2 6E10 VL MQAWHSPT 242 CDR3 12F9 VH QVQLVQSGAEVKKPGASVKVSCKASGYTF 130 aa TSYYMHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARGATYTMDYWGQGTLVTVS SAS 12F9_VH_ SYYMH 8 CDR1 12F9_VH_ IINPSGGSTSYAQKFQG 9 CDR2 12F9_VH_ GATYTMDY 243 CDR3 12F9 VL DIVMTQSPLSLPVTPGEPASISCRSSQSL 131 aa LHSNGYNYLDWYLQKPGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQALQTPITFGQGTKVEIKRT 12F9_VL_ RSSQSLLHSNGYNYLD 59 CDR1 12F9_VL_ LGSNRAS 60 CDR2 12F9_VL_ MQALQTPIT 244 CDR3
[0670] 10) 9D11 Glyscosite Variants: Variable Light Chain of Exemplary Embodiments (CDRs Underlined)
TABLE-US-00065 Variant Sequence Seq ID No N95S DIVMTQSPLSLPVTPGEPASISCRSSQS 132 LLHSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQASIMSRTFGQGTKVEIK 12F9_VL_ RSSQSLLHSNGYNYLD 59 CDR1 12F9_VL_ LGSNRAS 60 CDR2 12F9_VL_ MQASIMSRT 63 CDR3 N95Q DIVMTQSPLSLPVTPGEPASISCRSSQS 133 LLHSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQASIMQRTFGQGTKVEIK N95Q_VL_ RSSQSLLHSNGYNYLD 59 CDR1 N95Q_VL_ LGSNRAS 60 CDR2 N95Q_VL_ MQASIMQRT 65 CDR3 T97A DIVMTQSPLSLPVTPGEPASISCRSSQS 134 LLHSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQASIMNRAFGQGTKVEIK T97A_VL_ RSSQSLLHSNGYNYLD 59 CDR1 T97A_VL_ LGSNRAS 60 CDR2 T97A_VL_ MQASIMNRA 67 CDR3 T97N DIVMTQSPLSLPVTPGEPASISCRSSQS 135 LLHSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQASIMNRNFGQGTKVEIK T97N_VL_ RSSQSLLHSNGYNYLD 59 CDR1 T97N_VL_ LGSNRAS 60 CDR2 T97N_VL_ MQASIMNRN 69 CDR3
[0671] 11) Deamination Variants
TABLE-US-00066 Variant Sequence Seq ID No 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 248 VH_D52dE SKTEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW SWYDYWGQGTLVTVSS 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 249 VH_D52dQ SKTQGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW SWYDYWGQGTLVTVSS CD3_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIR 250 N100A SKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNF GASYVSWFAYWGQGTLVTVSS CD3_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIR 251 S100aA SKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNF GNAYVSWFAYWGQGTLVTVSS 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 252 [VHCH1]- SKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW CD3 SWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP [VHCH1- VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK N100A]- PSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASG Fcknob_ FTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSK PGLALA NTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 16D5- EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 253 Fchole- SKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW PGLALA SWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK CD3-CLC QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGG 254 TNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSS PVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS 16D5 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIK 255 [VHCH1]- SKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEW CD3 SWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP [VHCH1- VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK S100aA]- PSNTKVDKKVEPKSCDGGGGSGG Fcknob_ GGSEVQLLESGGGLVQPGGSLRLSCAASGETFSTYAMNWVRQAPGKGLEWVS PGLALA RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH GNFGNAYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPP CRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN 256 [VHCH1]- PSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWL CD3 DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV [VHCL- SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN N100A]- TKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTF Fcknob_ STYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTL PGLALA YLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 9D11- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN 257 Fchole PSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWL DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_LC DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLL 258 [N95Q] IYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMQRTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC CD3_ QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGG 259 VLCH1 TNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGT KLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSC 9D11 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN 260 [VHCH1]- PSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWL CD3 DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV [VHCH1- SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN S100aA]- TKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTF Fcknob_ STYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTL PGLALA YLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSSASVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
[0672] 12) Mov19 Based TCBs of Exemplary Embodiments (CDRs Underlined)
TABLE-US-00067 Name Sequence Seq ID No pETR11646 QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIH 136 Mov19 PYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRA VH-CH1- MDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT Fchole VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS PG/LALA NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQV SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK pETR11647 QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIH 137 Mov19 PYDGDTFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRA VH-CH1- MDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT CD3 VH- VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS CL- NTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFT Fcknob FNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSI PG/LALA LYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSAASVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL GAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK pETR11644 DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLI 138 Mov19 LC YRASNLEAGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGG GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Hu IgG1 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE 245 Fc VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK
[0673] 13) Additional FolR1 TCBs with Intermediate Affinity Binders (CDRs According to Kabat, Underlined):
TABLE-US-00068 Name Sequence Seq ID No 16D5 variant EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSK 274 W96Y/D52E TEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYD VH YWGQGTLVTVSS W96Y/D52E_ NAWMS 16 VH CDR1 W96Y/D52E_ RIKSKTEGGTTDYAAPVKG 275 VH CD R2 W96Y/D52E_ PYEWSWYDY 315 VH CD R3 16D5 variant QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA 31 W96Y/D52E FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC VL ALWYSNLWVFGGGTKLTVL W96Y/D52E_ EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSK 276 CD3- TEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYD VHCH1_Fc_ YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN knob_PGLALA SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK pETR14945 KVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNW VRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRA EDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLP PCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK W96Y/D52E_ EVQLVESGGGLVKPGGSLRLSCAASGETFSNAWMSWVRQAPGKGLEWVGRIKSK 277 Fc-hole_ TEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYD PGLALA_ YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN HYRF SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK pETR14946 KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMH EALHNRFTQKSLSLSPGK 14B1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS 278 VH GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDY WGQGTLVTVSS 14B1 SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRP 279 VL SGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRESPPTGLVVFGGGTKLTV L 14B1[EE]_ EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS 280 CD3[VLCH1]_ GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDY Fc-knob_ WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNS PGLALA GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEK pETR14976 VEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANW VQEKPGQAFRGLIGGTNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCA LWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALGAIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 14B1[EE]_ EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS 281 Fc-hole_ GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDY PGLALA WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNS pETR14977 GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEK VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHE ALHNHYTQKSLSLSPGK 14B1 LC SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRP 282 [KK] SGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRESPPTGLVVFGGGTKLTV Constant LGQPKAAPSVTLFPPSSKKLQANKATLVCLISDFYPGAVTVAWKADSSPVKAGV lambda ETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS pETR14979 9C7 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS 283 GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWSYYMDYW GQGTLVTVSS 9C7 VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY 284 LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPTFGQGTKV EIK 9C7[EE]_ QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS 285 CD3[VLCH1]_ GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWSYYMDYW Fe-knob_ GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSG PGLALA ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKV pETR14974 EPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWV QEKPGQAFRGLIGGTNKRAPGTPARESGSLLGGKAALTLSGAQPEDEAEYYCAL WYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 9C7[EE]_ QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS 286 Fc_hole_ GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWSYYMDYW PGLALA GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSG pETR14975 ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKV EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHEA LHNHYTQKSLSLSPGK 9C7 LC DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY 316 [RK] LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPTFGQGTKV pETR14980 EIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
[0674] 14) Antigen Sequences
TABLE-US-00069 Antigen Sequence Seq ID No hu FolR1 MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKL 139 HEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCL YECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKG WNWTSGENKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQM WFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS huFolR1 RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKD 140 ECD- VSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVL AcTev- NVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGENKCAVGAACQPFHFYFP Fcknob- TPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMVDE Avi tag QLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE Fchole DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV 141 KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFELVSKLTVDKSRWQQGNVESCSVMHEAL HNRFTQKSLSLSPGK MU MAHLMTVQLLLLVMWMAECAQSRATRARTELLNVCMDAKHHKEKPGPEDNLHD 142 FolR1 QCSPWKTNSCCSTNTSQEAHKDISYLYRFNWNHCGTMTSECKRHFIQDTCLYE CSPNLGPWIQQVDQSWRKERILDVPLCKEDCQQWWEDCQSSFTCKSNWHKGWN WSSGHNECPVGASCHPFTFYFPTSAALCEEIWSHSYKLSNYSRGSGRCIQMWF DPAQGNPNEEVARFYAEAMSGAGLHGTWPLLCSLSLVLLWVIS MU TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTNSCCSTNTSQEAHKDIS 143 FolR1 YLYRFNWNHCGTMTSECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERILDV ECD- PLCKEDCQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTS AcTev- AALCEEIWSHSYKLSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAEAMVDEQL Fcknob- YFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV Avitag VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE cy FolR1 MAQRMTTQLLLLLVWVAVVGEAQTRTARARTELLNVCMNAKHHKEKPGPEDKL 144 HEQCRPWKKNACCSTNTSQEAHKDVSYLYRENWNHCGEMAPACKRHFIQDTCL YECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCERWWEDCRTSYTCKSNWHKG WNWTSGENKCPVGAACQPFHEYEPTPTVLCNEIWTYSYKVSNYSRGSGRCIQM WFDPAQGNPNEEVARFYAAAMSGAGPWAAWPLLLSLALTLLWLLS cy FolR1 RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKD 145 ECD- VSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVL AcTev- NVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGENKCPVGAACQPFHFYFP Fcknob- TPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMVDE Avi tag QLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE hu FolR2 MVWKWMPLLLLLVCVATMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSP 146 WKKNACCTASTSQELHKDTSRLYNENWDHCGKMEPACKRHFIQDTCLYECSPN LGPWIQQVNQSWRKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSG VNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQ GNPNEEVARFYAAAMHVNAGEMLHGTGGLLLSLALMLQLWLLG hu FolR2 TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSPWKKNACCTASTSQELH 147 ECD- KDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKER AcTev- FLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESY Fcknob- FPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVARFYAAAMH Avi tag VVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE hu FolR3 MAWQMMQLLLLALVTAAGSAQPRSARARTDLLNVCMNAKHHKTQPSPEDELYG 148 QCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRHFIQDSCLYE CSPNLGPWIRQVNQSWRKERILNVPLCKEDCERWWEDCRTSYTCKSNWHKGWN WTSGINECPAGALCSTFESYFPTPAALCEGLWSHSEKVSNYSRGSGRCIQMWF DSAQGNPNEEVAKFYAAAMNAGAPSRGIIDS hu FolR3 SARARTDLLNVCMNAKHHKTQPSPEDELYGQCSPWKKNACCTASTSQELHKDT 149 ECD- SRLYNFNWDHCGKMEPTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKERILN AcTev- VPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAGALCSTFESYFPT Fcknob- PAALCEGLWSHSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGA Avi tag PSRGIIDSVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEW HE hu CD3.epsilon. MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYP 150 GSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPE DANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKA KPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI
[0675] 15) Nucleotide Sequences of Exemplary Embodiments
TABLE-US-00070 Seq Description Sequence ID No 16A3 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 151 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTACTACGCTGGTGTTACTCCGTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 15A1 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 152 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTACTACATCGGTGTTGTTACTTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 18D3 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 153 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTACTACACTGGTGGTTCTTCTGCTT TCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 19E5 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 154 NTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCGGTGAATGGCGTCGTTACACTTCTTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 19A4 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 155 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCGGTGGTTGGATCCGTTGGGAACATTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 15H7 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 156 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTACTACCTGTTCTCTACTTCTTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 15B6 CAGGTGCAATTGGTTCAATCTGGTGCTGAGGTAAAAAAACCGGGCG 157 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTACTACATCGGTATCGTTCCGTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 158 GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 15E12 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 159 GTTCCCNGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACCGAAGAC ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTACTTCG ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 21D1 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 160 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTACTACGTTGGTGTTTCTCCGTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 16F12 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 161 NTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCNTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCAACTTCACTGTTCTGCGTGTTCCGTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 21A5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 162 GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGGCTTGGTTCG ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 21G8 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 163 GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACCGAAGAC ACCGCAGTCTACTACTGTACTACCCCTTGGGAATGGGCTTACTTCG ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 19H3 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 164 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCACTGGTTGGTCTCGTTGGGGTTACATGG ACTATTGGGGCCAAGGCACCCTCGTAACGGTTTCTTCT 20G6 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 165 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCGGTGAATGGATCCGTTACTACCATTTCG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 20H7 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 166 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCGTTGGTTGGTACCGTTGGGGTTACATGG ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 11F8_VH CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGT 167 CCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAG CTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCAC AGAAGTTCCAGGGCAGGGTAACCATTACTGCAGACAAATCCACGAG CACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCC GTGTATTACTGTGCGAGAGCTGTTTTCTACCGTGCTTGGTACTCTT TCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 11F8_VL GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAG 168 GAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAG CTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTC CTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTT TCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAG CTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATACC AGCCCACCACCAACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 36F2_VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 169 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACT ATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 36F2_VL GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 170 GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAG CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG CTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACA GGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAG CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT ACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGGAAA TCAAA 9D11_VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 171 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGG GTCAAGGCACCCTCGTAACGGTTTCTTCT 9D11_VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 172 GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCA CTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGT CAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCG GTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCAC GTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTAC TGTATGCAGGCAAGCATTATGAACCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 173 N95S GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCA CTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGT CAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCG GTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCAC GTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTAC TGTATGCAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 174 N95Q GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCA CTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGT CAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCG GTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCAC GTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTAC TGTATGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 175 T97A GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCA CTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGT CAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCG GTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCAC GTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTAC TGTATGCAGGCAAGCATTATGAACCGGGCTTTTGGTCAAGGCACCA AGGTCGAAATTAAA 9D11_VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 176 T97N GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCA CTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGT CAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCG GTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCAC GTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTAC TGTATGCAGGCAAGCATTATGAACCGGAATTTTGGTCAAGGCACCA AGGTCGAAATTAAA 5D9_VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 177 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCTCTTACATCGACATGGACTATTGGGGTC AAGGCACCCTCGTAACGGTTTCTTCT 5D9_VL GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 178 GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAG CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG CTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAG CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGAT AACTGGAGCCCAACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA 6B6_VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 179 CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA TGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCTCTTACGTTGACATGGACTATTGGGGTC AAGGCACCCTCGTAACGGTTTCTTCT 6B6_VL GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 180 GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAG CAGCTACCTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG CTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACA GGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAG CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGAT ATTTGGAGCCCAACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA 14E4_VH GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG 181 GGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAG TTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG TGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAG ACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA CACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCC GTATATTACTGTGCGAAAGACTCTTCTTACGTTGAATGGTACGCTT TCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 14E4_VL GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 182 GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAG CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG CTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACA GGTTCAGTGGCAGTGGATCCGGGACAGACTCCACTCTCACCATCAG CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCCA ACCAGCAGCCCAATTACGTTCGGCCAGGGGACCAAAGTGGAAATCA AA CD3 heavy GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCT 183 chain GGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACC (VHCH1) TTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGC AAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAAC AACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTC ACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAG ATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGT GTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTT GCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCT AGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGC AAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTG AAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCT GGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTG CAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTG CCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTC AATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAG CCCAAGAGCTGC Crossed GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 184 CD3 heavy GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC chain CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA (VHC.kappa.) TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG CAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT GTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCA TCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGC TGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGA TAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAG GACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGT CTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT Mutagenesis GCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGG 185 primer GAB7734 N95Q Mutagenesis CAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGG 186 primer GAB7735 N95S Mutagenesis CATTATGAACCGGGCTTTTGGTCAAGGCACCAAGGTC 187 primer GAB7736 T97A Mutagenesis CATTATGAACCGGAATTTTGGTCAAGGCACCAAGGTC 188 primer GAB7737 T97N VHCH1[16D5]_ GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 189 VHCH1 GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA [CD3]_ CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG Fcknob_ TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT PGLALA ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pCON999 CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC (Inverted ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG TCB with ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC 16D5 2 + 1: AAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACA pCON999 + AGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCC pCON983 + CCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGG pETR13197) CGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCC CTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGA CAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGA GGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGG TGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTT CACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGC AAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACT ACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAG CCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTG CGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACT TCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCAC CCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTC CCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTC TTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCG TGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAA TCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAG AGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG CTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[16D5]_ GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 190 Fchole_ GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA PGLALA_ CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG HYRF TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT pCON983 ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC CAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACC AGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG CGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGC CTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC AAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCAC CCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTC TCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA CD3_common CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCG 191 light GCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCAC chain CAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC pETR13197 AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTG CCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTG TGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAG TCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCC CAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTG ATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCG ACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAA GCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACC CCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCC ACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAG C VHCH1[CD3]_ GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 192 VHCH1[16D5]_ GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC Fcknob_ CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA PGLALA TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT pETR13932 ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG (Classical CAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC TCB with ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT 16D5;2 + 1: ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT pETR13932 + GTCATCTGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCC pCON983 + AGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCG pETR13197) TGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGG AGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGC AGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCA GCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG CAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGC GGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAATTGGTTGAAT CTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTG CGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTT CGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGT CTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGG TCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTG CAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTA CTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCAC GCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTC CCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTC TTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCG TGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAA TCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAG AGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG CTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[CD3]_ GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 193 Fcknob_ GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC PGLALA CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA pETR13719 TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT (16D5 IgG ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG format 1 + 1: CAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC pETR13719 + ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT pCON983 + ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT pETR13197) GTCATCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC TCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAG AACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Fc_hole_ GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG 194 PGLALA_HYRF GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCT pETR10755 CATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG (16D5 Head- AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG to 1 + 1: TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA pCON999 + CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC pETR10755 + TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC pETR13197) TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCG CTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[9D11]_ CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 195 VHCL[CD3]_ CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC Fcknob_ CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA PGLALA TGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGC pCON1057 AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC (9D11 TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA inverted GTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGG format, GTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCC 2 + 1: CAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGA pCON1057 + ACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTG pCON1051 + TGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACAC pCON1063 + CTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGC pETR12940) GTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCT GCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGT GGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGA TCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTG GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAG CACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTG GAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCT ACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGA CAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAG GACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACA GCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGAC CGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCC CCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTC TGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGT GGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAA CAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACAC TGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGT CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG GGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTG AAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAA GGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACG TGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCA ACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGG GATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11_ CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 196 Fchole_ CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC PGLALA_HYRF CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAA pCON1051 TGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGC AGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTC TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA GTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGG GTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCC CTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGC ACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCG TGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACAC CTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGC GTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCT GCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT GGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCA GCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCC ATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCA GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 9D11_LC GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAG 197 pCON1063 GCGAACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCA CTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGT CAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCG GTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCAC GTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTAC TGTATGCAGGCAAGCATTATGAACCGGACTTTTGGTCAAGGCACCA AGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTT CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGT VLCH1[CD3] CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCG 198 pETR12940 GCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCAC CAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTG CCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTG TGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAG TGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGC TCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGC CTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATA GCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCA GTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGC AGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGC CCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT VHCL[CD3]_ GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCG 199 Fcknob_ GATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC PGLALA CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA pETR13378 TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT (9D11 ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAG CrossMab CAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC format, ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCT 1 + 1: ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT pETR13378 + GTCATCTGCTAGCGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCA pCON1051 + TCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGC pCON1063 + TGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGA pETR12940) TAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAG GACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGT CTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAG CTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGA CACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTG GATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGG ACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 200 inverted GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA 2 + 1 with CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG N100A in TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT CDR H3 ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pETR14096 CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC (pETR14096 + ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG pCON983 + ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC pETR13197) AAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACA AGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCC CCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGG CGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCC CTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGA CAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGA GGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGG TGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTT CACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGC AAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACT ACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAG CCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTG CGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACT TCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCAC CCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTC CCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTC TTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCG TGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAA TCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAG AGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG CTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 201 inverted GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA 2 + 1 with CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG S100aA in TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT CDR H3 ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pETR14097 CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC (pETR14097 + ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG pCON983 + ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCAC pETR13197) AAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACA AGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCC CCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGG CGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCC CTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGA CAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGA GGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGG TGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTT CACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGC AAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACT ACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAG CCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTG CGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACT TCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCAC CCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTC CCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTC TTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCG TGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAA TCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAG AGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG CTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3 light CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCG 202 chain fused GCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCAC to CH1; CAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC Fc_PGLALA; AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTG pETR13862 CCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT (Kappa- GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTG lambda TGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAG antibody with TGCTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC CD3 common ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGC light chain CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT fused to CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA CH1 + GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC Fc_PGLALA. AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC VHs fused to CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGA kappa or CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGG lambda GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA constant TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG chain CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG pETR13859 + GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA pETR13860 + GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG pETR13862) GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5 VH GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCG 203 fused to GTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAA constant CGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAG kappa TGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATT chain; ACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAG pETR13859 CAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGAC ACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACG ATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCGT GGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTG AAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACC CCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTC CGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACG AGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTC TAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGC CD3 VH GAAGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCG 204 fused to GATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAC constant CTACGCCATGAACTGGGTGCGACAGGCTCCTGGCAAGGGCCTGGAA lambda TGGGTGTCCCGGATCAGATCCAAGTACAACAACTACGCCACCTACT chain; ACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCTCGGGACGACTC pETR13860 CAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGAC ACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACTCCT ATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGT GTCATCTGCTAGCCCCAAGGCTGCCCCCAGCGTGACCCTGTTTCCC CCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGC CGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGC AAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGA CCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGAC CCACGAGGGCAGCACC GTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC VHCH1[36F2]_ CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 246 VHCL[CD3]_ GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG Fcknob_ CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT PGLALA AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC pCON1056 ACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACT GGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT GCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGC ACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCC GAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCAC ACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTG GTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG AACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAG AGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTG CTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGC TGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGC CAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTAC AACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGG GCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAAC AGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTG TCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGAT GAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTT TACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCC GGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTAC TCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA AAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGT CCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAG CCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGAC GGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG TGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 36F2-Fchole CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 247 PGLALA GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG pCON1050 CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC ACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACT GGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT GCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGC ACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCC GAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCAC ACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTG GTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG AACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG AGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCA GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCC CGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGC TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA 36F2 LC GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 97 pCON1062 AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTA GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGA GCATCCAGCAGGGCCACTGGCATCCCnAGACAGGTTCAGTGGCAGTGGATC CGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTCGGCCA GGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCANGGCCTGAG CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT CD3 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 198 VLCH1 GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTAC pETR1294O GCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGC GGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTG CTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAG GCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGA GGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTG TTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTC GGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAAT AGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCC TCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTG GGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAG GTGGACAAGAAGGTGGAACCCAAGTCTTGT Seq Name Sequence ID No K53A CAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 205 nt GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTAC GCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGC GGCACCAACGCCAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTG CTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAG GCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGA GGCACCAAGCTGACAGTCCTA S93A CAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 206 nt GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTAC GCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGC GGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTG CTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAG GCCGAGTACTACTGCGCCCTGTGGTACGCCAACCTGTGGGTGTTCGGCGGA GGCACCAAGCTGACAGTCCTA S35H GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 207 nt CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATG CACTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATC AAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGC CGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGG GAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCTGCTAGC G49S GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 208 nt CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATG AGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGTCCCGGATC AAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGC CGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGG GAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCTGCTAGC R50S GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 209 nt CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATG AGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGATCTATC AAGAGCAAGACCGACGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGC CGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGG GAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCT GCTAGC W96Y GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 210 nt CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATG AGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATC AAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGC CGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTAC GAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT GCTAGC W98Y GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 211 nt CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATG AGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATC AAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGC CGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGG GAGTACTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT GCTAGC 90D7 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 212 nt GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC ACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACACTATC GTTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 90C1 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 213 nt GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC
ACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTTCATC GGTTCTGTTGCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 5E8 VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 214 nt GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC ACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTCTGACTTAC TCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGC 5E8 VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 215 nt CCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGC TACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTG CTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGC GGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCA GAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGATTCCAAACACT TTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 12A4 VH GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC 216 nt CTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT AGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTC ACCATCTCCAGAGACAATCCAAGAACACGCTGTATCTGCAGATGAACAGC CTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATACGCTTACGCT CTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGC 12A4 VL GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 217 nt AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTA GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGA GCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCC GGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCA GTGTATTACTGTCAGCAGCATGGCAGCAGCAGCACGTTCGGCCAGGGGACC AAAGTGGAAATCAAACGTACG 7A3 VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 218 nt GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC ACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCTCT GCTGGTCGTCTGATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 7A3 VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 219 nt CCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGC TACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTG CTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGC GGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCA GAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCACCAATT ACCTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 6E10 VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 220 nt GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC ACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTACAAC GCTTTCGACTATTGGGGTCACGGCACCCTCGTAACGGTTTCTTCTGCTAGC 6E10 VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 221 nt CCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGC TACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTG CTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGC GGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCA GAAGACGTGGGCGTTTATTACTGTATGCAGGCATGGCATAGCCCAACTTTT GGTCAAGGCACCAAGGTCGAAATTAAACGTACG 12F9 VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 222 nt GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT AACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTC ACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGC CTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGCTACTTAC ACTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGC 12F9 VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 223 nt CCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGC TACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTG CTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGC GGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCA GAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCAATTACT TTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG pETR11646 CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTCC 224 Mov19 VH- GTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCATG CH1-Fchole AACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGAATC PG/LALA CACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAAGGCC ACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGCTGAGC CTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGACGGCAGC CGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCT AGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACC AGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAG CCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC TTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTC ACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGC GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTG AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA pETR11647 CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTCC 225 Mov19 VH- GTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCATG CH1-CD3 AACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGAATC VH-CL- CACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAAGGCC Fcknob ACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGCTGAGC PG/LALA CTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGACGGCAGC CGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCT AGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACA TCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAG CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACC TTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTC ACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGT GATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAAGTGCAGCTGGTGGAA AGCGGCGGAGGCCTGGTGCAGCCTAAGGGCTCTCTGAAGCTGAGCTGTGCC GCCAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGCGCCAGGCC CCTGGCAAAGGCCTGGAATGGGTGGCCCGGATCAGAAGCAAGTACAACAAT TACGCCACCTACTACGCCGACAGCGTGAAGGACCGGTTCACCATCAGCCGG GACGACAGCCAGAGCATCCTGTACCTGCAGATGAACAACCTGAAAACCGAG GACACCGCCATGTACTACTGCGTGCGGCACGGCAACTTCGGCAACAGCTAT GTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTGCT GCTAGCGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAG CTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCT AGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAAC TCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTG TCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC AACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCT GAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGAC ACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTG TCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAA GTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTG GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA pETR11644 GACATCGAGCTGACCCAGAGCCCTGCCTCTCTGGCCGTGTCTCTGGGACAG 226 Mov19 LC AGAGCCATCATCAGCTGCAAGGCCAGCCAGAGCGTGTCCTTTGCCGGCACC TCTCTGATGCACTGGTATCACCAGAAGCCCGGCCAGCAGCCCAAGCTGCTG ATCTACAGAGCCAGCAACCTGGAAGCCGGCGTGCCCACAAGATTTTCCGGC AGCGGCAGCAAGACCGACTTCACCCTGAACATCCACCCCGTGGAAGAAGAG GACGCCGCCACCTACTACTGCCAGCAGAGCAGAGAGTACCCCTACACCTTC GGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTC TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Seq Variant Sequence ID No 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 261 VH_D52dE TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG TCTAAAACTGAGGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGG TCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 262 VH_D52dQ TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG TCTAAAACTCAGGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGG TCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC CD3_VH GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTC 263 N100A TGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAA CTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGA AGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGT TCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAG CCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTC GGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA CCGTGTCAAGC CD3_VH GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTC 264 S100aA TGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAA CTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGA AGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGT TCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAG CCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTC GGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA CCGTGTCAAGC 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 265 [VHCH1]- TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG CD3[VHC CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG H1- TCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT N100A]- TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC Fcknob_ TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGG PGLALA TCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTA GCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAG CGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCC GTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCC CTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGT GCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCG GAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGG CGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGC TTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAG GCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTA CTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAG AACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGT ACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGC CTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGC CCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAG CCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTC TTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTG CAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACAC CAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGC CCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG TGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5- GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 266 Fchole- TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG PGLALA CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG TCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT
TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGG TCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTA GCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAG CGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCC GTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCC CCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGT GCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG CCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC CAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCG TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGAC AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3-CLC CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCG 267 TGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGC CAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGC ACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGG GAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGA GTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACC AAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCC CCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGAT CAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGC CCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACA AGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCA CAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACC GTGGCCCCCACCGAGTGCAGC 16D5 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 268 [VHCH1]- TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG CD3[VHCH1- CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG S100aA]- TCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTT Fcknob_ TTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTC PGLALA TCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGG TCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTA GCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAG CGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCC GTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCC CTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGT GCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCG GAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGG CGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGC TTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAG GCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTA CTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAG AACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGT ACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGC CTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGC CCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAG CCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTC TTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTG CAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACAC CAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGC CCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG TGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 269 [VHCH1]- TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA CD3[VHCL- CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC N100A]- CCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGA Fcknob_ TGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCG PGLALA TTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTG GACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGG GCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAAC AGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTG TCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGC TGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAG CAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC ACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGT CCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGT GCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTC AGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAAT GGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGA CAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTG TACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTG TGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGG CCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTG TTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCG TGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGT GGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGAC TCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTG ATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACC TGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGT TCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGAC CTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGG TACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGC GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC AGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAG CCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAG GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11- CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 270 Fchole TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC CCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGA TGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCG TTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTG GACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGG GCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC AGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGC TGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAG CAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC ACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA GGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGC CTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11_LC GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAAC 271 [N95Q] CGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTA CAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCG ATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCT CTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGA CGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAGCGGACTTTTGGT CAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTA CGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT CD3_VLCH1 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCG 272 TGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGC CAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGC ACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGG GAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGA GTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACC AAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTC TGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCT CGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCA CTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGT ACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGAC CTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAG GTGGAACCCAAGTCTTGT 9D11 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 273 [VHCH1]- TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA CD3[VHCH1- CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC S100aA]- CCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGA Fcknob_ TGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCG PGLALA TTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTG GACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGG GCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAAC AGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTG TCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGC TGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAG CAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC ACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGT CCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGT GCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTC AGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAAT GGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGA CAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTG TACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTG TGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGG CCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTG TTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCG TGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGT GGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGAC TCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTG ATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACC TGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGT TCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGAC CTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGG TACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGC GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC AGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAG CCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAG GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Seq Name Sequence ID No 16D5 GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTCTG 287 variant AGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGG W96Y/D52E GTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAG VH ACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATC AGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACC GAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGAC TACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCT W96Y/D52E_ GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTCTG 288 CD3- AGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGG VHCH1_ GTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAG Fc-knob_ ACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATC PGLALA AGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACC pETR14945 GAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGAC TACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACAAAGGGCCCT AGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCC CTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAAC AGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGC GGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACC CAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAG AAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCC GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTG AGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGG GTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAG TACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCC GAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTAT GTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCT AGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCT GGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTG ACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCT AGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC
ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGC CCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA W96Y/D52E_ GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTCTG 289 Fc-hole_ AGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGG PGLALA_ GTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAG HYRF ACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATC pETR14946 AGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACC GAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGAC TACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCC TCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCT CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAAC AGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCT GGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACC CAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAG AAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCA CCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAG CTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG 290 VH AGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGG GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT GGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA GACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTAC TGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 14B1 TCTTCTGAACTGACTCAAGATCCAGCTGTTAGCGTGGCTCTGGGTCAGACTGTA 291 VL CGTATCACCTGCCAAGGCGATTCTCTGCGCTCCTACTACGCAAGCTGGTACCAG CAGAAACCGGGTCAGGCCCCAGTTCTGGTGATTTACGGCAAAAACAACCGTCCG TCTGGGATCCCGGACCGTTTCTCCGGCAGCTCTTCCGGTAACACGGCGAGCCTC ACCATCACTGGCGCTCAAGCAGAAGACGAGGCCGACTATTACTGTAACTCTCGG GAAAGCCCACCAACCGGCCTGGTTGTCTTCGGTGGCGGTACCAAGCTGACCGTC CTA 14B1[EE]_ GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG 292 CD3[VLCH1]_ AGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGG Fc- GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT knob_ GGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA PGLALA GACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGAC pETR14976 ACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTAC TGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCC GTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTG GGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCT GGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGC CTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAG GTGGAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAG GCTGTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACC CTGACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGG GTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAG AGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCT GCCCTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCC CTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTG TCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAG TCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCT GAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACA TTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACC GTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAG CCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACC CACACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTC CTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGG TACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAG TACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGG CTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCC ATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTAC ACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGC CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAA 14B1[EE]_ GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG 293 Fc- AGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGG hole_ GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT PGLALA GGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA pETR14977 GACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTAC TGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCC GTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTG GGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGC GGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGC CTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAG GTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCT GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA GGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1 LC TCTTCTGAACTGACTCAAGATCCAGCTGTTAGCGTGGCTCTGGGTCAGACTGTA 294 [KK] CGTATCACCTGCCAAGGCGATTCTCTGCGCTCCTACTACGCAAGCTGGTACCAG Constant CAGAAACCGGGTCAGGCCCCAGTTCTGGTGATTTACGGCAAAAACAACCGTCCG lambda TCTGGGATCCCGGACCGTTTCTCCGGCAGCTCTTCCGGTAACACGGCGAGCCTC pETR14979 ACCATCACTGGCGCTCAAGCAGAAGACGAGGCCGACTATTACTGTAACTCTCGG GAAAGCCCACCAACCGGCCTGGTTGTCTTCGGTGGCGGTACCAAGCTGACCGTC CTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCAAG AAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCA GGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTG GAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTAC CTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTG ACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 9C7 VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT 295 AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGG GTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGC GGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGT GACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGAT ACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGG GGTCAAGGCACCCTCGTAACGGTTTCTTCT 9C7 VL GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAACCG 296 GCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAAC TATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTAC CTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCC GGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTT TATTACTGTATGCAGGCACGGCAGACCCCAACTTTTGGTCAAGGCACCAAGGTC GAAATTAAA 9C7[EE]_ CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT 297 CD3[VLCH1]_ AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGG Fc- GTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGC knob_ GGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGT PGLALA GACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGAT pETR14974 ACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGG GGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTG TTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTGGGC TGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGC GCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTG TACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACC TACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAGGTG GAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAGGCT GTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTG ACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGTG CAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGA GCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCC CTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTG TGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCC AGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCT ACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAG CCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTC CCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTG CCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCC AGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCAC ACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTCCTG TTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACC TGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTAC AACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTG AACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATC GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACC CTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTG GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCGGGTAAA 9C7[EE]_ CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT 298 Fc- AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGG hole_ GTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGC PGLALA GGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGT pETR14975 GACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGAT ACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGG GGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTG TTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGC TGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTG TATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACC TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTG GAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA GCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACC AAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGAC AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9C7 LC GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAACCG 299 [RK] GCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAAC pETR14980 TATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTAC CTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCC GGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTT TATTACTGTATGCAGGCACGGCAGACCCCAACTTTTGGTCAAGGCACCAAGGTC GAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT CGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT
[0676] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
Sequence CWU
1
1
3161120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ser Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Asn
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Tyr Tyr Ala Gly Val Thr Pro Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 2120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 2Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Tyr Thr Gly Gly Ser Ser Ala Phe Asp Tyr Trp Gly 100
105 110 Gln Gly Thr Leu Val Thr Val Ser
115 120 3120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 3Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Tyr Leu Phe Ser Thr Ser Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 4120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Tyr Ile Gly Ile Val Pro Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 5120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 5Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Tyr Val Gly Val Ser Pro Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 6120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 6Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Phe
Thr Val Leu Arg Val Pro Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 7120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Tyr Ile Gly Val Val Thr Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 85PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 8Ser Tyr Tyr Met His 1 5 917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 9Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln
1 5 10 15 Gly
1011PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 10Asn Tyr Tyr Ile Gly Val Val Thr Phe Asp Tyr 1
5 10 11120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 11Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Glu
Trp Arg Arg Tyr Thr Ser Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 1211PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Gly Glu Trp Arg Arg Tyr Thr Ser Phe Asp Tyr 1 5
10 13120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 13Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Gly
Trp Ile Arg Trp Glu His Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 1411PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 14Gly Gly Trp Ile Arg Trp Glu His Phe Asp Tyr 1 5
10 15120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 165PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 16Asn Ala Trp Met Ser 1 5 1719PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 17Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
Pro 1 5 10 15 Val
Lys Gly 189PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 18Pro Trp Glu Trp Ser Trp Tyr Asp Tyr 1
5 19120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Tyr Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 209PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Pro Trp Glu Trp Ser Tyr Phe Asp Tyr 1 5
21120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 21Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ala Trp Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120 229PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 22Pro Trp Glu Trp Ala Trp Phe Asp Tyr 1 5
23120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 23Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ala Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120 249PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Pro Trp Glu Trp Ala Tyr Phe Asp Tyr 1 5
25120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 25Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25
30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly
Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Thr Gly Trp Ser Arg Trp Gly Tyr Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120 2611PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Thr Gly Trp Ser Arg Trp Gly Tyr Met Asp Tyr 1 5
10 27120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Glu
Trp Ile Arg Tyr Tyr His Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 2811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Gly Glu Trp Ile Arg Tyr Tyr His Phe Asp Tyr 1 5
10 29120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 29Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Val Gly
Trp Tyr Arg Trp Gly Tyr Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 3011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30Val Gly Trp Tyr Arg Trp Gly Tyr Met Asp Tyr 1 5
10 31109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 31Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro
Gly Gly 1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30 Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35
40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala
Pro Gly Thr Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala 65 70 75
80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95 Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
3214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 32Gly Ser Ser Thr Gly Ala Val
Thr Thr Ser Asn Tyr Ala Asn 1 5 10
337PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 33Gly Thr Asn Lys Arg Ala Pro 1
5 349PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 34Ala Leu Trp Tyr Ser Asn Leu
Trp Val 1 5 35215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 35Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro
Gly Gly 1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30 Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35
40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala
Pro Gly Thr Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala 65 70 75
80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95 Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100
105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu Leu 115 120
125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr Pro 130 135 140
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145
150 155 160 Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165
170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu
Gln Trp Lys Ser His Arg 180 185
190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
Thr 195 200 205 Val
Ala Pro Thr Glu Cys Ser 210 215 36125PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 36Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125
375PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 37Thr Tyr Ala Met Asn 1 5
3819PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 38Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp Ser 1 5 10
15 Val Lys Gly 3914PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 39His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr 1 5 10
40228PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 40Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Thr Tyr 20 25
30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50
55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110 Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115
120 125 Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135
140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu 145 150 155
160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175 Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180
185 190 Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys 195 200
205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu 210 215 220
Pro Lys Ser Cys 225 41121PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 41Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30 Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr
Ala Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Ala Val
Phe Tyr Arg Ala Trp Tyr Ser Phe Asp Tyr Trp Gly 100
105 110 Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 425PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Ser Tyr Ala Ile Ser 1 5 4317PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 43Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
Gln 1 5 10 15 Gly
4412PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 44Ala Val Phe Tyr Arg Ala Trp Tyr Ser Phe Asp Tyr
1 5 10 45107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 45Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75
80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Ser Pro Pro Pro
85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105
4611PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 46Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1
5 10 477PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 47Asp Ala Ser Ser Leu Glu Ser 1 5
489PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 48Gln Gln Tyr Thr Ser Pro Pro Pro Thr 1
5 49119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 49Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr His Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Ser Phe
Phe Thr Gly Phe His Leu Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser Ser
115 5010PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 50Ser Phe Phe Thr Gly Phe His
Leu Asp Tyr 1 5 10 51109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 51Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Thr Asn Glu His
85 90 95 Tyr Tyr Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
5212PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 52Arg Ala Ser Gln Ser Val Ser
Ser Ser Tyr Leu Ala 1 5 10
537PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 53Gly Ala Ser Ser Arg Ala Thr 1 5
5410PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 54Gln Gln Tyr Thr Asn Glu His Tyr Tyr
Thr 1 5 10 55117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 55Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Pro
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Asp
Phe Ala Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115
5617PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 56Ile Ile Asn Pro Ser Gly Gly Pro Thr
Ser Tyr Ala Gln Lys Phe Gln 1 5 10
15 Gly 578PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 57Gly Asp Phe Ala Trp Leu
Asp Tyr 1 5 58112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 58Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Ser Ile Met Asn
Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 110 5916PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 59Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu
Asp 1 5 10 15
607PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 60Leu Gly Ser Asn Arg Ala Ser 1 5
619PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 61Met Gln Ala Ser Ile Met Asn Arg Thr 1
5 62112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 62Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Ser Ile Met Ser
Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 110 639PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 63Met Gln Ala Ser Ile Met Ser Arg Thr 1 5
64112PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 64Asp Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90
95 Ser Ile Met Gln Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
659PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 65Met Gln Ala Ser Ile Met Gln Arg Thr 1
5 66112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 66Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Ser Ile Met Asn
Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 110 679PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 67Met Gln Ala Ser Ile Met Asn Arg Ala 1 5
68112PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 68Asp Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90
95 Ser Ile Met Asn Arg Asn Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
699PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 69Met Gln Ala Ser Ile Met Asn Arg Asn 1
5 70116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 70Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Ser Tyr
Ile Asp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser 115
717PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 71Ser Tyr Ile Asp Met Asp Tyr 1 5
72107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 72Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Ser 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu 65 70
75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp
Asn Trp Ser Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 738PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 73Gln Gln Asp Asn Trp Ser
Pro Thr 1 5 74116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 74Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Ser Tyr
Val Asp Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser 115
757PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 75Ser Tyr Val Asp Met Asp Tyr 1 5
76107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 76Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Ser 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu 65 70
75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp
Ile Trp Ser Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 778PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 77Gln Gln Asp Ile Trp Ser
Pro Thr 1 5 78121PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 78Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Asp Ser
Ser Tyr Val Glu Trp Tyr Ala Phe Asp Tyr Trp Gly 100
105 110 Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 795PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 79Ser Tyr Ala Met Ser 1 5 8017PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 80Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 1 5 10 15 Gly
8112PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 81Asp Ser Ser Tyr Val Glu Trp Tyr Ala Phe Asp Tyr
1 5 10 82108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 82Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser
Arg Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Pro Thr Ser Ser Pro
85 90 95 Ile Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105
839PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 83Gln Gln Pro Thr Ser Ser Pro Ile Thr 1
5 84103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 84Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val Glu Pro
Lys Ser Cys 100 85103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 85Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75
80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95 Lys Val Glu Pro
Lys Ser Cys 100 86214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 86Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro
Gly Gly 1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30 Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35
40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala
Pro Gly Thr Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala 65 70 75
80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95 Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala 100
105 110 Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser 115 120
125 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe 130 135 140
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 145
150 155 160 Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165
170 175 Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr 180 185
190 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys 195 200 205 Val
Glu Pro Lys Ser Cys 210 87232PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 87Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Val 115 120
125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys 130 135 140
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145
150 155 160 Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165
170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser 180 185
190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys 195 200 205 Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210
215 220 Lys Ser Phe Asn Arg
Gly Glu Cys 225 230 88105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 88Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu 1 5 10 15
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
20 25 30 Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35
40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr 50 55
60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 65 70 75
80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
85 90 95 Thr Lys Ser Phe
Asn Arg Gly Glu Cys 100 105
89689PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 89Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 225
230 235 240 Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 245
250 255 Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
Ala Met Asn Trp Val Arg 260 265
270 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg
Ser Lys 275 280 285
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 290
295 300 Thr Ile Ser Arg Asp
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305 310
315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg His Gly 325 330
335 Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln
Gly 340 345 350 Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 355
360 365 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 370 375
380 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 385 390 395
400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 420
425 430 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 435 440
445 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys 450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
465 470 475 480 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
485 490 495 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp 500
505 510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 515 520
525 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 530 535 540
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545
550 555 560 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys 565
570 575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 580 585
590 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp 595 600 605
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610
615 620 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 625 630
635 640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 645 650
655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 660 665 670 Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675
680 685 Lys 90450PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 90Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120
125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys 195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210
215 220 Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285 Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300 Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
Ala Pro Ile Glu 325 330
335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
340 345 350 Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365 Ser Cys Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn Arg Phe
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 Gly Lys 450 91689PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 91Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 115 120
125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser 130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145
150 155 160 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165
170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 180 185
190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys 195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210
215 220 Pro Lys Ser Cys
Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 225 230
235 240 Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly Ser 245 250
255 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Ala Trp 260 265 270
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly
275 280 285 Arg Ile Lys Ser
Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro 290
295 300 Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr Leu 305 310
315 320 Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr Tyr 325 330
335 Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln Gly
340 345 350 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 355
360 365 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 370 375
380 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 385 390 395
400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 420
425 430 Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 435 440
445 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 465
470 475 480 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 485
490 495 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 500 505
510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
530 535 540 Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545
550 555 560 Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly Ala Pro Ile Glu Lys 565
570 575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 580 585
590 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Trp 595 600 605 Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610
615 620 Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 625 630
635 640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 645 650
655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
660 665 670 Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675
680 685 Lys 92455PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 92Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 115 120
125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser 130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145
150 155 160 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165
170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 180 185
190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys 195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210
215 220 Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230
235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 245 250
255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285 Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290
295 300 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 305 310
315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 325 330
335 Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350 Glu Pro
Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys 355
360 365 Asn Gln Val Ser Leu Trp Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375
380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 385 390 395
400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420
425 430 Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 435 440
445 Leu Ser Leu Ser Pro Gly Lys 450
455 93227PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 93Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50
55 60 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70
75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115
120 125 Cys Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180
185 190 Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205 His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser
Leu Ser Leu Ser 210 215 220
Pro Gly Lys 225 94690PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 94Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Pro
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Asp
Phe Ala Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 115 120
125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly 210
215 220 Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly 225 230
235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser 245 250
255 Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln
Ala Pro 260 265 270
Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys Tyr Asn Asn
275 280 285 Tyr Ala Thr Tyr
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 290
295 300 Arg Asp Asp Ser Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg 305 310
315 320 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His
Gly Asn Phe Gly 325 330
335 Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
340 345 350 Thr Val
Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro 355
360 365 Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu 370 375
380 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp 385 390 395
400 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
405 410 415 Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 420
425 430 Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln 435 440
445 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys Asp 450 455 460
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 465
470 475 480 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 485
490 495 Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu 500 505
510 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 515 520 525
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
530 535 540 Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 545
550 555 560 Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Gly Ala Pro Ile Glu 565
570 575 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 580 585
590 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu 595 600 605 Trp
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 610
615 620 Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 625 630
635 640 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 645 650
655 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
660 665 670 Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 675
680 685 Gly Lys 690
95447PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 95Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Asn
Pro Ser Gly Gly Pro Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Phe Ala Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser 145 150 155
160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175 Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His 210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225
230 235 240 Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245
250 255 Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu 260 265
270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300 Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile Glu Lys Thr Ile 325
330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr Leu Pro 340 345
350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
Ala 355 360 365 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370
375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390
395 400 Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
Val Asp Lys Ser Arg 405 410
415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430 His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
440 445 96219PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 96Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Ser Ile Met Asn
Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120
125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145
150 155 160 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165
170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 180 185
190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser 195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
97648DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 97gaaatcgtgt
taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcttgca
gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatacca acgaacatta ttatacgttc 300ggccagggga
ccaaagtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc 360ccgccatctg
atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480tcccaggaga
gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540ctgacgctga
gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600canggcctga
gctcgcccgt cacaaagagc ttcaacaggg gagagtgt
64898459PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 98Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Thr Tyr 20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Arg Ile Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110 Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val 115
120 125 Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135
140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg 145 150 155
160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
165 170 175 Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180
185 190 Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200
205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 210 215 220
Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro 225
230 235 240 Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 245
250 255 Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr 260 265
270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300 Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305
310 315 320 Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 325
330 335 Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 340 345
350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
Asp 355 360 365 Glu
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 370
375 380 Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390
395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe 405 410
415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 435
440 445 Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 450 455
99689PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 99Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 225
230 235 240 Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 245
250 255 Ala Ala Ser Gly Phe Thr Phe Ser
Thr Tyr Ala Met Asn Trp Val Arg 260 265
270 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg
Ile Arg Ser Lys 275 280 285
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
290 295 300 Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305
310 315 320 Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Val Arg His Gly 325
330 335 Asn Phe Gly Ala Ser Tyr Val Ser Trp Phe Ala
Tyr Trp Gly Gln Gly 340 345
350 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 355 360 365 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 370
375 380 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 385 390
395 400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 405 410
415 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
420 425 430 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 435
440 445 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 450 455
460 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro 465 470 475
480 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
485 490 495 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 500
505 510 Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 515 520
525 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val 530 535 540
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545
550 555 560 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys 565
570 575 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 580 585
590 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Trp 595 600 605
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
610 615 620 Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 625
630 635 640 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 645
650 655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 660 665
670 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 675 680 685 Lys
100689PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 100Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 225
230 235 240 Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 245
250 255 Ala Ala Ser Gly Phe Thr Phe Ser
Thr Tyr Ala Met Asn Trp Val Arg 260 265
270 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg
Ile Arg Ser Lys 275 280 285
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
290 295 300 Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305
310 315 320 Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Val Arg His Gly 325
330 335 Asn Phe Gly Asn Ala Tyr Val Ser Trp Phe Ala
Tyr Trp Gly Gln Gly 340 345
350 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 355 360 365 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 370
375 380 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 385 390
395 400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 405 410
415 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
420 425 430 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 435
440 445 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 450 455
460 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro 465 470 475
480 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
485 490 495 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 500
505 510 Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 515 520
525 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val 530 535 540
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545
550 555 560 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys 565
570 575 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 580 585
590 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Trp 595 600 605
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
610 615 620 Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 625
630 635 640 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 645
650 655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 660 665
670 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 675 680 685 Lys
101441PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 101Gln Ala Val Val Thr Gln Glu Pro
Ser Leu Thr Val Ser Pro Gly Gly 1 5 10
15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
Thr Thr Ser 20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly
35 40 45 Leu Ile Gly Gly
Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50
55 60 Ser Gly Ser Leu Leu Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Ala 65 70
75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn 85 90
95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala
100 105 110 Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 115
120 125 Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135
140 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly 145 150 155
160 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
165 170 175 Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 180
185 190 Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys 195 200
205 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro 210 215 220
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225
230 235 240 Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245
250 255 Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr 260 265
270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 275 280 285
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
290 295 300 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305
310 315 320 Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 325
330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu 340 345
350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 355 360 365 Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370
375 380 Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390
395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val 405 410
415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
420 425 430 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440
102227PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 102Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val 115
120 125 Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser 130 135
140 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys Val Gln 145 150 155
160 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175 Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 180
185 190 Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu 195 200
205 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg 210 215 220
Gly Glu Cys 225 103231PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 103Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Val Arg
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Pro 115 120
125 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
Glu Glu Leu 130 135 140
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 145
150 155 160 Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 165
170 175 Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln Ser Asn Asn Lys Tyr Ala 180 185
190 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
Ser His Arg 195 200 205
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 210
215 220 Val Ala Pro Thr
Glu Cys Ser 225 230 104118PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 104Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Gly
Ser Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ser 115
105118PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 105Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25
30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly
Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Ser Gly Gly Ser Met Asp Ala Trp Gly Gln Gly Thr
100 105 110 Thr Val
Thr Val Ser Ser 115 106120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 106Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Gly Gly Ser Gly Gly Ser Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 107118PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 107Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Gly Gly
Ser Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ser
115 108117PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 108Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5
10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Gly Ser Ile Ser Ser Tyr 20 25
30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly
Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50
55 60 Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95 Arg Gly Gly Ser Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr
Val Ser Ser 115 109118PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 109Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30 Trp Ile Gly Trp Val
Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp
Thr Arg Tyr Ser Pro Ser Phe 50 55
60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser
Thr Ala Tyr 65 70 75
80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95 Ala Arg Gly Gly
Ser Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ser 115
110109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 110Gln Thr Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5
10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser 20 25
30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg
Gly 35 40 45 Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50
55 60 Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70
75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala
Leu Trp Tyr Ser Asn 85 90
95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 111107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 111Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu
85 90 95 Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105
112109PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 112Gln Ala Val Val Thr Gln Glu Pro
Ser Leu Thr Val Ser Pro Gly Gly 1 5 10
15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
Thr Thr Ser 20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly
35 40 45 Leu Ile Gly Gly
Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50
55 60 Ser Gly Ser Leu Leu Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Ala 65 70
75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn 85 90
95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 113109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 113Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro
Gly Gly 1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30 Asn Tyr Ala Asn Trp
Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35
40 45 Leu Ile Gly Gly Thr Asn Ala Arg Ala
Pro Gly Thr Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
Ser Gly Val 65 70 75
80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95 Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
114109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 114Gln Thr Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5
10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser 20 25
30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg
Gly 35 40 45 Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50
55 60 Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70
75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala
Leu Trp Tyr Ala Asn 85 90
95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 115122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 115Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 116122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 116Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 117122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 117Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Ser Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 118122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 118Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Tyr Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 119122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 119Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Tyr Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 120122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 120Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Thr Ile Val Val Ser Pro Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 121122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 121Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr
Phe Ile Gly Ser Val Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser 115 120 122119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 122Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Leu
Thr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser Ala Ser
115 123114PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 123Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90
95 Leu Gln Ile Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Thr
124118PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 124Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Lys Tyr Ala Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110 Thr Val Ser
Ser Ala Ser 115 125109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 125Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Gly Ser Ser Ser
85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
126122PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 126Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25
30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly
Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Phe Ser Ala Gly Arg Leu Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser 115 120
127115PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 127Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90
95 Leu Gln Thr Pro Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110 Lys Arg Thr
115 128119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 128Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25
30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly
Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Tyr Asn Ala Phe Asp Tyr Trp Gly His Gly Thr Leu
100 105 110 Val Thr
Val Ser Ser Ala Ser 115 129113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 129Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Trp His Ser Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105 110 Thr 130119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 130Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Ala
Thr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser Ala Ser
115 131114PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 131Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90
95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Thr
132112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 132Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90
95 Ser Ile Met Ser Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
133112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 133Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90
95 Ser Ile Met Gln Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
134112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 134Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90
95 Ser Ile Met Asn Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
135112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 135Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90
95 Ser Ile Met Asn Arg Asn Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
136448PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 136Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Val Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30
Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45 Gly Arg Ile His
Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Asn Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Asn Thr Ala His 65 70
75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala
Val Tyr Tyr Cys 85 90
95 Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115
120 125 Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn 145 150 155
160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175 Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser 195 200
205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225
230 235 240 Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245
250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265
270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300 Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr 325 330
335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
340 345 350 Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 355
360 365 Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 385 390 395
400 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
405 410 415 Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445 137691PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 137Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30 Phe Met Asn Trp Val
Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Arg Ile His Pro Tyr Asp Gly Asp
Thr Phe Tyr Asn Gln Asn Phe 50 55
60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn
Thr Ala His 65 70 75
80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys
85 90 95 Thr Arg Tyr Asp
Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145
150 155 160 Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165
170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185
190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser 195 200 205 Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly 210
215 220 Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly 225 230
235 240 Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys
Leu Ser Cys Ala Ala 245 250
255 Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala
260 265 270 Pro Gly
Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn 275
280 285 Asn Tyr Ala Thr Tyr Tyr Ala
Asp Ser Val Lys Asp Arg Phe Thr Ile 290 295
300 Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln
Met Asn Asn Leu 305 310 315
320 Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe
325 330 335 Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 340
345 350 Val Thr Val Ser Ala Ala Ser Val
Ala Ala Pro Ser Val Phe Ile Phe 355 360
365 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys 370 375 380
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 385
390 395 400 Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 405
410 415 Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser 420 425
430 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His 435 440 445
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 450
455 460 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 465 470
475 480 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 485 490
495 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 500 505 510 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 515
520 525 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 530 535
540 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 545 550 555
560 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
565 570 575 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 580
585 590 Tyr Thr Leu Pro Pro Cys Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 595 600
605 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 610 615 620
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 625
630 635 640 Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 645
650 655 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 660 665
670 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 675 680 685
Pro Gly Lys 690 138218PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 138Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
Leu Gly 1 5 10 15
Gln Arg Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala
20 25 30 Gly Thr Ser Leu Met
His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35
40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn
Leu Glu Ala Gly Val Pro Thr 50 55
60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu
Asn Ile His 65 70 75
80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
85 90 95 Glu Tyr Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
105 110 Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln 115 120
125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145
150 155 160 Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165
170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185
190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 195 200 205 Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
139257PRTHomo sapiens 139Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu
Leu Leu Val Trp Val 1 5 10
15 Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu
20 25 30 Leu Leu
Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35
40 45 Pro Glu Asp Lys Leu His Glu
Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55
60 Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys
Asp Val Ser Tyr 65 70 75
80 Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys
85 90 95 Lys Arg His
Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100
105 110 Leu Gly Pro Trp Ile Gln Gln Val
Asp Gln Ser Trp Arg Lys Glu Arg 115 120
125 Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln
Trp Trp Glu 130 135 140
Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp 145
150 155 160 Asn Trp Thr Ser
Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165
170 175 Pro Phe His Phe Tyr Phe Pro Thr Pro
Thr Val Leu Cys Asn Glu Ile 180 185
190 Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser
Gly Arg 195 200 205
Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210
215 220 Val Ala Arg Phe Tyr
Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala 225 230
235 240 Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu
Met Leu Leu Trp Leu Leu 245 250
255 Ser 140468PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 140Arg Ile Ala Trp Ala
Arg Thr Glu Leu Leu Asn Val Cys Met Asn Ala 1 5
10 15 Lys His His Lys Glu Lys Pro Gly Pro Glu
Asp Lys Leu His Glu Gln 20 25
30 Cys Arg Pro Trp Arg Lys Asn Ala Cys Cys Ser Thr Asn Thr Ser
Gln 35 40 45 Glu
Ala His Lys Asp Val Ser Tyr Leu Tyr Arg Phe Asn Trp Asn His 50
55 60 Cys Gly Glu Met Ala Pro
Ala Cys Lys Arg His Phe Ile Gln Asp Thr 65 70
75 80 Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro
Trp Ile Gln Gln Val 85 90
95 Asp Gln Ser Trp Arg Lys Glu Arg Val Leu Asn Val Pro Leu Cys Lys
100 105 110 Glu Asp
Cys Glu Gln Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr Cys 115
120 125 Lys Ser Asn Trp His Lys Gly
Trp Asn Trp Thr Ser Gly Phe Asn Lys 130 135
140 Cys Ala Val Gly Ala Ala Cys Gln Pro Phe His Phe
Tyr Phe Pro Thr 145 150 155
160 Pro Thr Val Leu Cys Asn Glu Ile Trp Thr His Ser Tyr Lys Val Ser
165 170 175 Asn Tyr Ser
Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Pro 180
185 190 Ala Gln Gly Asn Pro Asn Glu Glu
Val Ala Arg Phe Tyr Ala Ala Ala 195 200
205 Met Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser Pro
Lys Ser Ala 210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225
230 235 240 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245
250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265
270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290
295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310
315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 325 330
335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 340 345 350 Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355
360 365 Leu Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395
400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420
425 430 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445 Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu
Ala Gln Lys Ile 450 455 460
Glu Trp His Glu 465 141227PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 141Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75
80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100
105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120
125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 195 200 205 His
Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225
142255PRTMus musculus 142Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu
Val Met Trp Met 1 5 10
15 Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu
20 25 30 Asn Val Cys
Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35
40 45 Asp Asn Leu His Asp Gln Cys Ser
Pro Trp Lys Thr Asn Ser Cys Cys 50 55
60 Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser
Tyr Leu Tyr 65 70 75
80 Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg
85 90 95 His Phe Ile Gln
Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100
105 110 Pro Trp Ile Gln Gln Val Asp Gln Ser
Trp Arg Lys Glu Arg Ile Leu 115 120
125 Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu
Asp Cys 130 135 140
Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp 145
150 155 160 Ser Ser Gly His Asn
Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165
170 175 Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu
Cys Glu Glu Ile Trp Ser 180 185
190 His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys
Ile 195 200 205 Gln
Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210
215 220 Arg Phe Tyr Ala Glu Ala
Met Ser Gly Ala Gly Leu His Gly Thr Trp 225 230
235 240 Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu
Trp Val Ile Ser 245 250
255 143466PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 143Thr Arg Ala Arg Thr Glu Leu Leu
Asn Val Cys Met Asp Ala Lys His 1 5 10
15 His Lys Glu Lys Pro Gly Pro Glu Asp Asn Leu His Asp
Gln Cys Ser 20 25 30
Pro Trp Lys Thr Asn Ser Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala
35 40 45 His Lys Asp Ile
Ser Tyr Leu Tyr Arg Phe Asn Trp Asn His Cys Gly 50
55 60 Thr Met Thr Ser Glu Cys Lys Arg
His Phe Ile Gln Asp Thr Cys Leu 65 70
75 80 Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln
Gln Val Asp Gln 85 90
95 Ser Trp Arg Lys Glu Arg Ile Leu Asp Val Pro Leu Cys Lys Glu Asp
100 105 110 Cys Gln Gln
Trp Trp Glu Asp Cys Gln Ser Ser Phe Thr Cys Lys Ser 115
120 125 Asn Trp His Lys Gly Trp Asn Trp
Ser Ser Gly His Asn Glu Cys Pro 130 135
140 Val Gly Ala Ser Cys His Pro Phe Thr Phe Tyr Phe Pro
Thr Ser Ala 145 150 155
160 Ala Leu Cys Glu Glu Ile Trp Ser His Ser Tyr Lys Leu Ser Asn Tyr
165 170 175 Ser Arg Gly Ser
Gly Arg Cys Ile Gln Met Trp Phe Asp Pro Ala Gln 180
185 190 Gly Asn Pro Asn Glu Glu Val Ala Arg
Phe Tyr Ala Glu Ala Met Val 195 200
205 Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala
Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350 Leu Pro
Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp 355
360 365 Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys
Ile Glu Trp 450 455 460
His Glu 465 144257PRTMacaca fascicularis 144Met Ala Gln Arg Met Thr
Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5
10 15 Ala Val Val Gly Glu Ala Gln Thr Arg Thr Ala
Arg Ala Arg Thr Glu 20 25
30 Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro
Gly 35 40 45 Pro
Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Lys Lys Asn Ala 50
55 60 Cys Cys Ser Thr Asn Thr
Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70
75 80 Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu
Met Ala Pro Ala Cys 85 90
95 Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn
100 105 110 Leu Gly
Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115
120 125 Val Leu Asn Val Pro Leu Cys
Lys Glu Asp Cys Glu Arg Trp Trp Glu 130 135
140 Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp
His Lys Gly Trp 145 150 155
160 Asn Trp Thr Ser Gly Phe Asn Lys Cys Pro Val Gly Ala Ala Cys Gln
165 170 175 Pro Phe His
Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180
185 190 Trp Thr Tyr Ser Tyr Lys Val Ser
Asn Tyr Ser Arg Gly Ser Gly Arg 195 200
205 Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro
Asn Glu Glu 210 215 220
Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala 225
230 235 240 Ala Trp Pro Leu
Leu Leu Ser Leu Ala Leu Thr Leu Leu Trp Leu Leu 245
250 255 Ser 145468PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 145Arg Thr Ala Arg Ala Arg Thr Glu Leu Leu Asn Val Cys Met
Asn Ala 1 5 10 15
Lys His His Lys Glu Lys Pro Gly Pro Glu Asp Lys Leu His Glu Gln
20 25 30 Cys Arg Pro Trp Lys
Lys Asn Ala Cys Cys Ser Thr Asn Thr Ser Gln 35
40 45 Glu Ala His Lys Asp Val Ser Tyr Leu
Tyr Arg Phe Asn Trp Asn His 50 55
60 Cys Gly Glu Met Ala Pro Ala Cys Lys Arg His Phe Ile
Gln Asp Thr 65 70 75
80 Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val
85 90 95 Asp Gln Ser Trp
Arg Lys Glu Arg Val Leu Asn Val Pro Leu Cys Lys 100
105 110 Glu Asp Cys Glu Gln Trp Trp Glu Asp
Cys Arg Thr Ser Tyr Thr Cys 115 120
125 Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser Gly Phe
Asn Lys 130 135 140
Cys Pro Val Gly Ala Ala Cys Gln Pro Phe His Phe Tyr Phe Pro Thr 145
150 155 160 Pro Thr Val Leu Cys
Asn Glu Ile Trp Thr Tyr Ser Tyr Lys Val Ser 165
170 175 Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile
Gln Met Trp Phe Asp Pro 180 185
190 Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala Ala
Ala 195 200 205 Met
Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala 210
215 220 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230
235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250
255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275
280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 305 310 315
320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360
365 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385
390 395 400 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405
410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425
430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445
Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile 450
455 460 Glu Trp His Glu 465
146255PRTHomo sapiens 146Met Val Trp Lys Trp Met Pro Leu Leu
Leu Leu Leu Val Cys Val Ala 1 5 10
15 Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val
Cys Met 20 25 30
Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His
35 40 45 Asp Gln Cys Ser
Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50
55 60 Ser Gln Glu Leu His Lys Asp Thr
Ser Arg Leu Tyr Asn Phe Asn Trp 65 70
75 80 Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg
His Phe Ile Gln 85 90
95 Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln
100 105 110 Gln Val Asn
Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 115
120 125 Cys Lys Glu Asp Cys Gln Arg Trp
Trp Glu Asp Cys His Thr Ser His 130 135
140 Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr
Ser Gly Val 145 150 155
160 Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe
165 170 175 Pro Thr Pro Ala
Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180
185 190 Val Ser Asn Tyr Ser Arg Gly Ser Gly
Arg Cys Ile Gln Met Trp Phe 195 200
205 Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe
Tyr Ala 210 215 220
Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly 225
230 235 240 Leu Leu Leu Ser Leu
Ala Leu Met Leu Gln Leu Trp Leu Leu Gly 245
250 255 147472PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 147Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val
Cys Met 1 5 10 15
Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His
20 25 30 Asp Gln Cys Ser Pro
Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 35
40 45 Ser Gln Glu Leu His Lys Asp Thr Ser
Arg Leu Tyr Asn Phe Asn Trp 50 55
60 Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His
Phe Ile Gln 65 70 75
80 Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln
85 90 95 Gln Val Asn Gln
Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 100
105 110 Cys Lys Glu Asp Cys Gln Arg Trp Trp
Glu Asp Cys His Thr Ser His 115 120
125 Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser
Gly Val 130 135 140
Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 145
150 155 160 Pro Thr Pro Ala Ala
Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 165
170 175 Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg
Cys Ile Gln Met Trp Phe 180 185
190 Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr
Ala 195 200 205 Ala
Ala Met His Val Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser 210
215 220 Pro Lys Ser Ala Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230
235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 245 250
255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270 Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275
280 285 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295
300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 305 310 315
320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335 Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340
345 350 Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys Arg Asp Glu Leu Thr Lys 355 360
365 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385
390 395 400 Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405
410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 420 425
430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 435 440 445
Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu 450
455 460 Ala Gln Lys Ile Glu
Trp His Glu 465 470 148243PRTHomo sapiens 148Met
Ala Trp Gln Met Met Gln Leu Leu Leu Leu Ala Leu Val Thr Ala 1
5 10 15 Ala Gly Ser Ala Gln Pro
Arg Ser Ala Arg Ala Arg Thr Asp Leu Leu 20
25 30 Asn Val Cys Met Asn Ala Lys His His Lys
Thr Gln Pro Ser Pro Glu 35 40
45 Asp Glu Leu Tyr Gly Gln Cys Ser Pro Trp Lys Lys Asn Ala
Cys Cys 50 55 60
Thr Ala Ser Thr Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr 65
70 75 80 Asn Phe Asn Trp Asp
His Cys Gly Lys Met Glu Pro Thr Cys Lys Arg 85
90 95 His Phe Ile Gln Asp Ser Cys Leu Tyr Glu
Cys Ser Pro Asn Leu Gly 100 105
110 Pro Trp Ile Arg Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Ile
Leu 115 120 125 Asn
Val Pro Leu Cys Lys Glu Asp Cys Glu Arg Trp Trp Glu Asp Cys 130
135 140 Arg Thr Ser Tyr Thr Cys
Lys Ser Asn Trp His Lys Gly Trp Asn Trp 145 150
155 160 Thr Ser Gly Ile Asn Glu Cys Pro Ala Gly Ala
Leu Cys Ser Thr Phe 165 170
175 Glu Ser Tyr Phe Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser
180 185 190 His Ser
Phe Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195
200 205 Gln Met Trp Phe Asp Ser Ala
Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215
220 Lys Phe Tyr Ala Ala Ala Met Asn Ala Gly Ala Pro
Ser Arg Gly Ile 225 230 235
240 Ile Asp Ser 149479PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 149Ser Ala Arg Ala Arg
Thr Asp Leu Leu Asn Val Cys Met Asn Ala Lys 1 5
10 15 His His Lys Thr Gln Pro Ser Pro Glu Asp
Glu Leu Tyr Gly Gln Cys 20 25
30 Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr Ser Gln
Glu 35 40 45 Leu
His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp Asp His Cys 50
55 60 Gly Lys Met Glu Pro Thr
Cys Lys Arg His Phe Ile Gln Asp Ser Cys 65 70
75 80 Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp
Ile Arg Gln Val Asn 85 90
95 Gln Ser Trp Arg Lys Glu Arg Ile Leu Asn Val Pro Leu Cys Lys Glu
100 105 110 Asp Cys
Glu Arg Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr Cys Lys 115
120 125 Ser Asn Trp His Lys Gly Trp
Asn Trp Thr Ser Gly Ile Asn Glu Cys 130 135
140 Pro Ala Gly Ala Leu Cys Ser Thr Phe Glu Ser Tyr
Phe Pro Thr Pro 145 150 155
160 Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Phe Lys Val Ser Asn
165 170 175 Tyr Ser Arg
Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser Ala 180
185 190 Gln Gly Asn Pro Asn Glu Glu Val
Ala Lys Phe Tyr Ala Ala Ala Met 195 200
205 Asn Ala Gly Ala Pro Ser Arg Gly Ile Ile Asp Ser Val
Asp Glu Gln 210 215 220
Leu Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala Asp Lys Thr His Thr 225
230 235 240 Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245
250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 260 265
270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val 275 280 285
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290
295 300 Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310
315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys 325 330
335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser 340 345 350 Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355
360 365 Cys Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Trp Cys Leu Val 370 375
380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly 385 390 395
400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
405 410 415 Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420
425 430 Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 435 440
445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys Ser Gly 450 455 460
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 465
470 475 150207PRTHomo sapiens
150Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1
5 10 15 Val Gly Val Trp
Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20
25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser
Gly Thr Thr Val Ile Leu Thr 35 40
45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn
Asp Lys 50 55 60
Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65
70 75 80 His Leu Ser Leu Lys
Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85
90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu
Asp Ala Asn Phe Tyr Leu 100 105
110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val
Met 115 120 125 Ser
Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130
135 140 Leu Leu Leu Val Tyr Tyr
Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys 145 150
155 160 Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg
Gln Arg Gly Gln Asn 165 170
175 Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg
180 185 190 Lys Gly
Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195
200 205 151360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 151caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcaactac 300tacgctggtg ttactccgtt cgactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360152360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 152caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcaactac 300tacatcggtg
ttgttacttt cgactattgg ggtcaaggca ccctcgtaac ggtttcttct
360153363DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 153caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcaactac 300tacactggtg gttcttctgc
tttcgactat tggggtcaag gcaccctcgt aacggtttct 360tct
363154360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 154caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgnttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcggtgaa 300tggcgtcgtt acacttcttt cgactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360155360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 155caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtggt 300tggatccgtt
gggaacattt cgactattgg ggtcaaggca ccctcgtaac ggtttcttct
360156360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 156caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcaactac 300tacctgttct ctacttcttt
cgactattgg ggtcaaggca ccctcgtaac ggtttcttct 360157360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 157caggtgcaat tggttcaatc tggtgctgag gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcaactac 300tacatcggta tcgttccgtt cgactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360158360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 158gaggtgcaat
tggttgaatc tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg
cttccggatt caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag
gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg
ctccagttaa aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc
agatgaactc tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat
ggtcttggta cgattattgg ggccagggca cgctggttac ggtgtcttcc
360159360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 159gaggtgcaat tggttgaatc
tggtggtggt ctggtaaaac cgggcggttc ccngcgtctg 60agctgcgcgg cttccggatt
caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag gcctcgagtg
ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg ctccagttaa
aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc agatgaactc
tctgaaaacc gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttactt
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360160360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 160caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcaactac 300tacgttggtg tttctccgtt cgactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360161360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 161caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgnttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcntac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcaacttc 300actgttctgc
gtgttccgtt cgactattgg ggtcaaggca ccctcgtaac ggtttcttct
360162360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 162gaggtgcaat tggttgaatc
tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg cttccggatt
caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag gcctcgagtg
ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg ctccagttaa
aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc agatgaactc
tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat gggcttggtt
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360163360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 163gaggtgcaat tggttgaatc tggtggtggt ctggtaaaac cgggcggttc
cctgcgtctg 60agctgcgcgg cttccggatt caccttctcc aacgcgtgga tgagctgggt
tcgccaggcc 120ccgggcaaag gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg
tggcaccacg 180gattacgcgg ctccagttaa aggtcgtttt accatttccc gcgacgatag
caaaaacact 240ctgtatctgc agatgaactc tctgaaaacc gaagacaccg cagtctacta
ctgtactacc 300ccttgggaat gggcttactt cgattattgg ggccagggca cgctggttac
ggtgtcttcc 360164360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 164caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcactggt 300tggtctcgtt
ggggttacat ggactattgg ggccaaggca ccctcgtaac ggtttcttct
360165360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 165caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcggtgaa 300tggatccgtt actaccattt
cgactattgg ggtcaaggca ccctcgtaac ggtttcttct 360166360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 166caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcgttggt 300tggtaccgtt ggggttacat ggactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360167363DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 167caggtgcaat
tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg
cctccggagg cacattcagc agctacgcta taagctgggt gcgacaggcc 120cctggacaag
ggctcgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180gcacagaagt
tccagggcag ggtaaccatt actgcagaca aatccacgag cacagcctac 240atggagctga
gcagcctgag atctgaggac accgccgtgt attactgtgc gagagctgtt 300ttctaccgtg
cttggtactc tttcgactac tggggccaag ggaccaccgt gaccgtctcc 360tca
363168321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 168gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gtgccagtca
gagtattagt agctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180cgtttcagcg gcagtggatc
cgggacagaa ttcactctca ccatcagcag cttgcagcct 240gatgattttg caacttatta
ctgccaacag tataccagcc caccaccaac gtttggccag 300ggcaccaaag tcgagatcaa g
321169357DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 169caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccatgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgctctttc 300ttcactggtt tccatctgga ctattggggt caaggcaccc tcgtaacggt
ttcttct 357170327DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 170gaaatcgtgt
taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcttgca
gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatacca acgaacatta ttatacgttc 300ggccagggga
ccaaagtgga aatcaaa
327171351DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 171caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggccc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcggtgac 300ttcgcttggc tggactattg
gggtcaaggc accctcgtaa cggtttcttc t 351172336DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 172gatattgtta tgactcaatc tccactgtct ctgccggtga ctccaggcga
accggcgagc 60atttcttgcc gttccagcca gtctctgctg cactccaacg gctacaacta
tctcgattgg 120tacctgcaaa aaccgggtca gagccctcag ctgctgatct acctgggctc
taaccgcgct 180tccggtgtac cggaccgttt cagcggctct ggatccggca ccgatttcac
gttgaaaatc 240agccgtgttg aagcagaaga cgtgggcgtt tattactgta tgcaggcaag
cattatgaac 300cggacttttg gtcaaggcac caaggtcgaa attaaa
336173336DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 173gatattgtta
tgactcaatc tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc
gttccagcca gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa
aaccgggtca gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac
cggaccgttt cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg
aagcagaaga cgtgggcgtt tattactgta tgcaggcaag cattatgagc 300cggacttttg
gtcaaggcac caaggtcgaa attaaa
336174336DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 174gatattgtta tgactcaatc
tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc gttccagcca
gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa aaccgggtca
gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac cggaccgttt
cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg aagcagaaga
cgtgggcgtt tattactgta tgcaggcaag cattatgcag 300cggacttttg gtcaaggcac
caaggtcgaa attaaa 336175336DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 175gatattgtta tgactcaatc tccactgtct ctgccggtga ctccaggcga
accggcgagc 60atttcttgcc gttccagcca gtctctgctg cactccaacg gctacaacta
tctcgattgg 120tacctgcaaa aaccgggtca gagccctcag ctgctgatct acctgggctc
taaccgcgct 180tccggtgtac cggaccgttt cagcggctct ggatccggca ccgatttcac
gttgaaaatc 240agccgtgttg aagcagaaga cgtgggcgtt tattactgta tgcaggcaag
cattatgaac 300cgggcttttg gtcaaggcac caaggtcgaa attaaa
336176336DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 176gatattgtta
tgactcaatc tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc
gttccagcca gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa
aaccgggtca gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac
cggaccgttt cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg
aagcagaaga cgtgggcgtt tattactgta tgcaggcaag cattatgaac 300cggaattttg
gtcaaggcac caaggtcgaa attaaa
336177348DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 177caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgctcttac 300atcgacatgg actattgggg
tcaaggcacc ctcgtaacgg tttcttct 348178321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 178gaaatcgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcttgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggagcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg atccgggaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgcagtgta ttactgtcag caggataact ggagcccaac
gttcggccag 300gggaccaaag tggaaatcaa a
321179348DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 179caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgctcttac 300gttgacatgg
actattgggg tcaaggcacc ctcgtaacgg tttcttct
348180321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 180gaaatcgtgt taacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcttgca gggccagtca
gagtgttagc agcagctacc tagcctggta ccagcagaaa 120cctggccagg ctcccaggct
cctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
atccgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag caggatattt ggagcccaac gttcggccag 300gggaccaaag tggaaatcaa a
321181363DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 181gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc
gaaagactct 300tcttacgttg aatggtacgc tttcgactac tggggccaag gaaccctggt
caccgtctcg 360agt
363182324DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 182gaaatcgtgt
taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcttgca
gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg atccgggaca gactccactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagccaacca gcagcccaat tacgttcggc 300caggggacca
aagtggaaat caaa
324183684DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 183gaggtgcagc tgctggaatc
tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60agctgtgccg ccagcggctt
caccttcagc acctacgcca tgaactgggt gcgccaggcc 120cctggcaaag gcctggaatg
ggtgtcccgg atcagaagca agtacaacaa ctacgccacc 180tactacgccg acagcgtgaa
gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc agatgaacag
cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact tcggcaacag
ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt caagcgctag
taccaagggc cccagcgtgt tccccctggc acccagcagc 420aagagcacat ctggcggaac
agccgctctg ggctgtctgg tgaaagacta cttccccgag 480cccgtgaccg tgtcttggaa
ctctggcgcc ctgaccagcg gcgtgcacac ctttccagcc 540gtgctgcaga gcagcggcct
gtactccctg tcctccgtgg tcaccgtgcc ctctagctcc 600ctgggaacac agacatatat
ctgtaatgtc aatcacaagc cttccaacac caaagtcgat 660aagaaagtcg agcccaagag
ctgc 684184696DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 184gaggtgcagc tgctggaatc tggcggcgga ctggtgcagc ctggcggatc
tctgagactg 60agctgtgccg ccagcggctt caccttcagc acctacgcca tgaactgggt
gcgccaggcc 120cctggcaaag gcctggaatg ggtgtcccgg atcagaagca agtacaacaa
ctacgccacc 180tactacgccg acagcgtgaa gggccggttc accatcagcc gggacgacag
caagaacacc 240ctgtacctgc agatgaacag cctgcgggcc gaggacaccg ccgtgtacta
ttgtgtgcgg 300cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca
gggcaccctc 360gtgaccgtgt caagcgctag tgtggccgct ccctccgtgt ttatctttcc
cccatccgat 420gaacagctga aaagcggcac cgcctccgtc gtgtgtctgc tgaacaattt
ttaccctagg 480gaagctaaag tgcagtggaa agtggataac gcactgcagt ccggcaactc
ccaggaatct 540gtgacagaac aggactccaa ggacagcacc tactccctgt cctccaccct
gacactgtct 600aaggctgatt atgagaaaca caaagtctac gcctgcgaag tcacccatca
gggcctgagc 660tcgcccgtca caaagagctt caacagggga gagtgt
69618536DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 185gcaggcaagc attatgcagc
ggacttttgg tcaagg 3618635DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 186caggcaagca ttatgagccg gacttttggt caagg
3518737DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 187cattatgaac cgggcttttg
gtcaaggcac caaggtc 3718837DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 188cattatgaac cggaattttg gtcaaggcac caaggtc
371892067DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 189gaggtgcaat
tggttgaatc tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg
cttccggatt caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag
gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg
ctccagttaa aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc
agatgaactc tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat
ggtcttggta cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacaa
agggccctag cgtgttccct ctggccccca gcagcaagag cacaagcggc 420ggaacagccg
ccctgggctg cctcgtgaag gactacttcc ccgagcccgt gacagtgtct 480tggaacagcg
gagccctgac aagcggcgtg cacactttcc ctgccgtgct gcagagcagc 540ggcctgtact
ccctgagcag cgtggtcacc gtgcctagca gcagcctggg cacccagacc 600tacatctgca
acgtgaacca caagcccagc aacaccaaag tggacaagaa ggtggagccc 660aagagctgtg
atggcggagg agggtccgga ggcggaggat ccgaggtgca gctgctggaa 720tctggcggcg
gactggtgca gcctggcgga tctctgagac tgagctgtgc cgccagcggc 780ttcaccttca
gcacctacgc catgaactgg gtgcgccagg cccctggcaa aggcctggaa 840tgggtgtccc
ggatcagaag caagtacaac aactacgcca cctactacgc cgacagcgtg 900aagggccggt
tcaccatcag ccgggacgac agcaagaaca ccctgtacct gcagatgaac 960agcctgcggg
ccgaggacac cgccgtgtac tattgtgtgc ggcacggcaa cttcggcaac 1020agctatgtgt
cttggtttgc ctactggggc cagggcaccc tcgtgaccgt gtcaagcgct 1080agtaccaagg
gccccagcgt gttccccctg gcacccagca gcaagagcac atctggcgga 1140acagccgctc
tgggctgtct ggtgaaagac tacttccccg agcccgtgac cgtgtcttgg 1200aactctggcg
ccctgaccag cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1260ctgtactccc
tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac acagacatat 1320atctgtaatg
tcaatcacaa gccttccaac accaaagtcg ataagaaagt cgagcccaag 1380agctgcgaca
aaactcacac atgcccaccg tgcccagcac ctgaagctgc agggggaccg 1440tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1500gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 1560gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1620acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1680tacaagtgca
aggtctccaa caaagccctc ggcgccccca tcgagaaaac catctccaaa 1740gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatgccg ggatgagctg 1800accaagaacc
aggtcagcct gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1860gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1920gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1980caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2040aagagcctct
ccctgtctcc gggtaaa
20671901350DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 190gaggtgcaat tggttgaatc
tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg cttccggatt
caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag gcctcgagtg
ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg ctccagttaa
aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc agatgaactc
tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttggta
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacca agggcccctc
cgtgttcccc ctggccccca gcagcaagag caccagcggc 420ggcacagccg ctctgggctg
cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480tggaacagcg gagccctgac
ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540ggcctgtata gcctgagcag
cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600tacatctgca acgtgaacca
caagcccagc aacaccaagg tggacaagaa ggtggagccc 660aagagctgcg acaaaactca
cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc
caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg
agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080ctgaccaaga accaggtcag
cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa
tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccgcttcacg 1320cagaagagcc tctccctgtc
tccgggtaaa 1350191645DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 191caggccgtcg tgacccagga acccagcctg acagtgtctc ctggcggcac
cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc accagcaact acgccaactg
ggtgcaggaa 120aagcccggcc aggccttcag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg ccctgacact
gtctggcgcc 240cagccagaag atgaggccga gtactactgc gccctgtggt acagcaacct
gtgggtgttc 300ggcggaggca ccaagctgac agtcctaggt caacccaagg ctgcccccag
cgtgaccctg 360ttccccccca gcagcgagga actgcaggcc aacaaggcca ccctggtctg
cctgatcagc 420gacttctacc caggcgccgt gaccgtggcc tggaaggccg acagcagccc
cgtgaaggcc 480ggcgtggaga ccaccacccc cagcaagcag agcaacaaca agtacgccgc
cagcagctac 540ctgagcctga cccccgagca gtggaagagc cacaggtcct acagctgcca
ggtgacccac 600gagggcagca ccgtggagaa aaccgtggcc cccaccgagt gcagc
6451922067DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 192gaggtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60agctgtgccg
ccagcggctt caccttcagc acctacgcca tgaactgggt gcgccaggcc 120cctggcaaag
gcctggaatg ggtgtcccgg atcagaagca agtacaacaa ctacgccacc 180tactacgccg
acagcgtgaa gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc
agatgaacag cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact
tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt
catctgctag cacaaagggc cctagcgtgt tccctctggc ccccagcagc 420aagagcacaa
gcggcggaac agccgccctg ggctgcctcg tgaaggacta cttccccgag 480cccgtgacag
tgtcttggaa cagcggagcc ctgacaagcg gcgtgcacac cttccctgcc 540gtgctgcaga
gcagcggcct gtactccctg agcagcgtgg tcaccgtgcc tagcagcagc 600ctgggcaccc
agacctacat ctgcaacgtg aaccacaagc ccagcaacac caaagtggac 660aagaaggtgg
agcccaagag ctgtgatggc ggaggagggt ccggaggcgg aggatccgag 720gtgcaattgg
ttgaatctgg tggtggtctg gtaaaaccgg gcggttccct gcgtctgagc 780tgcgcggctt
ccggattcac cttctccaac gcgtggatga gctgggttcg ccaggccccg 840ggcaaaggcc
tcgagtgggt tggtcgtatc aagtctaaaa ctgacggtgg caccacggat 900tacgcggctc
cagttaaagg tcgttttacc atttcccgcg acgatagcaa aaacactctg 960tatctgcaga
tgaactctct gaaaactgaa gacaccgcag tctactactg tactaccccg 1020tgggaatggt
cttggtacga ttattggggc cagggcacgc tggttacggt gtctagcgct 1080agtaccaagg
gccccagcgt gttccccctg gcacccagca gcaagagcac atctggcgga 1140acagccgctc
tgggctgtct ggtgaaagac tacttccccg agcccgtgac cgtgtcttgg 1200aactctggcg
ccctgaccag cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1260ctgtactccc
tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac acagacatat 1320atctgtaatg
tcaatcacaa gccttccaac accaaagtcg ataagaaagt cgagcccaag 1380agctgcgaca
aaactcacac atgcccaccg tgcccagcac ctgaagctgc agggggaccg 1440tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1500gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 1560gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1620acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1680tacaagtgca
aggtctccaa caaagccctc ggcgccccca tcgagaaaac catctccaaa 1740gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatgccg ggatgagctg 1800accaagaacc
aggtcagcct gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1860gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1920gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1980caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2040aagagcctct
ccctgtctcc gggtaaa
20671931365DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 193gaggtgcagc tgctggaatc
tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60agctgtgccg ccagcggctt
caccttcagc acctacgcca tgaactgggt gcgccaggcc 120cctggcaaag gcctggaatg
ggtgtcccgg atcagaagca agtacaacaa ctacgccacc 180tactacgccg acagcgtgaa
gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc agatgaacag
cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact tcggcaacag
ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt catctgctag
caccaagggc ccatcggtct tccccctggc accctcctcc 420aagagcacct ctgggggcac
agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480ccggtgacgg tgtcgtggaa
ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540gtcctacagt cctcaggact
ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600ttgggcaccc agacctacat
ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660aagaaagttg agcccaaatc
ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720gaagctgcag ggggaccgtc
agtcttcctc ttccccccaa aacccaagga caccctcatg 780atctcccgga cccctgaggt
cacatgcgtg gtggtggacg tgagccacga agaccctgag 840gtcaagttca actggtacgt
ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900gaggagcagt acaacagcac
gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960tggctgaatg gcaaggagta
caagtgcaag gtctccaaca aagccctcgg cgcccccatc 1020gagaaaacca tctccaaagc
caaagggcag ccccgagaac cacaggtgta caccctgccc 1080ccatgccggg atgagctgac
caagaaccag gtcagcctgt ggtgcctggt caaaggcttc 1140tatcccagcg acatcgccgt
ggagtgggag agcaatgggc agccggagaa caactacaag 1200accacgcctc ccgtgctgga
ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260gacaagagca ggtggcagca
ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320cacaaccact acacgcagaa
gagcctctcc ctgtctccgg gtaaa 1365194681DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 194gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 60ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc
tgaggtcaca 120tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 180ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
cagcacgtac 240cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 300tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 360gggcagcccc gagaaccaca ggtgtgcacc ctgcccccat cccgggatga
gctgaccaag 420aaccaggtca gcctctcgtg cgcagtcaaa ggcttctatc ccagcgacat
cgccgtggag 480tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt
gctggactcc 540gacggctcct tcttcctcgt gagcaagctc accgtggaca agagcaggtg
gcagcagggg 600aacgtcttct catgctccgt gatgcatgag gctctgcaca accgcttcac
gcagaagagc 660ctctccctgt ctccgggtaa a
6811952070DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 195caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggccc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtgac 300ttcgcttggc
tggactattg gggtcaaggc accctcgtaa cggtttcttc tgctagcaca 360aagggcccca
gcgtgttccc tctggcccct agcagcaaga gcacatctgg cggaacagcc 420gccctgggct
gcctcgtgaa ggactacttt cccgagcctg tgaccgtgtc ctggaactct 480ggcgccctga
caagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 540tctctgagca
gcgtggtcac cgtgcctagc agcagcctgg gcacccagac ctacatctgc 600aacgtgaacc
acaagcccag caacaccaaa gtggacaaga aggtggagcc caagagctgt 660gatggcggag
gagggtccgg aggcggagga tccgaggtgc agctgctgga atctggcggc 720ggactggtgc
agcctggcgg atctctgaga ctgagctgtg ccgccagcgg cttcaccttc 780agcacctacg
ccatgaactg ggtgcgccag gcccctggca aaggcctgga atgggtgtcc 840cggatcagaa
gcaagtacaa caactacgcc acctactacg ccgacagcgt gaagggccgg 900ttcaccatca
gccgggacga cagcaagaac accctgtacc tgcagatgaa cagcctgcgg 960gccgaggaca
ccgccgtgta ctattgtgtg cggcacggca acttcggcaa cagctatgtg 1020tcttggtttg
cctactgggg ccagggcacc ctcgtgaccg tgtcaagcgc tagtgtggcc 1080gctccctccg
tgtttatctt tcccccatcc gatgaacagc tgaaaagcgg caccgcctcc 1140gtcgtgtgtc
tgctgaacaa tttttaccct agggaagcta aagtgcagtg gaaagtggat 1200aacgcactgc
agtccggcaa ctcccaggaa tctgtgacag aacaggactc caaggacagc 1260acctactccc
tgtcctccac cctgacactg tctaaggctg attatgagaa acacaaagtc 1320tacgcctgcg
aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 1380ggagagtgtg
acaagaccca cacctgtccc ccttgtcctg cccctgaagc tgctggcggc 1440ccttctgtgt
tcctgttccc cccaaagccc aaggacaccc tgatgatcag ccggaccccc 1500gaagtgacct
gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 1560tacgtggacg
gcgtggaagt gcacaacgcc aagacaaagc cgcgggagga gcagtacaac 1620agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1680gagtacaagt
gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1740aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1800ctgaccaaga
accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1860gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1920ctggactccg
acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1980cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 2040cagaagagcc
tctccctgtc tccgggtaaa
20701961341DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 196caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggccc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcggtgac 300ttcgcttggc tggactattg
gggtcaaggc accctcgtaa cggtttcttc tgctagcacc 360aagggcccct ccgtgttccc
cctggccccc agcagcaaga gcaccagcgg cggcacagcc 420gctctgggct gcctggtcaa
ggactacttc cccgagcccg tgaccgtgtc ctggaacagc 480ggagccctga cctccggcgt
gcacaccttc cccgccgtgc tgcagagttc tggcctgtat 540agcctgagca gcgtggtcac
cgtgccttct agcagcctgg gcacccagac ctacatctgc 600aacgtgaacc acaagcccag
caacaccaag gtggacaaga aggtggagcc caagagctgc 660gacaaaactc acacatgccc
accgtgccca gcacctgaag ctgcaggggg accgtcagtc 720ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960tgcaaggtct ccaacaaagc
cctcggcgcc cccatcgaga aaaccatctc caaagccaaa 1020gggcagcccc gagaaccaca
ggtgtgcacc ctgcccccat cccgggatga gctgaccaag 1080aaccaggtca gcctctcgtg
cgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct tcttcctcgt
gagcaagctc accgtggaca agagcaggtg gcagcagggg 1260aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1320ctctccctgt ctccgggtaa a
1341197657DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 197gatattgtta tgactcaatc tccactgtct ctgccggtga ctccaggcga
accggcgagc 60atttcttgcc gttccagcca gtctctgctg cactccaacg gctacaacta
tctcgattgg 120tacctgcaaa aaccgggtca gagccctcag ctgctgatct acctgggctc
taaccgcgct 180tccggtgtac cggaccgttt cagcggctct ggatccggca ccgatttcac
gttgaaaatc 240agccgtgttg aagcagaaga cgtgggcgtt tattactgta tgcaggcaag
cattatgaac 300cggacttttg gtcaaggcac caaggtcgaa attaaacgta cggtggctgc
accatctgtc 360ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt
tgtgtgcctg 420ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa
cgccctccaa 480tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac
ctacagcctc 540agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta
cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg
agagtgt 657198642DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 198caggccgtcg
tgacccagga acccagcctg acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca
gttctacagg cgccgtgacc accagcaact acgccaactg ggtgcaggaa 120aagcccggcc
aggccttcag aggactgatc ggcggcacca acaagagagc ccctggcacc 180cctgccagat
tcagcggatc tctgctggga ggaaaggccg ccctgacact gtctggcgcc 240cagccagaag
atgaggccga gtactactgc gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca
ccaagctgac agtgctgagc agcgcttcca ccaaaggccc ttccgtgttt 360cctctggctc
ctagctccaa gtccacctct ggaggcaccg ctgctctcgg atgcctcgtg 420aaggattatt
ttcctgagcc tgtgacagtg tcctggaata gcggagcact gacctctgga 480gtgcatactt
tccccgctgt gctgcagtcc tctggactgt acagcctgag cagcgtggtg 540acagtgccca
gcagcagcct gggcacccag acctacatct gcaacgtgaa ccacaagccc 600agcaacacca
aggtggacaa gaaggtggaa cccaagtctt gt
6421991377DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 199gaggtgcagc tgctggaatc
tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60agctgtgccg ccagcggctt
caccttcagc acctacgcca tgaactgggt gcgccaggcc 120cctggcaaag gcctggaatg
ggtgtcccgg atcagaagca agtacaacaa ctacgccacc 180tactacgccg acagcgtgaa
gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc agatgaacag
cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact tcggcaacag
ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt catctgctag
cgtggccgct ccctccgtgt ttatctttcc cccatccgat 420gaacagctga aaagcggcac
cgcctccgtc gtgtgtctgc tgaacaattt ttaccctagg 480gaagctaaag tgcagtggaa
agtggataac gcactgcagt ccggcaactc ccaggaatct 540gtgacagaac aggactccaa
ggacagcacc tactccctgt cctccaccct gacactgtct 600aaggctgatt atgagaaaca
caaagtctac gcctgcgaag tcacccatca gggcctgagc 660tcgcccgtca caaagagctt
caacagggga gagtgtgaca agacccacac ctgtccccct 720tgtcctgccc ctgaagctgc
tggcggccct tctgtgttcc tgttcccccc aaagcccaag 780gacaccctga tgatcagccg
gacccccgaa gtgacctgcg tggtggtgga tgtgtcccac 840gaggaccctg aagtgaagtt
caattggtac gtggacggcg tggaagtgca caacgccaag 900acaaagccgc gggaggagca
gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 960ctgcaccagg actggctgaa
tggcaaggag tacaagtgca aggtctccaa caaagccctc 1020ggcgccccca tcgagaaaac
catctccaaa gccaaagggc agccccgaga accacaggtg 1080tacaccctgc ccccatgccg
ggatgagctg accaagaacc aggtcagcct gtggtgcctg 1140gtcaaaggct tctatcccag
cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1200aacaactaca agaccacgcc
tcccgtgctg gactccgacg gctccttctt cctctacagc 1260aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 1320catgaggctc tgcacaacca
ctacacgcag aagagcctct ccctgtctcc gggtaaa 13772002067DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 200gaggtgcaat tggttgaatc tggtggtggt ctggtaaaac cgggcggttc
cctgcgtctg 60agctgcgcgg cttccggatt caccttctcc aacgcgtgga tgagctgggt
tcgccaggcc 120ccgggcaaag gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg
tggcaccacg 180gattacgcgg ctccagttaa aggtcgtttt accatttccc gcgacgatag
caaaaacact 240ctgtatctgc agatgaactc tctgaaaact gaagacaccg cagtctacta
ctgtactacc 300ccgtgggaat ggtcttggta cgattattgg ggccagggca cgctggttac
ggtgtcttcc 360gctagcacaa agggccctag cgtgttccct ctggccccca gcagcaagag
cacaagcggc 420ggaacagccg ccctgggctg cctcgtgaag gactacttcc ccgagcccgt
gacagtgtct 480tggaacagcg gagccctgac aagcggcgtg cacactttcc ctgccgtgct
gcagagcagc 540ggcctgtact ccctgagcag cgtggtcacc gtgcctagca gcagcctggg
cacccagacc 600tacatctgca acgtgaacca caagcccagc aacaccaaag tggacaagaa
ggtggagccc 660aagagctgtg atggcggagg agggtccgga ggcggaggat ccgaggtgca
gctgctggaa 720tctggcggcg gactggtgca gcctggcgga tctctgagac tgagctgtgc
cgccagcggc 780ttcaccttca gcacctacgc catgaactgg gtgcgccagg cccctggcaa
aggcctggaa 840tgggtgtccc ggatcagaag caagtacaac aactacgcca cctactacgc
cgacagcgtg 900aagggccggt tcaccatcag ccgggacgac agcaagaaca ccctgtacct
gcagatgaac 960agcctgcggg ccgaggacac cgccgtgtac tattgtgtgc ggcacggcaa
cttcggcgcc 1020agctatgtgt cttggtttgc ctactggggc cagggcaccc tcgtgaccgt
gtcaagcgct 1080agtaccaagg gccccagcgt gttccccctg gcacccagca gcaagagcac
atctggcgga 1140acagccgctc tgggctgtct ggtgaaagac tacttccccg agcccgtgac
cgtgtcttgg 1200aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctgca
gagcagcggc 1260ctgtactccc tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac
acagacatat 1320atctgtaatg tcaatcacaa gccttccaac accaaagtcg ataagaaagt
cgagcccaag 1380agctgcgaca aaactcacac atgcccaccg tgcccagcac ctgaagctgc
agggggaccg 1440tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 1500gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 1560gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 1620acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 1680tacaagtgca aggtctccaa caaagccctc ggcgccccca tcgagaaaac
catctccaaa 1740gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatgccg
ggatgagctg 1800accaagaacc aggtcagcct gtggtgcctg gtcaaaggct tctatcccag
cgacatcgcc 1860gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc
tcccgtgctg 1920gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1980caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 2040aagagcctct ccctgtctcc gggtaaa
20672012067DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 201gaggtgcaat
tggttgaatc tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg
cttccggatt caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag
gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg
ctccagttaa aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc
agatgaactc tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat
ggtcttggta cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacaa
agggccctag cgtgttccct ctggccccca gcagcaagag cacaagcggc 420ggaacagccg
ccctgggctg cctcgtgaag gactacttcc ccgagcccgt gacagtgtct 480tggaacagcg
gagccctgac aagcggcgtg cacactttcc ctgccgtgct gcagagcagc 540ggcctgtact
ccctgagcag cgtggtcacc gtgcctagca gcagcctggg cacccagacc 600tacatctgca
acgtgaacca caagcccagc aacaccaaag tggacaagaa ggtggagccc 660aagagctgtg
atggcggagg agggtccgga ggcggaggat ccgaggtgca gctgctggaa 720tctggcggcg
gactggtgca gcctggcgga tctctgagac tgagctgtgc cgccagcggc 780ttcaccttca
gcacctacgc catgaactgg gtgcgccagg cccctggcaa aggcctggaa 840tgggtgtccc
ggatcagaag caagtacaac aactacgcca cctactacgc cgacagcgtg 900aagggccggt
tcaccatcag ccgggacgac agcaagaaca ccctgtacct gcagatgaac 960agcctgcggg
ccgaggacac cgccgtgtac tattgtgtgc ggcacggcaa cttcggcaac 1020gcctatgtgt
cttggtttgc ctactggggc cagggcaccc tcgtgaccgt gtcaagcgct 1080agtaccaagg
gccccagcgt gttccccctg gcacccagca gcaagagcac atctggcgga 1140acagccgctc
tgggctgtct ggtgaaagac tacttccccg agcccgtgac cgtgtcttgg 1200aactctggcg
ccctgaccag cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1260ctgtactccc
tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac acagacatat 1320atctgtaatg
tcaatcacaa gccttccaac accaaagtcg ataagaaagt cgagcccaag 1380agctgcgaca
aaactcacac atgcccaccg tgcccagcac ctgaagctgc agggggaccg 1440tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1500gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 1560gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1620acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1680tacaagtgca
aggtctccaa caaagccctc ggcgccccca tcgagaaaac catctccaaa 1740gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatgccg ggatgagctg 1800accaagaacc
aggtcagcct gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1860gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1920gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1980caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2040aagagcctct
ccctgtctcc gggtaaa
20672021323DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 202caggccgtcg tgacccagga
acccagcctg acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca gttctacagg
cgccgtgacc accagcaact acgccaactg ggtgcaggaa 120aagcccggcc aggccttcag
aggactgatc ggcggcacca acaagagagc ccctggcacc 180cctgccagat tcagcggatc
tctgctggga ggaaaggccg ccctgacact gtctggcgcc 240cagccagaag atgaggccga
gtactactgc gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac
agtgctgagc agcgctagca ccaagggccc atcggtcttc 360cccctggcac cctcctccaa
gagcacctct gggggcacag cggccctggg ctgcctggtc 420aaggactact tccccgaacc
ggtgacggtg tcgtggaact caggcgccct gaccagcggc 480gtgcacacct tcccggctgt
cctacagtcc tcaggactct actccctcag cagcgtggtg 540accgtgccct ccagcagctt
gggcacccag acctacatct gcaacgtgaa tcacaagccc 600agcaacacca aggtggacaa
gaaagttgag cccaaatctt gtgacaaaac tcacacatgc 660ccaccgtgcc cagcacctga
agctgcaggg ggaccgtcag tcttcctctt ccccccaaaa 720cccaaggaca ccctcatgat
ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 780agccacgaag accctgaggt
caagttcaac tggtacgtgg acggcgtgga ggtgcataat 840gccaagacaa agccgcggga
ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 900accgtcctgc accaggactg
gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 960gccctcggcg cccccatcga
gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1020caggtgtaca ccctgccccc
atcccgggat gagctgacca agaaccaggt cagcctgacc 1080tgcctggtca aaggcttcta
tcccagcgac atcgccgtgg agtgggagag caatgggcag 1140ccggagaaca actacaagac
cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200tacagcaagc tcaccgtgga
caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1260gtgatgcatg aggctctgca
caaccactac acgcagaaga gcctctccct gtctccgggt 1320aaa
1323203681DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 203gaggtgcaat tggttgaatc tggtggtggt ctggtaaaac cgggcggttc
cctgcgtctg 60agctgcgcgg cttccggatt caccttctcc aacgcgtgga tgagctgggt
tcgccaggcc 120ccgggcaaag gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg
tggcaccacg 180gattacgcgg ctccagttaa aggtcgtttt accatttccc gcgacgatag
caaaaacact 240ctgtatctgc agatgaactc tctgaaaact gaagacaccg cagtctacta
ctgtactacc 300ccgtgggaat ggtcttggta cgattattgg ggccagggca cgctggttac
ggtgtcttcc 360gctagcgtgg ccgctccctc cgtgttcatc ttcccacctt ccgacgagca
gctgaagtcc 420ggcaccgctt ctgtcgtgtg cctgctgaac aacttctacc cccgcgaggc
caaggtgcag 480tggaaggtgg acaacgccct gcagtccggc aacagccagg aatccgtgac
cgagcaggac 540tccaaggaca gcacctactc cctgtcctcc accctgaccc tgtccaaggc
cgactacgag 600aagcacaagg tgtacgcctg cgaagtgacc caccagggcc tgtctagccc
cgtgaccaag 660tctttcaacc ggggcgagtg c
681204693DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 204gaagtgcagc
tgctggaatc cggcggagga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt caccttctcc acctacgcca tgaactgggt gcgacaggct 120cctggcaagg
gcctggaatg ggtgtcccgg atcagatcca agtacaacaa ctacgccacc 180tactacgccg
actccgtgaa gggccggttc accatctctc gggacgactc caagaacacc 240ctgtacctgc
agatgaactc cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact
tcggcaactc ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt
catctgctag ccccaaggct gcccccagcg tgaccctgtt tccccccagc 420agcgaggaac
tgcaggccaa caaggccacc ctggtctgcc tgatcagcga cttctaccca 480ggcgccgtga
ccgtggcctg gaaggccgac agcagccccg tgaaggccgg cgtggagacc 540accaccccca
gcaagcagag caacaacaag tacgccgcca gcagctacct gagcctgacc 600cccgagcagt
ggaagagcca caggtcctac agctgccagg tgacccacga gggcagcacc 660gtggagaaaa
ccgtggcccc caccgagtgc agc
693205327DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 205cagaccgtcg tgacccagga
acccagcctg acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca gttctacagg
cgccgtgacc accagcaact acgccaactg ggtgcagcag 120aagccaggcc aggctcccag
aggactgatc ggcggcacca acgccagagc ccctggcacc 180cctgccagat tcagcggatc
tctgctggga ggaaaggccg ccctgacact gtctggcgtg 240cagcctgaag atgaggccga
gtactactgc gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac
agtccta 327206327DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 206cagaccgtcg tgacccagga acccagcctg acagtgtctc ctggcggcac
cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc accagcaact acgccaactg
ggtgcagcag 120aagccaggcc aggctcccag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg ccctgacact
gtctggcgtg 240cagcctgaag atgaggccga gtactactgc gccctgtggt acgccaacct
gtgggtgttc 300ggcggaggca ccaagctgac agtccta
327207366DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 207gaggtgcaat
tggtggaaag cggaggcggc ctcgtgaagc ctggcggatc tctgagactg 60agctgtgccg
ccagcggctt caccttcagc aacgcctgga tgcactgggt gcgccaggcc 120cctggaaaag
gactcgagtg ggtgggacgg atcaagagca agaccgatgg cggcaccacc 180gactatgccg
cccctgtgaa gggccggttc accatcagca gggacgacag caagaacacc 240ctgtacctgc
agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgcaccacc 300ccctgggagt
ggtcttggta cgactattgg ggccagggca ccctcgtgac cgtgtcctct 360gctagc
366208366DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 208gaggtgcaat tggtggaaag
cggaggcggc ctcgtgaagc ctggcggatc tctgagactg 60agctgtgccg ccagcggctt
caccttcagc aacgcctgga tgagctgggt gcgccaggcc 120cctggaaaag gactcgagtg
ggtgtcccgg atcaagagca agaccgatgg cggcaccacc 180gactatgccg cccctgtgaa
gggccggttc accatcagca gggacgacag caagaacacc 240ctgtacctgc agatgaacag
cctgaaaacc gaggacaccg ccgtgtacta ctgcaccacc 300ccctgggagt ggtcttggta
cgactattgg ggccagggca ccctcgtgac cgtgtcctct 360gctagc
366209366DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 209gaggtgcaat tggtggaaag cggaggcggc ctcgtgaagc ctggcggatc
tctgagactg 60agctgtgccg ccagcggctt caccttcagc aacgcctgga tgagctgggt
gcgccaggcc 120cctggaaaag gactcgagtg ggtgggatct atcaagagca agaccgacgg
cggcaccacc 180gactatgccg cccctgtgaa gggccggttc accatcagca gggacgacag
caagaacacc 240ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta
ctgcaccacc 300ccctgggagt ggtcttggta cgactattgg ggccagggca ccctcgtgac
cgtgtcctct 360gctagc
366210366DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 210gaggtgcaat
tggtggaaag cggaggcggc ctcgtgaagc ctggcggatc tctgagactg 60agctgtgccg
ccagcggctt caccttcagc aacgcctgga tgagctgggt gcgccaggcc 120cctggaaaag
gactcgagtg ggtgggacgg atcaagagca agaccgatgg cggcaccacc 180gactatgccg
cccctgtgaa gggccggttc accatcagca gggacgacag caagaacacc 240ctgtacctgc
agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgcaccacc 300ccctacgagt
ggtcttggta cgactactgg ggccagggca ccctcgtgac cgtgtcatct 360gctagc
366211366DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 211gaggtgcaat tggtggaaag
cggaggcggc ctcgtgaagc ctggcggatc tctgagactg 60agctgtgccg ccagcggctt
caccttcagc aacgcctgga tgagctgggt gcgccaggcc 120cctggaaaag gactcgagtg
ggtgggacgg atcaagagca agaccgatgg cggcaccacc 180gactatgccg cccctgtgaa
gggccggttc accatcagca gggacgacag caagaacacc 240ctgtacctgc agatgaacag
cctgaaaacc gaggacaccg ccgtgtacta ctgcaccacc 300ccctgggagt actcttggta
cgactactgg ggccagggca ccctcgtgac cgtgtcatct 360gctagc
366212366DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 212caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcaactac 300actatcgttg tttctccgtt cgactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360gctagc
366213366DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 213caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcaactac 300ttcatcggtt
ctgttgctat ggactattgg ggtcaaggca ccctcgtaac ggtttcttct 360gctagc
366214357DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 214caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcggtctg 300acttactcta tggactattg
gggtcaaggc accctcgtaa cggtttcttc tgctagc 357215342DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 215gatattgtta tgactcaatc tccactgtct ctgccggtga ctccaggcga
accggcgagc 60atttcttgcc gttccagcca gtctctgctg cactccaacg gctacaacta
tctcgattgg 120tacctgcaaa aaccgggtca gagccctcag ctgctgatct acctgggctc
taaccgcgct 180tccggtgtac cggaccgttt cagcggctct ggatccggca ccgatttcac
gttgaaaatc 240agccgtgttg aagcagaaga cgtgggcgtt tattactgta tgcaggcact
gcagattcca 300aacacttttg gtcaaggcac caaggtcgaa attaaacgta cg
342216354DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 216gaggtgcaat
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg
tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcagatga
acagcctgag agccgaggac acggccgtat attactgtgc gaaatacgct 300tacgctctgg
actactgggg ccaaggaacc ctggtcaccg tctcgagtgc tagc
354217327DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 217gaaatcgtgt taacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcttgca gggccagtca
gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct
cctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
atccgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagcatggca gcagcagcac gttcggccag 300gggaccaaag tggaaatcaa
acgtacg 327218366DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 218caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcggtgac 300ttctctgctg gtcgtctgat ggactattgg ggtcaaggca ccctcgtaac
ggtttcttct 360gctagc
366219345DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 219gatattgtta
tgactcaatc tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc
gttccagcca gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa
aaccgggtca gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac
cggaccgttt cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg
aagcagaaga cgtgggcgtt tattactgta tgcaggcact gcagacccca 300ccaattacct
ttggtcaagg caccaaggtc gaaattaaac gtacg
345220357DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 220caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgcggtgac 300tacaacgctt tcgactattg
gggtcacggc accctcgtaa cggtttcttc tgctagc 357221339DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 221gatattgtta tgactcaatc tccactgtct ctgccggtga ctccaggcga
accggcgagc 60atttcttgcc gttccagcca gtctctgctg cactccaacg gctacaacta
tctcgattgg 120tacctgcaaa aaccgggtca gagccctcag ctgctgatct acctgggctc
taaccgcgct 180tccggtgtac cggaccgttt cagcggctct ggatccggca ccgatttcac
gttgaaaatc 240agccgtgttg aagcagaaga cgtgggcgtt tattactgta tgcaggcatg
gcatagccca 300acttttggtc aaggcaccaa ggtcgaaatt aaacgtacg
339222357DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 222caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtgct 300acttacacta
tggactattg gggtcaaggc accctcgtaa cggtttcttc tgctagc
357223342DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 223gatattgtta tgactcaatc
tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc gttccagcca
gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa aaccgggtca
gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac cggaccgttt
cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg aagcagaaga
cgtgggcgtt tattactgta tgcaggcact gcagacccca 300attacttttg gtcaaggcac
caaggtcgaa attaaacgta cg 3422241344DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 224caggtgcagc tgcagcagtc tggcgccgag ctcgtgaaac ctggcgcctc
cgtgaagatc 60agctgcaagg ccagcggcta cagcttcacc ggctacttca tgaactgggt
caagcagagc 120cacggcaaga gcctggaatg gatcggcaga atccacccct acgacggcga
caccttctac 180aaccagaact tcaaggacaa ggccaccctg accgtggaca agagcagcaa
caccgcccac 240atggaactgc tgagcctgac cagcgaggac ttcgccgtgt actactgcac
cagatacgac 300ggcagccggg ccatggatta ttggggccag ggcaccaccg tgacagtgtc
cagcgctagc 360accaagggcc cctccgtgtt ccccctggcc cccagcagca agagcaccag
cggcggcaca 420gccgctctgg gctgcctggt caaggactac ttccccgagc ccgtgaccgt
gtcctggaac 480agcggagccc tgacctccgg cgtgcacacc ttccccgccg tgctgcagag
ttctggcctg 540tatagcctga gcagcgtggt caccgtgcct tctagcagcc tgggcaccca
gacctacatc 600tgcaacgtga accacaagcc cagcaacacc aaggtggaca agaaggtgga
gcccaagagc 660tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagg
gggaccgtca 720gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa
ctggtacgtg 840gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta
caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 960aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccat
ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtgc accctgcccc catcccggga
tgagctgacc 1080aagaaccagg tcagcctctc gtgcgcagtc aaaggcttct atcccagcga
catcgccgtg 1140gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 1200tccgacggct ccttcttcct cgtgagcaag ctcaccgtgg acaagagcag
gtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 1320agcctctccc tgtctccggg taaa
13442252073DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 225caggtgcagc
tgcagcagtc tggcgccgag ctcgtgaaac ctggcgcctc cgtgaagatc 60agctgcaagg
ccagcggcta cagcttcacc ggctacttca tgaactgggt caagcagagc 120cacggcaaga
gcctggaatg gatcggcaga atccacccct acgacggcga caccttctac 180aaccagaact
tcaaggacaa ggccaccctg accgtggaca agagcagcaa caccgcccac 240atggaactgc
tgagcctgac cagcgaggac ttcgccgtgt actactgcac cagatacgac 300ggcagccggg
ccatggatta ttggggccag ggcaccaccg tgacagtgtc cagcgctagc 360acaaagggcc
ccagcgtgtt ccctctggcc cctagcagca agagcacatc tggcggaaca 420gccgccctgg
gctgcctcgt gaaggactac tttcccgagc ctgtgaccgt gtcctggaac 480tctggcgccc
tgacaagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540tactctctga
gcagcgtggt caccgtgcct agcagcagcc tgggcaccca gacctacatc 600tgcaacgtga
accacaagcc cagcaacacc aaagtggaca agaaggtgga gcccaagagc 660tgtgatggcg
gaggagggtc cggaggcgga ggatccgaag tgcagctggt ggaaagcggc 720ggaggcctgg
tgcagcctaa gggctctctg aagctgagct gtgccgccag cggcttcacc 780ttcaacacct
acgccatgaa ctgggtgcgc caggcccctg gcaaaggcct ggaatgggtg 840gcccggatca
gaagcaagta caacaattac gccacctact acgccgacag cgtgaaggac 900cggttcacca
tcagccggga cgacagccag agcatcctgt acctgcagat gaacaacctg 960aaaaccgagg
acaccgccat gtactactgc gtgcggcacg gcaacttcgg caacagctat 1020gtgtcttggt
ttgcctactg gggccagggc accctcgtga cagtgtctgc tgctagcgtg 1080gccgctccct
ccgtgtttat ctttccccca tccgatgaac agctgaaaag cggcaccgcc 1140tccgtcgtgt
gtctgctgaa caatttttac cctagggaag ctaaagtgca gtggaaagtg 1200gataacgcac
tgcagtccgg caactcccag gaatctgtga cagaacagga ctccaaggac 1260agcacctact
ccctgtcctc caccctgaca ctgtctaagg ctgattatga gaaacacaaa 1320gtctacgcct
gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac 1380aggggagagt
gtgacaagac ccacacctgt cccccttgtc ctgcccctga agctgctggc 1440ggcccttctg
tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 1500cccgaagtga
cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 1560tggtacgtgg
acggcgtgga agtgcacaac gccaagacaa agccgcggga ggagcagtac 1620aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1680aaggagtaca
agtgcaaggt ctccaacaaa gccctcggcg cccccatcga gaaaaccatc 1740tccaaagcca
aagggcagcc ccgagaacca caggtgtaca ccctgccccc atgccgggat 1800gagctgacca
agaaccaggt cagcctgtgg tgcctggtca aaggcttcta tcccagcgac 1860atcgccgtgg
agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1920gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1980tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 2040acgcagaaga
gcctctccct gtctccgggt aaa
2073226654DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 226gacatcgagc tgacccagag
ccctgcctct ctggccgtgt ctctgggaca gagagccatc 60atcagctgca aggccagcca
gagcgtgtcc tttgccggca cctctctgat gcactggtat 120caccagaagc ccggccagca
gcccaagctg ctgatctaca gagccagcaa cctggaagcc 180ggcgtgccca caagattttc
cggcagcggc agcaagaccg acttcaccct gaacatccac 240cccgtggaag aagaggacgc
cgccacctac tactgccagc agagcagaga gtacccctac 300accttcggcg gaggcaccaa
gctggaaatc aagcgtacgg tggctgcacc atctgtcttc 360atcttcccgc catctgatga
gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 480ggtaactccc aggagagtgt
cacagagcag gacagcaagg acagcaccta cagcctcagc 540agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga gtgt 654227209PRTHomo sapiens
227Arg Ile Ala Trp Ala Arg Thr Glu Leu Leu Asn Val Cys Met Asn Ala 1
5 10 15 Lys His His Lys
Glu Lys Pro Gly Pro Glu Asp Lys Leu His Glu Gln 20
25 30 Cys Arg Pro Trp Arg Lys Asn Ala Cys
Cys Ser Thr Asn Thr Ser Gln 35 40
45 Glu Ala His Lys Asp Val Ser Tyr Leu Tyr Arg Phe Asn Trp
Asn His 50 55 60
Cys Gly Glu Met Ala Pro Ala Cys Lys Arg His Phe Ile Gln Asp Thr 65
70 75 80 Cys Leu Tyr Glu Cys
Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val 85
90 95 Asp Gln Ser Trp Arg Lys Glu Arg Val Leu
Asn Val Pro Leu Cys Lys 100 105
110 Glu Asp Cys Glu Gln Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr
Cys 115 120 125 Lys
Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser Gly Phe Asn Lys 130
135 140 Cys Ala Val Gly Ala Ala
Cys Gln Pro Phe His Phe Tyr Phe Pro Thr 145 150
155 160 Pro Thr Val Leu Cys Asn Glu Ile Trp Thr His
Ser Tyr Lys Val Ser 165 170
175 Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Pro
180 185 190 Ala Gln
Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala Ala Ala 195
200 205 Met 228214PRTHomo sapiens
228Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys Met 1
5 10 15 Asp Ala Lys His
His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His 20
25 30 Asp Gln Cys Ser Pro Trp Lys Lys Asn
Ala Cys Cys Thr Ala Ser Thr 35 40
45 Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe
Asn Trp 50 55 60
Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 65
70 75 80 Asp Thr Cys Leu Tyr
Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln 85
90 95 Gln Val Asn Gln Ser Trp Arg Lys Glu Arg
Phe Leu Asp Val Pro Leu 100 105
110 Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser
His 115 120 125 Thr
Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val 130
135 140 Asn Lys Cys Pro Ala Gly
Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 145 150
155 160 Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp
Ser His Ser Tyr Lys 165 170
175 Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe
180 185 190 Asp Ser
Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 195
200 205 Ala Ala Met His Val Asn
210 229220PRTHomo sapiens 229Ser Ala Arg Ala Arg Thr Asp
Leu Leu Asn Val Cys Met Asn Ala Lys 1 5
10 15 His His Lys Thr Gln Pro Ser Pro Glu Asp Glu
Leu Tyr Gly Gln Cys 20 25
30 Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr Ser Gln
Glu 35 40 45 Leu
His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp Asp His Cys 50
55 60 Gly Lys Met Glu Pro Thr
Cys Lys Arg His Phe Ile Gln Asp Ser Cys 65 70
75 80 Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp
Ile Arg Gln Val Asn 85 90
95 Gln Ser Trp Arg Lys Glu Arg Ile Leu Asn Val Pro Leu Cys Lys Glu
100 105 110 Asp Cys
Glu Arg Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr Cys Lys 115
120 125 Ser Asn Trp His Lys Gly Trp
Asn Trp Thr Ser Gly Ile Asn Glu Cys 130 135
140 Pro Ala Gly Ala Leu Cys Ser Thr Phe Glu Ser Tyr
Phe Pro Thr Pro 145 150 155
160 Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Phe Lys Val Ser Asn
165 170 175 Tyr Ser Arg
Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser Ala 180
185 190 Gln Gly Asn Pro Asn Glu Glu Val
Ala Lys Phe Tyr Ala Ala Ala Met 195 200
205 Asn Ala Gly Ala Pro Ser Arg Gly Ile Ile Asp Ser
210 215 220 230208PRTMus musculus 230Thr
Arg Ala Arg Thr Glu Leu Leu Asn Val Cys Met Asp Ala Lys His 1
5 10 15 His Lys Glu Lys Pro Gly
Pro Glu Asp Asn Leu His Asp Gln Cys Ser 20
25 30 Pro Trp Lys Thr Asn Ser Cys Cys Ser Thr
Asn Thr Ser Gln Glu Ala 35 40
45 His Lys Asp Ile Ser Tyr Leu Tyr Arg Phe Asn Trp Asn His
Cys Gly 50 55 60
Thr Met Thr Ser Glu Cys Lys Arg His Phe Ile Gln Asp Thr Cys Leu 65
70 75 80 Tyr Glu Cys Ser Pro
Asn Leu Gly Pro Trp Ile Gln Gln Val Asp Gln 85
90 95 Ser Trp Arg Lys Glu Arg Ile Leu Asp Val
Pro Leu Cys Lys Glu Asp 100 105
110 Cys Gln Gln Trp Trp Glu Asp Cys Gln Ser Ser Phe Thr Cys Lys
Ser 115 120 125 Asn
Trp His Lys Gly Trp Asn Trp Ser Ser Gly His Asn Glu Cys Pro 130
135 140 Val Gly Ala Ser Cys His
Pro Phe Thr Phe Tyr Phe Pro Thr Ser Ala 145 150
155 160 Ala Leu Cys Glu Glu Ile Trp Ser His Ser Tyr
Lys Leu Ser Asn Tyr 165 170
175 Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Pro Ala Gln
180 185 190 Gly Asn
Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala Glu Ala Met Ser 195
200 205 231213PRTMacaca
fascicularis 231Glu Ala Gln Thr Arg Thr Ala Arg Ala Arg Thr Glu Leu Leu
Asn Val 1 5 10 15
Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly Pro Glu Asp Lys
20 25 30 Leu His Glu Gln Cys
Arg Pro Trp Lys Lys Asn Ala Cys Cys Ser Thr 35
40 45 Asn Thr Ser Gln Glu Ala His Lys Asp
Val Ser Tyr Leu Tyr Arg Phe 50 55
60 Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys Lys
Arg His Phe 65 70 75
80 Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp
85 90 95 Ile Gln Gln Val
Asp Gln Ser Trp Arg Lys Glu Arg Val Leu Asn Val 100
105 110 Pro Leu Cys Lys Glu Asp Cys Glu Arg
Trp Trp Glu Asp Cys Arg Thr 115 120
125 Ser Tyr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr
Ser Gly 130 135 140
Phe Asn Lys Cys Pro Val Gly Ala Ala Cys Gln Pro Phe His Phe Tyr 145
150 155 160 Phe Pro Thr Pro Thr
Val Leu Cys Asn Glu Ile Trp Thr Tyr Ser Tyr 165
170 175 Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly
Arg Cys Ile Gln Met Trp 180 185
190 Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe
Tyr 195 200 205 Ala
Ala Ala Met Ser 210 2329PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 232Pro Trp Glu Tyr Ser Trp Tyr Asp Tyr 1 5
23311PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 233Asn Tyr Thr Ile Val Val
Ser Pro Phe Asp Tyr 1 5 10
23411PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 234Asn Tyr Phe Ile Gly Ser Val Ala Met
Asp Tyr 1 5 10 2358PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 235Gly Leu Thr Tyr Ser Met Asp Tyr 1 5
2369PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 236Met Gln Ala Leu Gln Ile Pro Asn Thr 1
5 2377PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 237Tyr Ala Tyr Ala Leu Asp Tyr 1 5
2388PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 238Gln Gln His Gly Ser Ser Ser Thr 1
5 23911PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 239Gly Asp Phe Ser Ala Gly
Arg Leu Met Asp Tyr 1 5 10
24010PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 240Met Gln Ala Leu Gln Thr Pro Pro Ile
Thr 1 5 10 2418PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 241Gly Asp Tyr Asn Ala Phe Asp Tyr 1 5
2428PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 242Met Gln Ala Trp His Ser Pro Thr 1
5 2438PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 243Gly Ala Thr Tyr Thr Met Asp Tyr 1 5
2449PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 244Met Gln Ala Leu Gln Thr Pro Ile Thr 1
5 245227PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 245Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 50 55
60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65 70 75
80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100
105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120
125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165
170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys 225
2462076DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 246caggtgcaat tggttcaatc
tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag catccggata
caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg gtctggaatg
gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat tccagggtcg
cgtcacgatg acccatgaca ctagcacctc taccgtttat 240atggagctgt ccagcctgcg
ttctgaagat actgcagtgt actactgtgc acgctctttc 300ttcactggtt tccatctgga
ctattggggt caaggcaccc tcgtaacggt ttcttctgct 360agcacaaagg gccccagcgt
gttccctctg gcccctagca gcaagagcac atctggcgga 420acagccgccc tgggctgcct
cgtgaaggac tactttcccg agcctgtgac cgtgtcctgg 480aactctggcg ccctgacaag
cggcgtgcac acctttccag ccgtgctgca gagcagcggc 540ctgtactctc tgagcagcgt
ggtcaccgtg cctagcagca gcctgggcac ccagacctac 600atctgcaacg tgaaccacaa
gcccagcaac accaaagtgg acaagaaggt ggagcccaag 660agctgtgatg gcggaggagg
gtccggaggc ggaggatccg aggtgcagct gctggaatct 720ggcggcggac tggtgcagcc
tggcggatct ctgagactga gctgtgccgc cagcggcttc 780accttcagca cctacgccat
gaactgggtg cgccaggccc ctggcaaagg cctggaatgg 840gtgtcccgga tcagaagcaa
gtacaacaac tacgccacct actacgccga cagcgtgaag 900ggccggttca ccatcagccg
ggacgacagc aagaacaccc tgtacctgca gatgaacagc 960ctgcgggccg aggacaccgc
cgtgtactat tgtgtgcggc acggcaactt cggcaacagc 1020tatgtgtctt ggtttgccta
ctggggccag ggcaccctcg tgaccgtgtc aagcgctagt 1080gtggccgctc cctccgtgtt
tatctttccc ccatccgatg aacagctgaa aagcggcacc 1140gcctccgtcg tgtgtctgct
gaacaatttt taccctaggg aagctaaagt gcagtggaaa 1200gtggataacg cactgcagtc
cggcaactcc caggaatctg tgacagaaca ggactccaag 1260gacagcacct actccctgtc
ctccaccctg acactgtcta aggctgatta tgagaaacac 1320aaagtctacg cctgcgaagt
cacccatcag ggcctgagct cgcccgtcac aaagagcttc 1380aacaggggag agtgtgacaa
gacccacacc tgtccccctt gtcctgcccc tgaagctgct 1440ggcggccctt ctgtgttcct
gttcccccca aagcccaagg acaccctgat gatcagccgg 1500acccccgaag tgacctgcgt
ggtggtggat gtgtcccacg aggaccctga agtgaagttc 1560aattggtacg tggacggcgt
ggaagtgcac aacgccaaga caaagccgcg ggaggagcag 1620tacaacagca cgtaccgtgt
ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1680ggcaaggagt acaagtgcaa
ggtctccaac aaagccctcg gcgcccccat cgagaaaacc 1740atctccaaag ccaaagggca
gccccgagaa ccacaggtgt acaccctgcc cccatgccgg 1800gatgagctga ccaagaacca
ggtcagcctg tggtgcctgg tcaaaggctt ctatcccagc 1860gacatcgccg tggagtggga
gagcaatggg cagccggaga acaactacaa gaccacgcct 1920cccgtgctgg actccgacgg
ctccttcttc ctctacagca agctcaccgt ggacaagagc 1980aggtggcagc aggggaacgt
cttctcatgc tccgtgatgc atgaggctct gcacaaccac 2040tacacgcaga agagcctctc
cctgtctccg ggtaaa 20762471347DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 247caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccatgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgctctttc 300ttcactggtt tccatctgga ctattggggt caaggcaccc tcgtaacggt
ttcttctgct 360agcaccaagg gcccctccgt gttccccctg gcccccagca gcaagagcac
cagcggcggc 420acagccgctc tgggctgcct ggtcaaggac tacttccccg agcccgtgac
cgtgtcctgg 480aacagcggag ccctgacctc cggcgtgcac accttccccg ccgtgctgca
gagttctggc 540ctgtatagcc tgagcagcgt ggtcaccgtg ccttctagca gcctgggcac
ccagacctac 600atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagaaggt
ggagcccaag 660agctgcgaca aaactcacac atgcccaccg tgcccagcac ctgaagctgc
agggggaccg 720tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 780gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 840gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 900acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 960tacaagtgca aggtctccaa caaagccctc ggcgccccca tcgagaaaac
catctccaaa 1020gccaaagggc agccccgaga accacaggtg tgcaccctgc ccccatcccg
ggatgagctg 1080accaagaacc aggtcagcct ctcgtgcgca gtcaaaggct tctatcccag
cgacatcgcc 1140gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc
tcccgtgctg 1200gactccgacg gctccttctt cctcgtgagc aagctcaccg tggacaagag
caggtggcag 1260caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 1320aagagcctct ccctgtctcc gggtaaa
1347248120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 248Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Glu Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120
249120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 249Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Gln Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 250125PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 250Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Val Arg
His Gly Asn Phe Gly Ala Ser Tyr Val Ser Trp Phe 100
105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125
251125PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 251Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Thr Tyr 20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Arg Ile Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ala Tyr Val Ser Trp Phe
100 105 110 Ala Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125 252689PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 252Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 225
230 235 240 Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 245
250 255 Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr Ala Met Asn Trp Val Arg 260 265
270 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg
Ser Lys 275 280 285
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 290
295 300 Thr Ile Ser Arg Asp
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305 310
315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg His Gly 325 330
335 Asn Phe Gly Ala Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln
Gly 340 345 350 Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 355
360 365 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 370 375
380 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 385 390 395
400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 420
425 430 Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 435 440
445 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 465
470 475 480 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 485
490 495 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 500 505
510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 530
535 540 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545 550
555 560 Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile Glu Lys 565 570
575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 580 585 590
Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
595 600 605 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610
615 620 Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 625 630
635 640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 645 650
655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
660 665 670 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675
680 685 Lys 253450PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 253Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120
125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205 Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210
215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315
320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
325 330 335 Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340
345 350 Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 355 360
365 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp 370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385
390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425
430 Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445
Gly Lys 450 254215PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 254Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5
10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser 20 25
30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg
Gly 35 40 45 Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50
55 60 Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70
75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala
Leu Trp Tyr Ser Asn 85 90
95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110 Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115
120 125 Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135
140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro Val Lys Ala 145 150 155
160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
165 170 175 Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180
185 190 Ser Tyr Ser Cys Gln Val Thr His
Glu Gly Ser Thr Val Glu Lys Thr 195 200
205 Val Ala Pro Thr Glu Cys Ser 210
215 255689PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 255Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 225
230 235 240 Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 245
250 255 Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
Ala Met Asn Trp Val Arg 260 265
270 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser
Lys 275 280 285 Tyr
Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 290
295 300 Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305 310
315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Val Arg His Gly 325 330
335 Asn Phe Gly Asn Ala Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly
340 345 350 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 355
360 365 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 370 375
380 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 385 390 395
400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 420
425 430 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 435 440
445 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 465
470 475 480 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 485
490 495 Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 500 505
510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 530
535 540 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545 550
555 560 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Gly Ala Pro Ile Glu Lys 565 570
575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr 580 585 590 Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp 595
600 605 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610 615
620 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 625 630 635
640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
645 650 655 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 660
665 670 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 675 680
685 Lys 256690PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 256Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Pro
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Asp
Phe Ala Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120
125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly 210
215 220 Gly Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Leu Glu Ser Gly Gly 225 230
235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser 245 250
255 Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro
260 265 270 Gly Lys
Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys Tyr Asn Asn 275
280 285 Tyr Ala Thr Tyr Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser 290 295
300 Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg 305 310 315
320 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly
325 330 335 Ala Ser Tyr
Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 340
345 350 Thr Val Ser Ser Ala Ser Val Ala
Ala Pro Ser Val Phe Ile Phe Pro 355 360
365 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu 370 375 380
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 385
390 395 400 Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 405
410 415 Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys 420 425
430 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln 435 440 445
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp 450
455 460 Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 465 470
475 480 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile 485 490
495 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 500 505 510 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 515
520 525 Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 530 535
540 Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 545 550 555
560 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
565 570 575 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 580
585 590 Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu 595 600
605 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 610 615 620
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 625
630 635 640 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 645
650 655 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 660 665
670 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 675 680 685
Gly Lys 690 257447PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 257Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25
30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly
Ile Ile Asn Pro Ser Gly Gly Pro Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Phe Ala Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser 145 150 155
160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175 Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200
205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225
230 235 240 Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245
250 255 Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265
270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290
295 300 Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310
315 320 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu
Pro 340 345 350 Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 355
360 365 Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 385 390 395
400 Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415 Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430 His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445 258219PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 258Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90
95 Ser Ile Met Gln Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115
120 125 Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 145 150 155
160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180
185 190 Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200
205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 259214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 259Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro
Gly Gly 1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30 Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35
40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala
Pro Gly Thr Pro Ala Arg Phe 50 55
60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala 65 70 75
80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95 Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala 100
105 110 Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser 115 120
125 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe 130 135 140
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 145
150 155 160 Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165
170 175 Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr 180 185
190 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys 195 200 205 Val
Glu Pro Lys Ser Cys 210 260690PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 260Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Pro
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Asp
Phe Ala Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120
125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly 210
215 220 Gly Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Leu Glu Ser Gly Gly 225 230
235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser 245 250
255 Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro
260 265 270 Gly Lys
Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys Tyr Asn Asn 275
280 285 Tyr Ala Thr Tyr Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser 290 295
300 Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg 305 310 315
320 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly
325 330 335 Asn Ala Tyr
Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 340
345 350 Thr Val Ser Ser Ala Ser Val Ala
Ala Pro Ser Val Phe Ile Phe Pro 355 360
365 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu 370 375 380
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 385
390 395 400 Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 405
410 415 Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys 420 425
430 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln 435 440 445
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp 450
455 460 Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 465 470
475 480 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile 485 490
495 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 500 505 510 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 515
520 525 Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 530 535
540 Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 545 550 555
560 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
565 570 575 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 580
585 590 Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu 595 600
605 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 610 615 620
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 625
630 635 640 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 645
650 655 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 660 665
670 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 675 680 685
Gly Lys 690 261360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 261gaggtgcaat
tggttgaatc tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg
cttccggatt caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag
gcctcgagtg ggttggtcgt atcaagtcta aaactgaggg tggcaccacg 180gattacgcgg
ctccagttaa aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc
agatgaactc tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat
ggtcttggta cgattattgg ggccagggca cgctggttac ggtgtcttcc
360262360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 262gaggtgcaat tggttgaatc
tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg cttccggatt
caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag gcctcgagtg
ggttggtcgt atcaagtcta aaactcaggg tggcaccacg 180gattacgcgg ctccagttaa
aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc agatgaactc
tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttggta
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360263375DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 263gaggtgcagc tgctggaatc tggcggcgga ctggtgcagc ctggcggatc
tctgagactg 60agctgtgccg ccagcggctt caccttcagc acctacgcca tgaactgggt
gcgccaggcc 120cctggcaaag gcctggaatg ggtgtcccgg atcagaagca agtacaacaa
ctacgccacc 180tactacgccg acagcgtgaa gggccggttc accatcagcc gggacgacag
caagaacacc 240ctgtacctgc agatgaacag cctgcgggcc gaggacaccg ccgtgtacta
ttgtgtgcgg 300cacggcaact tcggcgccag ctatgtgtct tggtttgcct actggggcca
gggcaccctc 360gtgaccgtgt caagc
375264375DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 264gaggtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60agctgtgccg
ccagcggctt caccttcagc acctacgcca tgaactgggt gcgccaggcc 120cctggcaaag
gcctggaatg ggtgtcccgg atcagaagca agtacaacaa ctacgccacc 180tactacgccg
acagcgtgaa gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc
agatgaacag cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact
tcggcaacgc ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt
caagc
3752652067DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 265gaggtgcaat tggttgaatc
tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg cttccggatt
caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag gcctcgagtg
ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg ctccagttaa
aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc agatgaactc
tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttggta
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacaa agggccctag
cgtgttccct ctggccccca gcagcaagag cacaagcggc 420ggaacagccg ccctgggctg
cctcgtgaag gactacttcc ccgagcccgt gacagtgtct 480tggaacagcg gagccctgac
aagcggcgtg cacactttcc ctgccgtgct gcagagcagc 540ggcctgtact ccctgagcag
cgtggtcacc gtgcctagca gcagcctggg cacccagacc 600tacatctgca acgtgaacca
caagcccagc aacaccaaag tggacaagaa ggtggagccc 660aagagctgtg atggcggagg
agggtccgga ggcggaggat ccgaggtgca gctgctggaa 720tctggcggcg gactggtgca
gcctggcgga tctctgagac tgagctgtgc cgccagcggc 780ttcaccttca gcacctacgc
catgaactgg gtgcgccagg cccctggcaa aggcctggaa 840tgggtgtccc ggatcagaag
caagtacaac aactacgcca cctactacgc cgacagcgtg 900aagggccggt tcaccatcag
ccgggacgac agcaagaaca ccctgtacct gcagatgaac 960agcctgcggg ccgaggacac
cgccgtgtac tattgtgtgc ggcacggcaa cttcggcgcc 1020agctatgtgt cttggtttgc
ctactggggc cagggcaccc tcgtgaccgt gtcaagcgct 1080agtaccaagg gccccagcgt
gttccccctg gcacccagca gcaagagcac atctggcgga 1140acagccgctc tgggctgtct
ggtgaaagac tacttccccg agcccgtgac cgtgtcttgg 1200aactctggcg ccctgaccag
cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1260ctgtactccc tgtcctccgt
ggtcaccgtg ccctctagct ccctgggaac acagacatat 1320atctgtaatg tcaatcacaa
gccttccaac accaaagtcg ataagaaagt cgagcccaag 1380agctgcgaca aaactcacac
atgcccaccg tgcccagcac ctgaagctgc agggggaccg 1440tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 1500gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 1560gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 1620acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1680tacaagtgca aggtctccaa
caaagccctc ggcgccccca tcgagaaaac catctccaaa 1740gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatgccg ggatgagctg 1800accaagaacc aggtcagcct
gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1860gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1920gactccgacg gctccttctt
cctctacagc aagctcaccg tggacaagag caggtggcag 1980caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2040aagagcctct ccctgtctcc
gggtaaa 20672661350DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 266gaggtgcaat tggttgaatc tggtggtggt ctggtaaaac cgggcggttc
cctgcgtctg 60agctgcgcgg cttccggatt caccttctcc aacgcgtgga tgagctgggt
tcgccaggcc 120ccgggcaaag gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg
tggcaccacg 180gattacgcgg ctccagttaa aggtcgtttt accatttccc gcgacgatag
caaaaacact 240ctgtatctgc agatgaactc tctgaaaact gaagacaccg cagtctacta
ctgtactacc 300ccgtgggaat ggtcttggta cgattattgg ggccagggca cgctggttac
ggtgtcttcc 360gctagcacca agggcccctc cgtgttcccc ctggccccca gcagcaagag
caccagcggc 420ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt
gaccgtgtcc 480tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct
gcagagttct 540ggcctgtata gcctgagcag cgtggtcacc gtgccttcta gcagcctggg
cacccagacc 600tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa
ggtggagccc 660aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaagc
tgcaggggga 720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa
aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc
ccgggatgag 1080ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 1200ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccgcttcacg 1320cagaagagcc tctccctgtc tccgggtaaa
1350267645DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 267caggccgtcg
tgacccagga acccagcctg acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca
gttctacagg cgccgtgacc accagcaact acgccaactg ggtgcaggaa 120aagcccggcc
aggccttcag aggactgatc ggcggcacca acaagagagc ccctggcacc 180cctgccagat
tcagcggatc tctgctggga ggaaaggccg ccctgacact gtctggcgcc 240cagccagaag
atgaggccga gtactactgc gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca
ccaagctgac agtcctaggt caacccaagg ctgcccccag cgtgaccctg 360ttccccccca
gcagcgagga actgcaggcc aacaaggcca ccctggtctg cctgatcagc 420gacttctacc
caggcgccgt gaccgtggcc tggaaggccg acagcagccc cgtgaaggcc 480ggcgtggaga
ccaccacccc cagcaagcag agcaacaaca agtacgccgc cagcagctac 540ctgagcctga
cccccgagca gtggaagagc cacaggtcct acagctgcca ggtgacccac 600gagggcagca
ccgtggagaa aaccgtggcc cccaccgagt gcagc
6452682067DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 268gaggtgcaat tggttgaatc
tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg cttccggatt
caccttctcc aacgcgtgga tgagctgggt tcgccaggcc 120ccgggcaaag gcctcgagtg
ggttggtcgt atcaagtcta aaactgacgg tggcaccacg 180gattacgcgg ctccagttaa
aggtcgtttt accatttccc gcgacgatag caaaaacact 240ctgtatctgc agatgaactc
tctgaaaact gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttggta
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacaa agggccctag
cgtgttccct ctggccccca gcagcaagag cacaagcggc 420ggaacagccg ccctgggctg
cctcgtgaag gactacttcc ccgagcccgt gacagtgtct 480tggaacagcg gagccctgac
aagcggcgtg cacactttcc ctgccgtgct gcagagcagc 540ggcctgtact ccctgagcag
cgtggtcacc gtgcctagca gcagcctggg cacccagacc 600tacatctgca acgtgaacca
caagcccagc aacaccaaag tggacaagaa ggtggagccc 660aagagctgtg atggcggagg
agggtccgga ggcggaggat ccgaggtgca gctgctggaa 720tctggcggcg gactggtgca
gcctggcgga tctctgagac tgagctgtgc cgccagcggc 780ttcaccttca gcacctacgc
catgaactgg gtgcgccagg cccctggcaa aggcctggaa 840tgggtgtccc ggatcagaag
caagtacaac aactacgcca cctactacgc cgacagcgtg 900aagggccggt tcaccatcag
ccgggacgac agcaagaaca ccctgtacct gcagatgaac 960agcctgcggg ccgaggacac
cgccgtgtac tattgtgtgc ggcacggcaa cttcggcaac 1020gcctatgtgt cttggtttgc
ctactggggc cagggcaccc tcgtgaccgt gtcaagcgct 1080agtaccaagg gccccagcgt
gttccccctg gcacccagca gcaagagcac atctggcgga 1140acagccgctc tgggctgtct
ggtgaaagac tacttccccg agcccgtgac cgtgtcttgg 1200aactctggcg ccctgaccag
cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1260ctgtactccc tgtcctccgt
ggtcaccgtg ccctctagct ccctgggaac acagacatat 1320atctgtaatg tcaatcacaa
gccttccaac accaaagtcg ataagaaagt cgagcccaag 1380agctgcgaca aaactcacac
atgcccaccg tgcccagcac ctgaagctgc agggggaccg 1440tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 1500gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 1560gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 1620acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1680tacaagtgca aggtctccaa
caaagccctc ggcgccccca tcgagaaaac catctccaaa 1740gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatgccg ggatgagctg 1800accaagaacc aggtcagcct
gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1860gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1920gactccgacg gctccttctt
cctctacagc aagctcaccg tggacaagag caggtggcag 1980caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2040aagagcctct ccctgtctcc
gggtaaa 20672692070DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 269caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggccc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcggtgac 300ttcgcttggc tggactattg gggtcaaggc accctcgtaa cggtttcttc
tgctagcaca 360aagggcccca gcgtgttccc tctggcccct agcagcaaga gcacatctgg
cggaacagcc 420gccctgggct gcctcgtgaa ggactacttt cccgagcctg tgaccgtgtc
ctggaactct 480ggcgccctga caagcggcgt gcacaccttt ccagccgtgc tgcagagcag
cggcctgtac 540tctctgagca gcgtggtcac cgtgcctagc agcagcctgg gcacccagac
ctacatctgc 600aacgtgaacc acaagcccag caacaccaaa gtggacaaga aggtggagcc
caagagctgt 660gatggcggag gagggtccgg aggcggagga tccgaggtgc agctgctgga
atctggcggc 720ggactggtgc agcctggcgg atctctgaga ctgagctgtg ccgccagcgg
cttcaccttc 780agcacctacg ccatgaactg ggtgcgccag gcccctggca aaggcctgga
atgggtgtcc 840cggatcagaa gcaagtacaa caactacgcc acctactacg ccgacagcgt
gaagggccgg 900ttcaccatca gccgggacga cagcaagaac accctgtacc tgcagatgaa
cagcctgcgg 960gccgaggaca ccgccgtgta ctattgtgtg cggcacggca acttcggcgc
cagctatgtg 1020tcttggtttg cctactgggg ccagggcacc ctcgtgaccg tgtcaagcgc
tagtgtggcc 1080gctccctccg tgtttatctt tcccccatcc gatgaacagc tgaaaagcgg
caccgcctcc 1140gtcgtgtgtc tgctgaacaa tttttaccct agggaagcta aagtgcagtg
gaaagtggat 1200aacgcactgc agtccggcaa ctcccaggaa tctgtgacag aacaggactc
caaggacagc 1260acctactccc tgtcctccac cctgacactg tctaaggctg attatgagaa
acacaaagtc 1320tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag
cttcaacagg 1380ggagagtgtg acaagaccca cacctgtccc ccttgtcctg cccctgaagc
tgctggcggc 1440ccttctgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatcag
ccggaccccc 1500gaagtgacct gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaa
gttcaattgg 1560tacgtggacg gcgtggaagt gcacaacgcc aagacaaagc cgcgggagga
gcagtacaac 1620agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 1680gagtacaagt gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa
aaccatctcc 1740aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg
ccgggatgag 1800ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc
cagcgacatc 1860gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 1920ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa
gagcaggtgg 1980cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacacg 2040cagaagagcc tctccctgtc tccgggtaaa
20702701341DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 270caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggccc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtgac 300ttcgcttggc
tggactattg gggtcaaggc accctcgtaa cggtttcttc tgctagcacc 360aagggcccct
ccgtgttccc cctggccccc agcagcaaga gcaccagcgg cggcacagcc 420gctctgggct
gcctggtcaa ggactacttc cccgagcccg tgaccgtgtc ctggaacagc 480ggagccctga
cctccggcgt gcacaccttc cccgccgtgc tgcagagttc tggcctgtat 540agcctgagca
gcgtggtcac cgtgccttct agcagcctgg gcacccagac ctacatctgc 600aacgtgaacc
acaagcccag caacaccaag gtggacaaga aggtggagcc caagagctgc 660gacaaaactc
acacatgccc accgtgccca gcacctgaag ctgcaggggg accgtcagtc 720ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780tgcgtggtgg
tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960tgcaaggtct
ccaacaaagc cctcggcgcc cccatcgaga aaaccatctc caaagccaaa 1020gggcagcccc
gagaaccaca ggtgtgcacc ctgcccccat cccgggatga gctgaccaag 1080aaccaggtca
gcctctcgtg cgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct
tcttcctcgt gagcaagctc accgtggaca agagcaggtg gcagcagggg 1260aacgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320ctctccctgt
ctccgggtaa a
1341271657DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 271gatattgtta tgactcaatc
tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc gttccagcca
gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa aaccgggtca
gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac cggaccgttt
cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg aagcagaaga
cgtgggcgtt tattactgta tgcaggcaag cattatgcag 300cggacttttg gtcaaggcac
caaggtcgaa attaaacgta cggtggctgc accatctgtc 360ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420ctgaataact tctatcccag
agaggccaaa gtacagtgga aggtggataa cgccctccaa 480tcgggtaact cccaggagag
tgtcacagag caggacagca aggacagcac ctacagcctc 540agcagcaccc tgacgctgag
caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600gtcacccatc agggcctgag
ctcgcccgtc acaaagagct tcaacagggg agagtgt 657272642DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 272caggccgtcg tgacccagga acccagcctg acagtgtctc ctggcggcac
cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc accagcaact acgccaactg
ggtgcaggaa 120aagcccggcc aggccttcag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg ccctgacact
gtctggcgcc 240cagccagaag atgaggccga gtactactgc gccctgtggt acagcaacct
gtgggtgttc 300ggcggaggca ccaagctgac agtgctgagc agcgcttcca ccaaaggccc
ttccgtgttt 360cctctggctc ctagctccaa gtccacctct ggaggcaccg ctgctctcgg
atgcctcgtg 420aaggattatt ttcctgagcc tgtgacagtg tcctggaata gcggagcact
gacctctgga 480gtgcatactt tccccgctgt gctgcagtcc tctggactgt acagcctgag
cagcgtggtg 540acagtgccca gcagcagcct gggcacccag acctacatct gcaacgtgaa
ccacaagccc 600agcaacacca aggtggacaa gaaggtggaa cccaagtctt gt
6422732070DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 273caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggccc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtgac 300ttcgcttggc
tggactattg gggtcaaggc accctcgtaa cggtttcttc tgctagcaca 360aagggcccca
gcgtgttccc tctggcccct agcagcaaga gcacatctgg cggaacagcc 420gccctgggct
gcctcgtgaa ggactacttt cccgagcctg tgaccgtgtc ctggaactct 480ggcgccctga
caagcggcgt gcacaccttt ccagccgtgc tgcagagcag cggcctgtac 540tctctgagca
gcgtggtcac cgtgcctagc agcagcctgg gcacccagac ctacatctgc 600aacgtgaacc
acaagcccag caacaccaaa gtggacaaga aggtggagcc caagagctgt 660gatggcggag
gagggtccgg aggcggagga tccgaggtgc agctgctgga atctggcggc 720ggactggtgc
agcctggcgg atctctgaga ctgagctgtg ccgccagcgg cttcaccttc 780agcacctacg
ccatgaactg ggtgcgccag gcccctggca aaggcctgga atgggtgtcc 840cggatcagaa
gcaagtacaa caactacgcc acctactacg ccgacagcgt gaagggccgg 900ttcaccatca
gccgggacga cagcaagaac accctgtacc tgcagatgaa cagcctgcgg 960gccgaggaca
ccgccgtgta ctattgtgtg cggcacggca acttcggcaa cgcctatgtg 1020tcttggtttg
cctactgggg ccagggcacc ctcgtgaccg tgtcaagcgc tagtgtggcc 1080gctccctccg
tgtttatctt tcccccatcc gatgaacagc tgaaaagcgg caccgcctcc 1140gtcgtgtgtc
tgctgaacaa tttttaccct agggaagcta aagtgcagtg gaaagtggat 1200aacgcactgc
agtccggcaa ctcccaggaa tctgtgacag aacaggactc caaggacagc 1260acctactccc
tgtcctccac cctgacactg tctaaggctg attatgagaa acacaaagtc 1320tacgcctgcg
aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 1380ggagagtgtg
acaagaccca cacctgtccc ccttgtcctg cccctgaagc tgctggcggc 1440ccttctgtgt
tcctgttccc cccaaagccc aaggacaccc tgatgatcag ccggaccccc 1500gaagtgacct
gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 1560tacgtggacg
gcgtggaagt gcacaacgcc aagacaaagc cgcgggagga gcagtacaac 1620agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1680gagtacaagt
gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1740aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1800ctgaccaaga
accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1860gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1920ctggactccg
acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1980cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 2040cagaagagcc
tctccctgtc tccgggtaaa
2070274120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 274Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Ala 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys
Ser Lys Thr Glu Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Tyr Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 27519PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 275Arg Ile Lys Ser Lys Thr Glu Gly Gly Thr Thr Asp Tyr Ala Ala
Pro 1 5 10 15 Val
Lys Gly 276689PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 276Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Glu Gly Gly Thr Thr Asp Tyr Ala Ala 50
55 60 Pro Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Thr Pro Tyr Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 225
230 235 240 Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 245
250 255 Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr Ala Met Asn Trp Val Arg 260 265
270 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg
Ser Lys 275 280 285
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 290
295 300 Thr Ile Ser Arg Asp
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 305 310
315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg His Gly 325 330
335 Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln
Gly 340 345 350 Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 355
360 365 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 370 375
380 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 385 390 395
400 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 420
425 430 Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 435 440
445 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 465
470 475 480 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 485
490 495 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 500 505
510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 530
535 540 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545 550
555 560 Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile Glu Lys 565 570
575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 580 585 590
Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
595 600 605 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610
615 620 Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 625 630
635 640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 645 650
655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
660 665 670 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675
680 685 Lys 277450PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 277Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Lys Ser Lys Thr Glu Gly
Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75
80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95 Tyr Cys Thr Thr
Pro Tyr Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120
125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205 Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210
215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315
320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
325 330 335 Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340
345 350 Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 355 360
365 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp 370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385
390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425
430 Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445
Gly Lys 450 278119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 278Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Tyr Arg Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu
Val Thr Val Ser Ser 115 279109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 279Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu
Gly Gln 1 5 10 15
Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala
20 25 30 Ser Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35
40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser Gly Ser 50 55
60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala
Gln Ala Glu 65 70 75
80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Glu Ser Pro Pro Thr Gly
85 90 95 Leu Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
280674PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 280Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Tyr Arg Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser
Cys Asp Gly 210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu 225
230 235 240 Pro Ser Leu Thr
Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly 245
250 255 Ser Ser Thr Gly Ala Val Thr Thr Ser
Asn Tyr Ala Asn Trp Val Gln 260 265
270 Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr
Asn Lys 275 280 285
Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 290
295 300 Lys Ala Ala Leu Thr
Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu 305 310
315 320 Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu
Trp Val Phe Gly Gly Gly 325 330
335 Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val 340 345 350 Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 355
360 365 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 370 375
380 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 385 390 395
400 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
405 410 415 Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 420
425 430 Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp 435 440
445 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly 450 455 460
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 465
470 475 480 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 485
490 495 Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 500 505
510 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg 515 520 525
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530
535 540 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 545 550
555 560 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 565 570
575 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 580 585 590
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
595 600 605 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610
615 620 Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp 625 630
635 640 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 645 650
655 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
660 665 670 Gly Lys
281449PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 281Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Tyr Arg Tyr Arg Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
340 345 350 Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser 355
360 365 Cys Ala Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 Lys 282215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 282Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu
Gly Gln 1 5 10 15
Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala
20 25 30 Ser Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35
40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser Gly Ser 50 55
60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala
Gln Ala Glu 65 70 75
80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Glu Ser Pro Pro Thr Gly
85 90 95 Leu Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100
105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Lys Lys Leu 115 120
125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr Pro 130 135 140
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145
150 155 160 Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165
170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu
Gln Trp Lys Ser His Arg 180 185
190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
Thr 195 200 205 Val
Ala Pro Thr Glu Cys Ser 210 215 283118PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 283Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Asp
Trp Ser Tyr Tyr Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ser 115
284111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 284Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90
95 Arg Gln Thr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
285673PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 285Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Asn
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Asp Trp Ser Tyr Tyr Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115
120 125 Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140 Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn 145 150 155
160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175 Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser 195 200
205 Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp
Gly Gly 210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro 225
230 235 240 Ser Leu Thr Val Ser
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser 245
250 255 Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr
Ala Asn Trp Val Gln Glu 260 265
270 Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys
Arg 275 280 285 Ala
Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys 290
295 300 Ala Ala Leu Thr Leu Ser
Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr 305 310
315 320 Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val
Phe Gly Gly Gly Thr 325 330
335 Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
340 345 350 Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 355
360 365 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 370 375
380 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 385 390 395
400 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
405 410 415 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 420
425 430 Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys 435 440
445 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro 450 455 460
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 465
470 475 480 Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 485
490 495 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 500 505
510 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 515 520 525
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 530
535 540 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys 545 550
555 560 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 565 570
575 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Trp 580 585 590 Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 595
600 605 Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 610 615
620 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 625 630 635
640 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
645 650 655 Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 660
665 670 Lys 286448PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 286Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser
Thr Ser Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Asp
Trp Ser Tyr Tyr Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145
150 155 160 Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165
170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185
190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser 195 200 205 Asn
Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220 His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230
235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250
255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270 Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285 Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 305 310 315
320 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
325 330 335 Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 340
345 350 Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Ser Cys 355 360
365 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385
390 395 400 Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 405
410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425
430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 445
287360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 287gaggtgcaat tggtggaaag
cggaggcggc ctcgtgaagc ctggcggatc tctgagactg 60agctgtgccg ccagcggctt
caccttcagc aacgcctgga tgagctgggt gcgccaggcc 120cctggaaaag gactcgagtg
ggtgggacgg atcaagagca agaccgaggg cggcaccacc 180gactatgccg cccctgtgaa
gggccggttc accatcagca gggacgacag caagaacacc 240ctgtacctgc agatgaacag
cctgaaaacc gaggacaccg ccgtgtacta ctgcaccacc 300ccctacgagt ggtcttggta
cgactactgg ggccagggca ccctcgtgac cgtgtcatct 3602882067DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 288gaggtgcaat tggtggaaag cggaggcggc ctcgtgaagc ctggcggatc
tctgagactg 60agctgtgccg ccagcggctt caccttcagc aacgcctgga tgagctgggt
gcgccaggcc 120cctggaaaag gactcgagtg ggtgggacgg atcaagagca agaccgaggg
cggcaccacc 180gactatgccg cccctgtgaa gggccggttc accatcagca gggacgacag
caagaacacc 240ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta
ctgcaccacc 300ccctacgagt ggtcttggta cgactactgg ggccagggca ccctcgtgac
cgtgtcatct 360gctagcacaa agggccctag cgtgttccct ctggccccca gcagcaagag
cacaagcggc 420ggaacagccg ccctgggctg cctcgtgaag gactacttcc ccgagcccgt
gacagtgtct 480tggaacagcg gagccctgac aagcggcgtg cacaccttcc ctgccgtgct
gcagagcagc 540ggcctgtact ccctgagcag cgtggtcacc gtgcctagca gcagcctggg
cacccagacc 600tacatctgca acgtgaacca caagcccagc aacaccaaag tggacaagaa
ggtggagccc 660aagagctgtg atggcggagg agggtccgga ggcggaggat ccgaggtgca
gctgctggaa 720tctggcggcg gactggtgca gcctggcgga tctctgagac tgagctgtgc
cgccagcggc 780ttcaccttca gcacctacgc catgaactgg gtgcgccagg cccctggcaa
aggcctggaa 840tgggtgtccc ggatcagaag caagtacaac aactacgcca cctactacgc
cgacagcgtg 900aagggccggt tcaccatcag ccgggacgac agcaagaaca ccctgtacct
gcagatgaac 960agcctgcggg ccgaggacac cgccgtgtac tattgtgtgc ggcacggcaa
cttcggcaac 1020agctatgtgt cttggtttgc ctactggggc cagggcaccc tcgtgaccgt
gtcaagcgct 1080agtaccaagg gccccagcgt gttccccctg gcacccagca gcaagagcac
atctggcgga 1140acagccgctc tgggctgtct ggtgaaagac tacttccccg agcccgtgac
cgtgtcttgg 1200aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctgca
gagcagcggc 1260ctgtactccc tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac
acagacatat 1320atctgtaatg tcaatcacaa gccttccaac accaaagtcg ataagaaagt
cgagcccaag 1380agctgcgaca aaactcacac atgcccaccg tgcccagcac ctgaagctgc
agggggaccg 1440tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 1500gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 1560gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 1620acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 1680tacaagtgca aggtctccaa caaagccctc ggcgccccca tcgagaaaac
catctccaaa 1740gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatgccg
ggatgagctg 1800accaagaacc aggtcagcct gtggtgcctg gtcaaaggct tctatcccag
cgacatcgcc 1860gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc
tcccgtgctg 1920gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1980caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 2040aagagcctct ccctgtctcc gggtaaa
20672891350DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 289gaggtgcaat
tggtggaaag cggaggcggc ctcgtgaagc ctggcggatc tctgagactg 60agctgtgccg
ccagcggctt caccttcagc aacgcctgga tgagctgggt gcgccaggcc 120cctggaaaag
gactcgagtg ggtgggacgg atcaagagca agaccgaggg cggcaccacc 180gactatgccg
cccctgtgaa gggccggttc accatcagca gggacgacag caagaacacc 240ctgtacctgc
agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgcaccacc 300ccctacgagt
ggtcttggta cgactactgg ggccagggca ccctcgtgac cgtgtcatct 360gctagcacca
agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc 420ggcacagccg
ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480tggaacagcg
gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagttct 540ggcctgtata
gcctgagcag cgtggtcacc gtgccttcta gcagcctggg cacccagacc 600tacatctgca
acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660aagagctgcg
acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga 720ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt
gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 1080ctgaccaaga
accaggtcag cctctcgtgc gcagtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200ctggactccg
acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccgcttcacg 1320cagaagagcc
tctccctgtc tccgggtaaa
1350290357DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 290gaggtgcaat tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctccggatt
cacctttagc agttatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcagatga acagcctgag
agccgaggac acggccgtat attactgtgc gcgtggtgac 300taccgttacc gttacttcga
ctactggggc caaggaaccc tggtcaccgt ctcgagt 357291327DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 291tcttctgaac tgactcaaga tccagctgtt agcgtggctc tgggtcagac
tgtacgtatc 60acctgccaag gcgattctct gcgctcctac tacgcaagct ggtaccagca
gaaaccgggt 120caggccccag ttctggtgat ttacggcaaa aacaaccgtc cgtctgggat
cccggaccgt 180ttctccggca gctcttccgg taacacggcg agcctcacca tcactggcgc
tcaagcagaa 240gacgaggccg actattactg taactctcgg gaaagcccac caaccggcct
ggttgtcttc 300ggtggcggta ccaagctgac cgtccta
3272922022DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 292gaggtgcaat
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg
tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcagatga
acagcctgag agccgaggac acggccgtat attactgtgc gcgtggtgac 300taccgttacc
gttacttcga ctactggggc caaggaaccc tggtcaccgt ctcgagtgct 360agcaccaagg
gcccctccgt gtttcctctg gccccttcca gcaagtccac ctctggcgga 420actgccgctc
tgggctgcct ggtggaagat tacttccccg agcccgtgac cgtgtcctgg 480aattctggcg
ctctgacctc cggcgtgcac acctttccag ctgtgctgca gtcctccggc 540ctgtactccc
tgtcctccgt cgtgacagtg ccctccagct ctctgggcac ccagacctac 600atctgcaacg
tgaaccacaa gccctccaac accaaggtgg acgagaaggt ggaacccaag 660tcctgcgacg
gtggcggagg ttccggaggc ggaggatccc aggctgtcgt gacccaggaa 720ccctccctga
cagtgtctcc tggcggcacc gtgaccctga cctgtggatc ttctaccggc 780gctgtgacca
cctccaacta cgccaattgg gtgcaggaaa agcccggcca ggccttcaga 840ggactgatcg
gcggcaccaa caagagagcc cctggcaccc ctgccagatt ctccggttct 900ctgctgggcg
gcaaggctgc cctgactctg tctggtgctc agcctgagga cgaggccgag 960tactactgcg
ccctgtggta ctccaacctg tgggtgttcg gcggaggcac caagctgacc 1020gtgctgtcca
gcgcttccac caagggaccc agtgtgttcc ccctggcccc cagctccaag 1080tctacatccg
gtggcacagc tgccctggga tgtctcgtga aggactactt tcctgagcct 1140gtgacagtgt
cttggaacag cggagccctg accagcggag tgcacacatt ccctgcagtg 1200ctgcagagca
gcggcctgta tagcctgagc agcgtcgtga ccgtgccttc ctctagcctg 1260ggaacacaga
catatatctg taatgtgaat cataagccca gtaataccaa agtggataag 1320aaagtggaac
ctaagagctg cgataagacc cacacctgtc ccccctgccc tgctcctgaa 1380gctgctggtg
gccctagcgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1440tcccggaccc
ccgaagtgac ctgcgtggtg gtggatgtgt cccacgagga ccctgaagtg 1500aagttcaatt
ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcctagagag 1560gaacagtaca
actccaccta ccgggtggtg tccgtgctga cagtgctgca ccaggactgg 1620ctgaacggca
aagagtacaa gtgcaaggtg tccaacaagg ccctgggcgc tcccatcgaa 1680aagaccatct
ccaaggccaa gggccagccc cgggaacccc aggtgtacac cctgccccca 1740tgccgggatg
agctgaccaa gaaccaggtc agcctgtggt gcctggtcaa aggcttctat 1800cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1860acgcctcccg
tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1920aagagcaggt
ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1980aaccactaca
cgcagaagag cctctccctg tctccgggta aa
20222931347DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 293gaggtgcaat tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctccggatt
cacctttagc agttatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcagatga acagcctgag
agccgaggac acggccgtat attactgtgc gcgtggtgac 300taccgttacc gttacttcga
ctactggggc caaggaaccc tggtcaccgt ctcgagtgct 360agcaccaagg gcccctccgt
gttccccctg gcccccagca gcaagagcac cagcggcggc 420acagccgctc tgggctgcct
ggtcgaggac tacttccccg agcccgtgac cgtgtcctgg 480aacagcggag ccctgacctc
cggcgtgcac accttccccg ccgtgctgca gagttctggc 540ctgtatagcc tgagcagcgt
ggtcaccgtg ccttctagca gcctgggcac ccagacctac 600atctgcaacg tgaaccacaa
gcccagcaac accaaggtgg acgagaaggt ggagcccaag 660agctgcgaca aaactcacac
atgcccaccg tgcccagcac ctgaagctgc agggggaccg 720tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 780gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 840gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 900acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960tacaagtgca aggtctccaa
caaagccctc ggcgccccca tcgagaaaac catctccaaa 1020gccaaagggc agccccgaga
accacaggtg tgcaccctgc ccccatcccg ggatgagctg 1080accaagaacc aggtcagcct
ctcgtgcgca gtcaaaggct tctatcccag cgacatcgcc 1140gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200gactccgacg gctccttctt
cctcgtgagc aagctcaccg tggacaagag caggtggcag 1260caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320aagagcctct ccctgtctcc
gggtaaa 1347294645DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 294tcttctgaac tgactcaaga tccagctgtt agcgtggctc tgggtcagac
tgtacgtatc 60acctgccaag gcgattctct gcgctcctac tacgcaagct ggtaccagca
gaaaccgggt 120caggccccag ttctggtgat ttacggcaaa aacaaccgtc cgtctgggat
cccggaccgt 180ttctccggca gctcttccgg taacacggcg agcctcacca tcactggcgc
tcaagcagaa 240gacgaggccg actattactg taactctcgg gaaagcccac caaccggcct
ggttgtcttc 300ggtggcggta ccaagctgac cgtcctaggt caacccaagg ctgcccccag
cgtgaccctg 360ttccccccca gcagcaagaa actgcaggcc aacaaggcca ccctggtctg
cctgatcagc 420gacttctacc caggcgccgt gaccgtggcc tggaaggccg acagcagccc
cgtgaaggcc 480ggcgtggaga ccaccacccc cagcaagcag agcaacaaca agtacgccgc
cagcagctac 540ctgagcctga cccccgagca gtggaagagc cacaggtcct acagctgcca
ggtgacccac 600gagggcagca ccgtggagaa aaccgtggcc cccaccgagt gcagc
645295354DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 295caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtgac 300tggtcttact
acatggacta ttggggtcaa ggcaccctcg taacggtttc ttct
354296333DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 296gatattgtta tgactcaatc
tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc gttccagcca
gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa aaccgggtca
gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac cggaccgttt
cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg aagcagaaga
cgtgggcgtt tattactgta tgcaggcacg gcagacccca 300acttttggtc aaggcaccaa
ggtcgaaatt aaa 3332972019DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 297caggtgcaat tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc
cgttaaagtg 60agctgcaaag catccggata caccttcact tcctattaca tgcactgggt
tcgtcaagcc 120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccgtgaca ctagcacctc
taccgtttat 240atggagctgt ccagcctgcg ttctgaagat actgcagtgt actactgtgc
acgcggtgac 300tggtcttact acatggacta ttggggtcaa ggcaccctcg taacggtttc
ttctgctagc 360accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc
tggcggaact 420gccgctctgg gctgcctggt ggaagattac ttccccgagc ccgtgaccgt
gtcctggaat 480tctggcgctc tgacctccgg cgtgcacacc tttccagctg tgctgcagtc
ctccggcctg 540tactccctgt cctccgtcgt gacagtgccc tccagctctc tgggcaccca
gacctacatc 600tgcaacgtga accacaagcc ctccaacacc aaggtggacg agaaggtgga
acccaagtcc 660tgcgacggtg gcggaggttc cggaggcgga ggatcccagg ctgtcgtgac
ccaggaaccc 720tccctgacag tgtctcctgg cggcaccgtg accctgacct gtggatcttc
taccggcgct 780gtgaccacct ccaactacgc caattgggtg caggaaaagc ccggccaggc
cttcagagga 840ctgatcggcg gcaccaacaa gagagcccct ggcacccctg ccagattctc
cggttctctg 900ctgggcggca aggctgccct gactctgtct ggtgctcagc ctgaggacga
ggccgagtac 960tactgcgccc tgtggtactc caacctgtgg gtgttcggcg gaggcaccaa
gctgaccgtg 1020ctgtccagcg cttccaccaa gggacccagt gtgttccccc tggcccccag
ctccaagtct 1080acatccggtg gcacagctgc cctgggatgt ctcgtgaagg actactttcc
tgagcctgtg 1140acagtgtctt ggaacagcgg agccctgacc agcggagtgc acacattccc
tgcagtgctg 1200cagagcagcg gcctgtatag cctgagcagc gtcgtgaccg tgccttcctc
tagcctggga 1260acacagacat atatctgtaa tgtgaatcat aagcccagta ataccaaagt
ggataagaaa 1320gtggaaccta agagctgcga taagacccac acctgtcccc cctgccctgc
tcctgaagct 1380gctggtggcc ctagcgtgtt cctgttcccc ccaaagccca aggacaccct
gatgatctcc 1440cggacccccg aagtgacctg cgtggtggtg gatgtgtccc acgaggaccc
tgaagtgaag 1500ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc
tagagaggaa 1560cagtacaact ccacctaccg ggtggtgtcc gtgctgacag tgctgcacca
ggactggctg 1620aacggcaaag agtacaagtg caaggtgtcc aacaaggccc tgggcgctcc
catcgaaaag 1680accatctcca aggccaaggg ccagccccgg gaaccccagg tgtacaccct
gcccccatgc 1740cgggatgagc tgaccaagaa ccaggtcagc ctgtggtgcc tggtcaaagg
cttctatccc 1800agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta
caagaccacg 1860cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac
cgtggacaag 1920agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc
tctgcacaac 1980cactacacgc agaagagcct ctccctgtct ccgggtaaa
20192981344DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 298caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc 120ccgggccagg
gtctggaatg gatgggcatc attaacccaa gcggtggctc tacctcctac 180gcgcagaaat
tccagggtcg cgtcacgatg acccgtgaca ctagcacctc taccgtttat 240atggagctgt
ccagcctgcg ttctgaagat actgcagtgt actactgtgc acgcggtgac 300tggtcttact
acatggacta ttggggtcaa ggcaccctcg taacggtttc ttctgctagc 360accaagggcc
cctccgtgtt ccccctggcc cccagcagca agagcaccag cggcggcaca 420gccgctctgg
gctgcctggt cgaggactac ttccccgagc ccgtgaccgt gtcctggaac 480agcggagccc
tgacctccgg cgtgcacacc ttccccgccg tgctgcagag ttctggcctg 540tatagcctga
gcagcgtggt caccgtgcct tctagcagcc tgggcaccca gacctacatc 600tgcaacgtga
accacaagcc cagcaacacc aaggtggacg agaaggtgga gcccaagagc 660tgcgacaaaa
ctcacacatg cccaccgtgc ccagcacctg aagctgcagg gggaccgtca 720gtcttcctct
tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780acatgcgtgg
tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960aagtgcaagg
tctccaacaa agccctcggc gcccccatcg agaaaaccat ctccaaagcc 1020aaagggcagc
cccgagaacc acaggtgtgc accctgcccc catcccggga tgagctgacc 1080aagaaccagg
tcagcctctc gtgcgcagtc aaaggcttct atcccagcga catcgccgtg 1140gagtgggaga
gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct
ccttcttcct cgtgagcaag ctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320agcctctccc
tgtctccggg taaa
1344299654DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 299gatattgtta tgactcaatc
tccactgtct ctgccggtga ctccaggcga accggcgagc 60atttcttgcc gttccagcca
gtctctgctg cactccaacg gctacaacta tctcgattgg 120tacctgcaaa aaccgggtca
gagccctcag ctgctgatct acctgggctc taaccgcgct 180tccggtgtac cggaccgttt
cagcggctct ggatccggca ccgatttcac gttgaaaatc 240agccgtgttg aagcagaaga
cgtgggcgtt tattactgta tgcaggcacg gcagacccca 300acttttggtc aaggcaccaa
ggtcgaaatt aaacgtacgg tggctgcacc atctgtcttc 360atcttcccgc catctgatcg
gaagttgaaa tctggaactg cctctgttgt gtgcctgctg 420aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 480ggtaactccc aggagagtgt
cacagagcag gacagcaagg acagcaccta cagcctcagc 540agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga gtgt 65430050PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 300Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30 Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35
40 45 Gly Ser 50 30150PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 301Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 1 5 10 15
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
20 25 30 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35
40 45 Gly Gly 50 30254PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 302Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30 Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35
40 45 Gly Ser Gly Gly Gly Gly 50
30310PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 303Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 1 5 10
30415PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 304Glu Pro Lys Ser Cys Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 1 5 10
15 30516PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 305Glu Pro Lys Ser Cys Asp
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5
10 15 3065PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Penta-His tag" 306His His His His His 1 5
307104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 307Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp 100
308692PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 308Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Asn
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr His
Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Phe Phe Thr Gly Phe His Leu Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Gly 210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser 225
230 235 240 Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 245
250 255 Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala
Met Asn Trp Val Arg Gln 260 265
270 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys
Tyr 275 280 285 Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 290
295 300 Ile Ser Arg Asp Asp Ser
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 305 310
315 320 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Val Arg His Gly Asn 325 330
335 Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr
340 345 350 Leu Val
Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile 355
360 365 Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val 370 375
380 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys 385 390 395
400 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
405 410 415 Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 420
425 430 Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala Cys Glu Val Thr 435 440
445 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu 450 455 460
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 465
470 475 480 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 485
490 495 Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 500 505
510 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 515 520 525
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 530
535 540 Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 545 550
555 560 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly Ala Pro 565 570
575 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln 580 585 590 Val
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val 595
600 605 Ser Leu Trp Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 610 615
620 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 625 630 635
640 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
645 650 655 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 660
665 670 Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 675 680
685 Ser Pro Gly Lys 690
309449PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 309Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Asn
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr His
Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Phe Phe Thr Gly Phe His Leu Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 260 265
270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
340 345 350 Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser 355
360 365 Cys Ala Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 Lys 310216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 310Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Thr Asn Glu His
85 90 95 Tyr Tyr Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100
105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys 115 120
125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg 130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145
150 155 160 Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165
170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys 180 185
190 Val Tyr Ala Cys Glu Val Thr His Xaa Gly Leu Ser Ser Pro Val
Thr 195 200 205 Lys
Ser Phe Asn Arg Gly Glu Cys 210 215
3117PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 311Gly Thr Asn Ala Arg Ala Pro 1 5
3129PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 312Ala Leu Trp Tyr Ala Asn Leu Trp Val 1
5 3135PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 313Asn Ala Trp Met His 1 5 31419PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 314Ser Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
Pro 1 5 10 15 Val
Lys Gly 3159PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 315Pro Tyr Glu Trp Ser Trp Tyr Asp Tyr 1
5 316218PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 316Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser
Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Arg Gln Thr Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105 110 Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Arg Lys 115 120
125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145
150 155 160 Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165
170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185
190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 195 200 205 Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
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