Patent application title: MONOCLONAL ANTIBODIES AGAINST ANTITHROMBIN BETA
Inventors:
Ye Jin (San Ramon, CA, US)
John E. Murphy (Berkeley, CA, US)
John E. Murphy (Berkeley, CA, US)
Terry Hermiston (Corte Madera, CA, US)
Terry Hermiston (Corte Madera, CA, US)
Timothy Myles (Sunnyvale, CA, US)
Frank Dittmer (Duesseldorf, DE)
Michael Strerath (Duesseldorf, DE)
Uwe Gritzan (Cologne, DE)
Assignees:
BAYER HEALTHCARE LLC
IPC8 Class: AC07K1636FI
USPC Class:
4241451
Class name: Immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material monoclonal antibody or fragment thereof (i.e., produced by any cloning technology) binds hormone or other secreted growth regulatory factor, differentiation factor, or intercellular mediator (e.g., cytokine, etc.); or binds serum protein, plasma protein (e.g., tpa, etc.), or fibrin
Publication date: 2014-09-18
Patent application number: 20140271660
Abstract:
This patent document relates to antibodies, antigen-binding antibody
fragments (Fabs), and other protein scaffolds, directed against human
antithrombin β complexed with heparin and/or heparin-like structure
(ATβH). These ATβH binding proteins can block the
anti-coagulant activity of ATβ to induce coagulation. Therapeutic
uses of these antibodies and binders are described herein as are methods
of panning and screening specific antibodies.Claims:
1. A monoclonal antibody capable of binding the antithrombin (β)
heparin complex (ATβH), wherein the heavy chain of said antibody
comprises: a CDR1 sequence of amino acids 31 to 35 (AYRMG) of SEQ ID NO:
2, a CDR2 sequence of amino acids 50 to 66 (RIYSSGGRTRYADSVKG) of SEQ ID
NO: 2, and a CDR3 sequence of amino acids 97 to 114 (AREKASDLSGSFSEALDY)
of SEQ ID NO: 2; and wherein the light chain of said antibody comprises:
a CDR1 sequence of amino acids 24 to 34 (QGDSLRSYYAS) of SEQ ID NO: 1, a
CDR2 sequence of amino acids 50 to 56 (GKNNRPS) of SEQ ID NO: 1; and a
CDR3 sequence of amino acids 89 to 99 (NSRDSSGNHLV) of SEQ ID NO: 1.
2. A monoclonal antibody capable of binding ATβH, wherein the heavy chain of said antibody comprises: a CDR1 sequence of amino acids 31 to 35 (KYKMD) of SEQ ID NO: 4, a CDR2 sequence of amino acids 50 to 66 (RIGPSGGKTM YADSVKG) of SEQ ID NO: 4, and a CDR3 sequence of amino acids 97 to 114 (AREKASDLSG TYSEALDY) of SEQ ID NO: 4; and wherein the light chain of said antibody comprises: a CDR1 sequence of amino acids 26 to 37 (RASQSVSSSYLA) of SEQ ID NO: 3, a CDR2 sequence of amino acids 53 to 59 (GASSRAT) of SEQ ID NO: 3, and a CDR3 sequence of amino acids 92 to 99 (QQYGSSRT) of SEQ ID NO: 3.
3. A monoclonal antibody capable of binding ATβH, wherein the heavy chain of said antibody comprises: a CDR1 sequence of amino acids 31 to 35 (KYRMD) of SEQ ID NO: 6, a CDR2 sequence of amino acids 50 to 66 (RIGPSGGKTT YADSVKG) of SEQ ID NO: 6, and a CDR3 sequence of amino acids 97 to 114 (AREKTSDLSG SYSEALDY) of SEQ ID NO: 6; and wherein the light chain of said antibody comprises: a CDR1 sequence of amino acids 26 to 36 (RASQNINRNLA) of SEQ ID NO: 5, a CDR2 sequence of amino acids 52 to 58 (TASTRAP) of SEQ ID NO: 5, and a CDR3 sequence of amino acids 91 to 99 (QQYASPPRT) of SEQ ID NO: 6.
4. A monoclonal antibody capable of binding ATβH, wherein the heavy chain of said antibody comprises: a CDR1 sequence of amino acids 31 to 35 (RYAMY) of SEQ ID NO: 8, a CDR2 sequence of amino acids 50 to 66 (RISPSGGKTH YADSVKG) of SEQ ID NO: 8, and a CDR3 sequence of amino acids 97 to 115 (ARLSQTGYYP HYHYYGMDV) of SEQ ID NO 8; and wherein the light chain of said antibody comprises: a CDR1 sequence of amino acids 26 to 37 (RASQRVSSSYLT) of SEQ ID NO: 7, a CDR2 sequence of amino acids 53 to 59 (GASSRAT) of SEQ ID NO: 7; and a CDR3 sequence of amino acids 92 to 101 (QQYDSTPPLT) of SEQ ID NO: 7.
5. An isolated monoclonal antibody that binds to ATβH and inhibits anticoagulant activity, wherein said antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, and amino acid sequences having substantial homology to SEQ ID NOS: 2, 4, 6, and 8.
6. The isolated monoclonal antibody of claim 6, further comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5 and 7, and amino acid sequences having homology to SEQ ID NOS: 1, 3, 5, and 7.
7. An isolated monoclonal antibody that binds to AtβH and inhibits anticoagulant activity, wherein said antibody further comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, and amino acid sequences having substantial homology to SEQ ID NOS: 1, 3, 5, and 7.
8. An isolated monoclonal antibody that binds to ATβH and inhibits anticoagulant activity, wherein said antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 39, 40, 41, and 42.
9. The isolated monoclonal antibody of claim 8, further comprising: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 31, 32, 33, and 34; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ. ID NOS: 35, 36, 37, and 38; or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ. ID NOS: 31, 32, 33, and 34 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 35, 36, 37, and 38.
10. An isolated monoclonal antibody that binds to ATβH and inhibits anticoagulant activity, wherein said antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 28, 29, and 30.
11. The isolated monoclonal antibody of claim 10, further comprising: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 19, 20, 21 and 22; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ. ID NOS: 23, 24, 25, and 26; or (c) both CDR1 comprising an amino acid sequence selected from the group consisting of SEQ. ID NOS: 19, 20, 21 and 22 and a CDR2 comprising and amino acid sequence selected from the group consisting of SEQ ID NOS: 23, 24, 25, and 26.
12. An isolated monoclonal antibody that binds to the active site of ATβH.
13. An isolated monoclonal antibody that binds to ATβH and provides anticoagulant activity, wherein the isolated monoclonal antibody exhibits minimal binding to AT and wherein said antibody is a fully human antibody.
14. The isolated monoclonal antibody of claim 1, wherein the antibody is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, a secretory IgA, an IgD, an IgE antibody, and an antibody fragment.
15. An isolated monoclonal antibody that binds to human ATβH.
16. The isolated monoclonal antibody of claim 15, wherein the antibody further binds to a nonhuman species of ATβH.
17. The isolated monoclonal antibody of claim 1, wherein blood clotting time in the presence of the antibody is shortened.
18. An antibody which would compete with the isolated monoclonal antibody of claim 1.
19. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier.
20. A method for treating a genetic or acquired deficiency or a defect in coagulation comprising administering a therapeutically effective amount of a pharmaceutical composition of claim 1 to a patient.
21. A method for treating coagulopathy comprising administering a therapeutically effective amount of a pharmaceutical composition of claim 19 to a patient.
22. The method of claim 21, wherein the coagulopathy is hemophilia A, hemophilia B, or hemophilia C.
23. The method of claim 21, wherein the coagulopathy is selected from the group consisting of trauma-induced coagulopathy and severe bleeding.
24. The method of claim 21, further comprising administering a clotting factor.
25. The method of claim 24, wherein the clotting factor is selected from the group consisting of Factor VIIa, Factor VIII, and Factor IX.
26. A method for shortening bleeding time comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 19 to a patient.
27. An isolated nucleic acid molecule encoding an antibody that binds to ATβH and inhibits anticoagulant activity, wherein the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7.
28. An isolated nucleic acid molecule encoding an antibody that binds to ATβH and inhibits anticoagulant activity, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8.
29. The method of claim 20, wherein the defect in coagulation is hemophilia A, hemophilia B or hemophilia C.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of U.S. Application No. 61/784,590, entitled "Monoclonal Antibodies against Antithrombin β" and filed Mar. 14, 2013, the entire disclosure of which is expressly incorporated herein by reference.
SEQUENCE LISTING SUBMISSION
[0002] The present application includes a Sequence Listing in electronic format as a txt file titled "Sequence_Listing--17207--0006USU1_ST25" which was created on Mar. 14, 2014 and which has a size of 56.1 kilobytes (KB). The contents of txt file "Sequence_Listing--17207--0006USU1_ST25" are incorporated by reference herein.
BACKGROUND
[0003] Current unmet medical needs in the hemophilia field are mainly: (1) treatment of hemophilia patients with inhibitors (˜30% of hemophilia patients); and (2) long acting and efficacious coagulant factors (FVIII/FIX) and/or their replacement (bypass drugs) (WFH report 2012, Paris). The most widely used bypass drug for treating hemophilia patients with inhibitors is rFVII, which has major drawbacks such as risk of thrombogenicity, short half-life in plasma and high production cost. Antibodies against anti-coagulant factors, such as Tissue Factor Protein Inhibitor (TFPI), APC (Activated Protein C) and Antithrombin (AT) represent a new treatment paradigm. These antibodies not only bypass or reduce the need for FVIII or FIX coagulation factors in hemophilia patients with inhibitors, but also exhibit longer plasma half-life (which reduces the dosing frequency) and, thus, increases patient compliance. To date, there have been several antibody-based procoagulant drugs at the preclinical development or research stage, such as anti-TFPI and anti-APC.
[0004] AT is a major anticoagulant in human plasma. It inhibits thrombin, FXa and other serine proteases functioning in the coagulation pathway. It consists of 432 amino acids, is produced by the liver hepatocyte and has a long plasma half-life of three days (Collen, Schetz et al. 1977). The amino acid sequence of AT is well-conserved and the homology among cow, sheep, rabbit, mouse and human is 84%-89% (Olson and Bjork 1994). Although the primary physiological targets of AT are thrombin and FXa, AT also inhibits FIXa, FXIa, FXIIa, as well as FVIIa to a lesser extent. AT exerts its inhibition together with heparin. In presence of heparin the inhibition rate of thrombin and FXa by AT increases by 3 to 4 orders of magnitude from 7-11×103M-s-1 to 1.5-4×107 M-1s-1 and from 2.5×10-3 M-1 s-1 to 1.25 -2.5 M-1s-1 respectively (Olson, Swanson et al. 2004).
[0005] Unlike TFPI and APC which inhibit coagulation solely at the initiating stage and the amplification stage respectively, AT exerts its inhibition on coagulation at both the initiation and amplification stage. Therefore, blocking AT could have more potent pro-coagulant effect than blocking either TFPI or APC alone. Decreased AT levels and activity have been shown to correlate with increased thrombosis in human. Patients with AT deficiency tend to show recurrent venous thrombosis and pulmonary embolisms (van Boven and Lane 1997). Furthermore, homozygous AT knockout mice die in the embryonic stage with an extreme hypercoagulable state (Ishiguro, Kojima et al. 2000). A recent study shows that heterozygous AT knockout hemA mice in which AT is reduced by 50% significantly have less blood loss and enhanced thrombin generation in a tail-clip bleeding model (Bolliger, Szlam et al. 2010).
[0006] AT is a glycoprotein with two isoforms based on differential glycosylation on Asn135, ATα and ATβ (Bjork 1997). ATβ lacks glycosylation at Asn135 and is a minor glyco-isoform representing 10% of human plasma AT. Asn135 is located adjacent to the initial heparin attachment site and constitutes part of extended heparin binding site after allosteric activation and D helix extension (dela Cruz, Jairajpuri et al. 2006). The lack of bulky-sized glycan at Asn135 affects ATβ activation profoundly in two ways: 1) a faster allosteric activation upon heparin binding required for inhibition of FXa and FIXa; and 2) extra accessible binding sites for higher affinity heparin binding for inhibition of FXa and thrombin by a bridging mechanism. Indeed, under physiological salt concentration, plasma-derived ATβ binds to heparin with a KD of 36+/-3 nm while ATα binds to heparin with a KD of 500+/-50 nm (Turk IV. et al., 1993). The higher affinity of ATβ for heparin leads to its preferential distribution to the sub-endothelial layer which is enriched in the heparin-like structure--glycosaminoglycan. Consequently, ATβ is proposed to play a major and potent role in inhibition of FXa and thrombin at the vascular injury sites (Carlson and Atencio 1982; McCoy A J, Pei X Y. et al. 2003; Turk B, Brieditis I. et al. 1997; Witmer M R, Hatton M W. 1991; Frebelius S, et al. 1996). The importance and stronger potency of ATβ relative to that of ATα is also reported in clinical studies. In patients, the severity of AT homozygous mutations defective in heparin-binding is ameliorated by the beta form of AT (Martinez-Martinez, Navarro-Fernandez et al. 2012). In another study, a borderline level (˜70% of normal AT antigen and activity) of AT is compensated by the 20%˜30% ATβ in plasma (Bayston, Tripodi et al. 1999).
SUMMARY
[0007] Monoclonal antibodies to human ATIβH (ATβ complexed with heparin and/or heparin-like structure) are provided. In at least one embodiment, the anti-ATβH monoclonal antibodies exhibit binding to ATβ complexed with Heparin.
[0008] In other embodiments, the monoclonal antibodies to ATIβH may be optimized, for example to have increased affinity or increased functional activity. Also provided are specific epitopes that may be on human ATβH and are bound by an isolated monoclonal antibody. Further provided are the isolated nucleic acid molecules encoding the same.
[0009] Pharmaceutical compositions comprising the anti-ATβH monoclonal antibodies and methods of treatment of genetic and acquired deficiencies or defects in coagulation such as hemophilia A and B are also provided.
[0010] Also provided are methods for shortening bleeding time by administering an anti-ATβH monoclonal antibody to a patient in need thereof. Methods for producing a monoclonal antibody that binds human ATβH are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings or claims in any way.
[0012] FIG. 1 shows a schematic representation of ATβ bound to heparin and the various binding domains of ATβ.
[0013] FIGS. 2A-2C show how ATβ is distinguished from ATα by lacking of one N-glycan.
[0014] FIGS. 3A-3D show ATβ with faster binding to heparin and more potent inhibition than ATα.
[0015] FIG. 4A shows biotinlayted hAT and rAT are functional in inhibition of Fxa generation (FIG. 4A). FIGS. 4B-4C show various strategies for antibody discovery by phage display.
[0016] FIG. 5 shows a screening method for identifying antibodies capable of functional inhibition of ATβ::heparin.
[0017] FIGS. 6A and 6B show the amino acid sequence alignment of antibodies with functional inhibition of ATβH including the light chain domain and heavy chain domain of antibodies TPP-2009 (SEQ ID NO:1 and SEQ ID NO: 2, respectively), TPP-2015 (SEQ ID NO:3 and SEQ ID NO:4, respectively), TPP-2016 (SEQ ID NO:5 and SEQ ID NO:6 respectively), TPP-2019 (SEQ ID NO:7 and SEQ ID NO:8, respectively), and the consensus sequence (SEQ ID NO:9 and SEQ ID NO:10, respectively).
[0018] FIGS. 7A-7C show antibody binding specificity determined by Biacore (FIG. 7A) and ELISA (FIG. 7B) tests, and antibody binding affinity to human AtβH (FIG. 7C).
[0019] FIG. 8A is a graphical representation of the effect of TPP antibodies on thrombin generation in human HEM-A plasma, and illustrates that antibody presence increases peak thrombin generation in human HEM-A plasma.
[0020] FIG. 8B is a table showing antibodies shorten clotting time in human HemA plasma and in human AT-deficient plasma spiked in with Atβ or Atα.
[0021] FIG. 9 is a graphical representation of the PK of antibody TPP 2009 in HEM-A mice using IV dosing at 0.3, 3 and 30 mg/kg, three mice per time point (10 time points over 21 days), and associated PK parameters.
[0022] FIGS. 10A and 10B show an experimental protocol for a tail vein transection (TVT) model in HemA and the efficacy of antibody TPP-2009 in the TVT model in HemA mice. FIG. 10B shows the antibody TPP-2009 has potent efficacy in the Tail Vein Transection (TVT) model of HemA mice.
[0023] FIGS. 11A and 11B shows a molecular model of the three-dimensional structures of native ATβcomplexed with/without heparin (FIG. 11A), and fully activated antibody TPP2009 bound to heparin (FIG. 11B) and its predicted epitope structure. Helix D is extended upon heparin binding B.
DETAILED DESCRIPTION
[0024] This disclosure provides antibodies, including monoclonal antibodies and other binding proteins that specifically bind to the activated form of ATβ, but exhibit comparatively little or no reactivity against the ATα form, either naive or activated.
DEFINITIONS
[0025] For the purpose of interpreting this specification, the following definitions will apply. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used).
[0026] Whenever appropriate, terms used in the singular will also include the plural and vice versa. The use of "a" herein means "one or more" unless stated otherwise or where the use of "one or more" is clearly inappropriate. The use of "or" means "and/or" unless stated otherwise. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not limiting. The term "such as" also is not intended to be limiting. For example, the term "including" shall mean "including, but not limited to."
[0027] As used herein, the term "about" refers to +/-10% of the unit value provided. As used herein, the term "substantially" refers to the qualitative condition of exhibiting a total or approximate degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, achieve or avoid an absolute result because of the many variables that affect testing, production, and storage of biological and chemical compositions and materials, and because of the inherent error in the instruments and equipment used in the testing, production, and storage of biological and chemical compositions and materials. The term substantially is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
[0028] The term "ATβ" or "ATβH" as used herein refers to any variant, isoform, and/or species homolog of AT in its form that is naturally expressed by cells and present in plasma and is distinct from ATα. Further, the term "ATβ" or "ATβH" as used herein can also refer to an activated form of ATβ complexed with heparin or a heparin-like structure.
[0029] The term "antibody" as used herein refers to a whole antibody and any antigen binding fragment (i.e., "antigen-binding portion") or single chain thereof. This term includes a full-length immunoglobulin molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes, or an immunologically active portion of an immunoglobulin molecule, such as an antibody fragment, that retains the specific binding activity. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody. For example, an anti-ATβH monoclonal antibody fragment binds to an epitope of ATβH. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR); (vii) minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); (viii) camel IgG; and (ix) IgNAR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are analyzed for utility in the same manner as are intact antibodies.
[0030] Furthermore, it is contemplated that an antigen binding fragment can be encompassed in an antibody mimetic. The term "antibody mimetic" or "mimetic" as used herein refers to a protein that exhibits binding activity similar to a particular antibody but is a smaller alternative antibody or a non-antibody protein. Such antibody mimetic can be comprised in a scaffold. The term "scaffold" refers to a polypeptide platform for the engineering of new products with tailored functions and characteristics.
[0031] The term "anti-ATβ antibody" as used herein refers to an antibody that specifically binds to an epitope of ATβ associated with heparin or heparin-like. When bound in vivo to an epitope of ATβH, the anti-ATβ antibodies disclosed herein augment one or more aspects of the blood clotting cascade.
[0032] The terms "inhibits binding" and "blocks binding" (e.g., referring to inhibition/blocking of binding of ATβ substrate to ATβH) as used herein are used interchangeably and encompass both partial and complete inhibition or blocking of a protein with its substrate, such as an inhibition or blocking by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.
[0033] In reference to the inhibition and/or blocking of binding of ATβ substrate to ATβ, the terms inhibition and blocking also include any measurable decrease in the binding affinity of ATβ and/or ATβH to a physiological substrate when in contact with an anti-ATβ antibody as compared to ATβ not in contact with an anti-ATβ antibody, e.g., the blocking of the interaction of ATβ with its substrates by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.
[0034] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" as used herein refers to antibodies displaying a single binding specificity that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
[0035] The term "isolated antibody" as used herein is intended to refer to an antibody which is substantially free of other biological molecules, including antibodies having different antigenic specificities (e.g., an isolated antibody that binds to ATβH is substantially free of antibodies that bind antigens other than ATβH). In some embodiments, the isolated antibody is at least about 75%, about 80%, about 90%, about 95%, about 97%, about 99%, about 99.9% or about 100% pure by dry weight. In some embodiments, purity can be measured by a method such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated antibody that binds to an epitope, isoform or variant of human ATβH can, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., ATβH species homologs). Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.
[0036] The term "specific binding" as used herein refers to antibody binding to a predetermined antigen. An antibody that exhibits specific binding typically binds to an antigen with an affinity of at least about 105M-1 and binds to that antigen with an affinity that is higher, for example at least two-fold greater, than its binding affinity for an irrelevant antigen (e.g., BSA, casein). The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen." As used herein, the term "minimal binding" refers to an antibody that does not bind to and/or exhibits low affinity to a specified antigen. Typically, an antibody having minimal binding to an antigen binds to that antigen with an affinity that is lower than about 102 M-1 and does not bind to a predetermined antigen with higher affinity than it binds to an irrelevant antigen.
[0037] When used herein for an antibody such as an IgG antibody, the term "high affinity" refers to a binding affinity of at least about 107 M-1, in at least one embodiment at least about 108 M-1, in some embodiments at least about 109 M-1, about 1010 M-1, about 1011 M-1 or greater, e.g., up to about 1013 M-1 or greater. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to a binding affinity of at least about 107 M-1.
[0038] The term "isotype" as used herein refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.
[0039] The terms "Complementarity-determining region" or "CDR" as used herein refers to one of three hypervariable regions within the variable region of the heavy chain or the variable region of the light chain of an antibody molecule that form the N-terminal antigen-binding surface that is complementary to the three-dimensional structure of the bound antigen. Proceeding from the N-terminus of a heavy or light chain, these complementarity-determining regions are denoted as "CDR1," "CDR2," and "CDR3," respectively [Wu T T, Kabat E A, Bilofsky H, Proc Natl Acad Sci USA. 1975 December; 72(12):5107 and Wu T T, Kabat E A, J Exp Med. 1970 Aug. 1; 132(2):211]. CDRs are involved in antigen-antibody binding, and the CDR3 comprises a unique region specific for antigen-antibody binding. An antigen-binding site, therefore, can include six CDRs, comprising the CDR regions from each of a heavy and a light chain V region. The term "epitope" refers to the area or region of an antigen to which an antibody specifically binds or interacts, which in some embodiments indicates where the antigen is in physical contact with the antibody. Conversely, the term "paratope" refers to the area or region of the antibody on which the antigen specifically binds. Epitopes characterized by competition binding are said to be overlapping if the binding of the corresponding antibodies are mutually exclusive, i.e. binding of one antibody excludes simultaneous binding of another antibody. The epitopes are said to be separate (unique) if the antigen is able to accommodate binding of both corresponding antibodies simultaneously.
[0040] The term "competing antibodies" as used herein refers to antibodies that bind to about the same, substantially the same, essentially the same, or even the same epitope as an antibody against ATβH as described herein. Competing antibodies include antibodies with overlapping epitope specificities. Competing antibodies are thus able to effectively compete with an antibody as described herein for binding to ATβH. In some embodiments, the competing antibody can bind to the same epitope as the antibody described herein. Alternatively viewed, the competing antibody has the same epitope specificity as the antibody described herein.
[0041] The term "conservative substitutions" as used herein refers to modifications of a polypeptide that involve the substitution of one or more amino acids for amino acids having similar biochemical properties that do not result in loss of a biological or biochemical function of the polypeptide. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Antibodies of the present disclosure can have one or more conservative amino acid substitutions yet retain antigen binding activity.
[0042] For nucleic acids and polypeptides, the term "substantial homology" as used herein indicates that two nucleic acids or two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide or amino acid insertions or deletions, in at least about 80% of the nucleotides or amino acids, usually at least about 85%, in some embodiments about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%, in at least one embodiment at least about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, or about 99.5% of the nucleotides or amino acids. Alternatively, substantial homology for nucleic acids exists when the segments will hybridize under selective hybridization conditions to the complement of the strand. Also included are nucleic acid sequences and polypeptide sequences having substantial homology to the specific nucleic acid sequences and amino acid sequences recited herein. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as without limitation the AlignX® module of VectorNTI® (Invitrogen Corp., Carlsbad, Calif.). For AlignX®, the default parameters of multiple alignment are: gap opening penalty: 10; gap extension penalty: 0.05; gap separation penalty range: 8; % identity for alignment delay: 40.
[0043] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as without limitation the AlignX® module of VectorNTI® (Invitrogen Corp., Carlsbad, Calif.). For AlignX®, the default parameters of multiple alignment are: gap opening penalty: 10; gap extension penalty: 0.05; gap separation penalty range: 8; % identity for alignment delay: 40. (further details found at http://www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEAComm- unities/Vector-NTI-Community/Sequence-analysis-and-data-management-softwar- e-for-PCs/AlignX-Module-for-Vector-NTI-Advance.reg.us.html).
[0044] Another method for determining the an overall match between a query sequence (a sequence of the present disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson et al., Nucleic Acids Research, 1994, 2(22): 4673-4680), which is based on the algorithm of Higgins et al., Computer Applications in the Biosciences (CABIOS), 1992, 8(2): 189-191. In a sequence alignment the query and subject sequences are both DNA sequences. The result of said global sequence alignment is in percent identity. Parameters that can be used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty=10, Gap Extension Penalty=0.1. For multiple alignments, the following CLUSTALW parameters can be used: Gap Opening Penalty=10, Gap Extension Parameter=0.05; Gap Separation Penalty=8; % Identity for Alignment Delay=40.
[0045] The nucleic acids can be present in whole cells, in a cell lysate, or in a partially ppurified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components with which it is normally associated in the natural environment. To isolate a nucleic acid, standard techniques such as the following can be used: alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art.
Monoclonal Antibodies Against ATβH
[0046] Bleeding disorders where homeostasis is deregulated in hemophilia or in trauma patients where the wound results in a temporary loss of hemostatsis, can be treated by AT inhibitors. Antibodies, antigen-binding fragments thereof, and other AT-specific protein scaffolds can be used to provide targeting specificity to inhibit a subset of AT protein functions while preserving the rest. Given the at least 10-fold difference in plasma concentration of ATβ (<12 ug/ml) versus ATα (120 ug/ml), increased specificity of any potential ATβ inhibitor therapeutics is helpful to block ATβ function in the presence of a high circulating excess of ATα. ATβ specific antibodies that block the anti-coagulant function of ATβ can be used as therapeutics for patients with bleeding disorders. Examples of bleeding disorders include hemophilia, hemophilia patients with inhibitors, trauma-induced coagulopathy, severe bleeding patients during sepsis treatment by AT, bleeding resulting from elective surgery such as transplantation, cardiac surgery, orthopedic surgery, and excessive bleeding from Menorrhagia. Anti-ATβH antibodies having long circulating half-live can be useful in treating chronic diseases like hemophilia. ATβH antibody fragments or ATβH-binding protein scaffolds with shorter half-lives can be more effective for acute use (e.g. therapeutic use in trauma). ATβH-binding antibodies were identified by panning and screening human antibody libraries against human ATβ in complex with heparin. The identified antibodies exhibited binding to human ATβH. The heavy chain variable region and light chain variable region of each monoclonal antibody isolated was sequenced and its CDR regions were identified. The sequence identifier numbers ("SEQ ID NO") that correspond to the heavy and light chain variable regions of the ATβH-specific monoclonal antibodies are summarized in Table 1.
TABLE-US-00001 TABLE 1 Human anti-ATβH (heparin complexed ATβ) antibodies SEQ Heavy Chain Variable SEQ Clone Light Chain Variable Region ID Region ID TPP2009 AQSVLTQDPAVSVALGQTVRITCQ No. 1 EVQLLESGGGLVQPG No. 2 GDSLRSYYASWYQQKPGQAPVLVI GSLRLSCAASGFTFSA YGKNNRPSGIPDRFSGSSSGNTASL YRMGWVRQAPGKGL TITGAQAEDEADYYCNSRDSSGNH EWVSRIYSSGGRTRY LVFGGGTKLTVLGQPKAAPSVTLF ADSVKGRFTISRDNSK PPSSEELQANKATLVCLISDFYPGA NTLYLQMNSLRAEDT VTVAWKADGSPVKAGVETTKPSK AVYYCAREKASDLSG QSNNKYAASSYLSLTPEQWKSHRS SFSEALDYWGQGTLV YSCQVTHEGSTVEKTVAPAECS TVSS TPP2015 AQDIQMTQSPGTLSLSPGERATLSC No. 3 EVQLLESGGGLVQPG No. 4 RASQSVSSSYLAWYQQKPGQAPRL GSLRLSCAASGFTFSK LIYGASSRATGIPDRFSGSGSGTDFT YKMDWVRQAPGKGL LTISRLEPEDFAVYYCQQYGSSRTF EWVSRIGPSGGKTMY GQGTKVEIRRTVAAPSVFIFPPSDE ADSVKGRFTISRDNSK QLKSGTASVVCLLNNFYPREAKVQ NTLYLQMNSLRAEDT WKVDNALQSGNSQESVTEQDSKD AVYYCAREKASDLSG STYSLSSTLTLSKADYEKHKVYAC TYSEALDYWGQGTLV EVTH QGLSSPVTKS FNRGEC TVSS TPP2016 AQDIQMTQSPATLSVSPGERATLSC No. 5 EVQLLESGGGLVQPG No. 6 RASQNINRNLAWYQQKPGRAPRLL GSLRLSCAASGFTFSK IHTASTRAPGVPVRITGSGSGTEFTL YRMDWVRQAPGKGL TISSLEPEDFAVYFCQQYASPPRTF EWVSRIGPSGGKTTY GQGTKVEIKRTVAAPSVFIFPPSDE ADSVKGRFTISRDNSK QLKSGTASVVCLLNNFYPREAKVQ NTLYLQMNSLRAEDT WKVDNALQSGNSQESVTEQDSKD AVYYCAREKTSDLSG STYSLSST LTLSKADYEK SYSEALDYWGQGTL HKVYACEVTH QGLSSPVTKS VTVSS FNRGEC TPP2019 AQDIQMTQSPATLSLSPGERATLSC No. 7 EVQLLESGGG No. 8 RASQRVSSSYLTWYQQKPGQAPRL LVQPGGSLRL LIYGASSRATGIPDRFSGSGSGTDFT SCAASGFTFS LTISRLEPEDFAVYYCQQYDSTPPL RYAMYWVRQA TFGGGTKVEIKRTVAAPSVFIFPPS PGKGLEWVSR DEQLKSGTASVVCLLNNFYPREAK ISPSGGKTHY VQWKVDNALQSGNSQESVTEQDS ADSVKGRFTI KDSTYSLS SRDNSKNTLY STLTLSKADYEKHKVYACEVTHQ LQMNSLRAED GLSSPVTKSFNRGEC TAVYYCARLS QTGYYPHYHY YGMDVWGQGT TVTVSS
[0047] In at least some possible embodiments, an isolated monoclonal antibody binds to human ATβH and inhibits anticoagulant activity, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8.
[0048] In at least some possible embodiments, an isolated monoclonal antibody binds to human ATβH and inhibits anticoagulant activity, wherein the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7.
[0049] In at least some possible embodiments an isolated monoclonal antibody binds to human ATβH and inhibits anticoagulant activity, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7.
[0050] In at least some possible embodiments, the antibody comprises heavy and light chain variable regions comprising:
[0051] (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 1;
[0052] (b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 3;
[0053] (c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 5; or
[0054] (d) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 7.
[0055] Table 1B shows heavy and light chain amino acid sequences for humanized IgG mAbs.
TABLE-US-00002 TABLE 1B Heavy and Light Chain Amino Acid Sequences for humanized IgG mAbs. TPP2009 |hIgG1| Light_Chain AQSVLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYG KNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVF GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPAECS SEQ ID NO: 43 TPP2009| hIgG|Heavy_Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMGWVRQAPGKGLEWVSR IYSSGGRTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREK ASDLSGSFSEALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO: 44 TPP-2015 | hIgG|Light_Chain AQDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSRTF GQGTKVEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQ ID NO: 45 TPP-2015|hIgG|Heavy_Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYKMDWVRQAPGKGLEWVSR IGPSGGKTMYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREK ASDLSGTYSEALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSG SEQ ID NO: 46 TPP-2016|hIgG|Light_Chain, Kappa AQDIQMTQSPATLSVSPGERATLSCRASQNINRNLAWYQQKPGRAPRLLI HTASTRAPGVPVRITGSGSGTEFTLTISSLEPEDFAVYFCQQYASPPRTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQ ID NO: 47 TPP-2016|hIgG|Heavy_Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYRMDWVRQAPGKGLEWVSR IGPSGGKTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREK TSDLSGSYSEALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO: 48 TPP-2019|hIgG|Light_Chain, Kappa AQDIQMTQSPATLSLSPGERATLSCRASQRVSSSYLTWYQQKPGQAPRLL IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSTPPL TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC SEQ ID NO: 49 TPP-2019|hIgG|Heavy_Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMYWVRQAPGKGLEWVSR ISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLS QTGYYPHYHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG SEQ ID NO: 50
[0056] Table 2A provides a summary of the SEQ ID NOS: for the CDR regions ("CDR1," "CDR2," and "CDR3") of heavy and light chains of monoclonal antibodies that bind to human ATβH.
TABLE-US-00003 TABLE 2A Sequence Identifiers for CDR Regions of Human Anti-ATβH Antibodies Light Chain Heavy Chain Variable Region Variable Region Clones CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 TPP2009 19 23 27 31 35 39 TPP2015 20 24 28 32 36 40 TPP2016 21 25 29 33 37 41 TPP2019 22 26 30 34 38 42
[0057] Table 2B provides sequences of the SEQ ID NOS: for the CDR regions ("CDR1," "CDR2," and "CDR3") of heavy and light chains of monoclonal antibodies that bind to human ATβH.
TABLE-US-00004 TABLE 2B Sequences for CDR Regions of Human Anti-ATβH Antibodies Sequence Clone CDR Identifier Amino Acid Sequence TPP2009 LCDR1 SEQ ID NO: 19 QGDSLRSYYAS TPP2015 LCDR1 SEQ ID NO: 20 RASQSVSSSYLA TPP2016 LCDR1 SEQ ID NO: 21 RASQNINRNLA TPP2019 LCDR1 SEQ ID NO: 22 RASQRVSSSYLT TPP2009 LCDR2 SEQ ID NO: 23 GKNNRPS TPP2015 LCDR2 SEQ ID NO: 24 GASSRAT TPP2016 LCDR2 SEQ ID NO: 25 TASTRAP TPP2019 LCDR2 SEQ ID NO: 26 GASSRAT TPP2009 LCDR3 SEQ ID NO: 27 NSRDSSGNHLV TPP2015 LCDR3 SEQ ID NO: 28 QQYGSSRT TPP2016 LCDR3 SEQ ID NO: 29 QQYASPPRT TPP2019 LCDR3 SEQ ID NO: 30 QQYDSTPPLT TPP2009 HCDR1 SEQ ID NO: 31 AYRMG TPP2015 HCDR1 SEQ ID NO: 32 KYKMD TPP2016 HCDR1 SEQ ID NO: 33 KYRMD TPP2019 HCDR1 SEQ ID NO: 34 RYAMY TPP2009 HCDR2 SEQ ID NO: 35 RIYSSGGRTRYADSVKG TPP2015 HCDR2 SEQ ID NO: 36 RIGPSGGKTM YADSVKG TPP2016 HCDR2 SEQ ID NO: 37 RIGPSGGKTT YADSVKG TPP2019 HCDR2 SEQ ID NO: 38 RISPSGGKTH YADSVKG TPP2009 HCDR3 SEQ ID NO: 39 AREKASDLSGSFSEALDY TPP2015 HCDR3 SEQ ID NO: 40 AREKASDLSG TYSEALDY TPP2016 HCDR3 SEQ ID NO: 41 AREKTSDLSG SYSEALDY TPP2019 HCDR3 SEQ ID NO: 42 ARLSQTGYYP HYHYYGMDV
[0058] In at least some possible embodiments, an isolated monoclonal antibody binds to human ATβH, wherein the antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 39, 40, 41, and 42. This CDR3 is from a heavy chain of the antibodies identified during panning and screening.
[0059] In a further embodiment, this antibody further comprises: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 31, 32, 33, and 34; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 35, 36, 37, and 38; or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 31, 32, 33, and 34 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 35, 36, 37, and 38.
[0060] In at least some possible embodiments, antibodies share a CDR3 from one of the light chains of the antibodies identified during panning and screening. Thus, also provided is an isolated monoclonal antibody, wherein said antibody binds to ATβH and inhibits anticoagulant activity, wherein said antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 28, 29, and 30. In further embodiments, the antibody further comprises (a) a CDRlcomprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 19, 20, 21, and 22; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 23, 24, 25, and 26, or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 19, 20, 21, and 22 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 23, 24, 25, and 26.
[0061] In at least some possible embodiments, the antibody comprises a CDR3 from a heavy chain and a light chain of the antibodies identified from screening and panning. Provided is an isolated monoclonal antibody, wherein said antibody binds to ATβH and inhibits anticoagulant activity, wherein said antibody comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 39, 40, 41, and 42 and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 28, 29 and 30. In a further embodiment, the antibody further comprises: (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 31, 32, 33, and 34; (b) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 23, 24, 25, and 26; (c) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 19, 20, 21, and 22; and/or (d) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, and 26.
[0062] In some embodiments, the antibody comprises heavy and light chain variable regions comprising:
[0063] (a) a light chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 19, 23, and 27 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 31, 35, and 39;
[0064] (b) a light chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 20, 26, and 30 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 32, 36, and 40;
[0065] (c) a light chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 21, 25, and 29 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 33, 37, and 41; or
[0066] (d) a light chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 22, 26, and 30 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOS: 34, 38, and 42.
[0067] Also provided is an isolated monoclonal antibody that binds to AtβH and inhibits anticoagulant activity, wherein said antibody comprises an amino acid sequence having at least about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 1-8.
[0068] The antibody can be species specific or can cross react with multiple species. In some embodiments, the antibody can specifically react or cross react with ATβH of human, mouse, rat, rabbit, guinea pig, monkey, pig, dog, cat or other mammalian species.
[0069] The antibody can be of any of the various classes of antibodies, such as without limitation an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, a secretory IgA, and IgD, and an IgE antibody.
[0070] In one embodiment, provided is an isolated fully human monoclonal antibody to human ATIII.
Optimized Variants of Anti-ATM Antibodies
[0071] In some embodiments, the antibodies can be panned, screened and optimized, for example to increase affinity to ATβH, to further decrease any affinity to ATα, to improve cross-reactivity to different species, or to improve blocking activity of ATβH. Such optimization can be performed for example by utilizing site saturation mutagenesis of the CDRs or amino acid residues in close proximity to the CDRs, i.e. about 3 or 4 residues adjacent to the CDRs, of the antibodies.
[0072] Also provided are monoclonal antibodies that may have increased or high affinity to ATβH. In some embodiments, the anti-ATβH antibodies may have a binding affinity of at least about 108M-1, in some other embodiments may have at least about 109M-1, about 1010M-1, about 1011M-1 or greater, e.g., up to about 1013M-1 or greater.
[0073] In some embodiments, additional amino acid modifications can be introduced to reduce divergence from the germ line sequence. In other embodiments, amino acid modifications can be introduced to facilitate antibody production for large scale production processes.
[0074] In some embodiments, provided are isolated anti-ATβH monoclonal antibodies that specifically bind to human ATβ, which antibodies may comprise one or more amino acid modifications. In some embodiments, the antibody may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 or more modifications.
Epitopes
[0075] Also provided is an isolated monoclonal antibody that can bind to a predicted epitope of human ATβH, wherein the epitope comprises one or more of residues from human ATβH as shown in FIG. 11.
[0076] In some embodiments, the epitope comprises the N135 site of human ATβH. In other embodiments, the site can comprise part of the amino acid residue sequence of RCL loop of human ATβH.
[0077] Also provided are antibodies that can compete with any of the antibodies described herein for binding to human ATβH. For example, such a competing antibody can bind to one or more epitopes described above.
Nucleic Acids, Vectors and Host Cells
[0078] Also provided are isolated nucleic acid molecules encoding any of the monoclonal antibodies described herein. Thus, provided is an isolated nucleic acid molecule encoding an antibody that binds to human ATβH. Table 3 shows the nucleotide sequences of some anti-ATβH antibodies.
TABLE-US-00005 TABLE 3 Nucleotide sequence of anti-ATβH antibodies. Light Chain Heavy Chain TPP 2009 GCACAGAGCGTCTTGACTCAGGA GAAGTTCAATTGTTAGAGTCTGGTG CCCTGCTGTGTCTGTGGCCTTGGG GCGGTCTTGTTCAGCCTGGTGGTTCT ACAGACAGTCAGGATCACATGCC TTACGTCTTTCTTGCGCTGCTTCCGG AAGGAGACAGCCTCAGAAGCTAT ATTCACTTTCTCTGCTTACCGTATGG TATGCAAGCTGGTACCAGCAGAA GTTGGGTTCGCCAAGCTCCTGGTAA GCCAGGACAGGCCCCTGTACTTG AGGTTTGGAGTGGGTTTCTCGTATCT TCATCTATGGTAAAAACAACCGG ATTCTTCTGGTGGCCGTACTCGTTAT CCCTCAGGGATCCCAGACCGATT GCTGACTCCGTTAAAGGTCGCTTCA CTCTGGCTCCAGCTCAGGAAACA CTATCTCTAGAGACAACTCTAAGAA CAGCTTCCTTGACCATCACTGGGG TACTCTCTACTTGCAGATGAACAGCT CTCAGGCGGAAGATGAGGCTGAC TAAGGGCTGAGGACACGGCCGTGTA TATTACTGTAACTCCCGGGACAG TTACTGTGCGAGAGAGAAAGCGTCG CAGTGGTAACCATCTGGTATTCG GATCTATCGGGGAGTTTTTCTGAGG GCGGAGGGACCAAGCTGACCGTC CCCTTGACTACTGGGGCCAGGGAAC CTAGGTCAGCCCAAGGCTGCCCC CCTGGTCACCGTCTCAAGCGCCTCC CTCGGTCACTCTGTTCCCGCCCTC ACCAAGGGCCCATCGGTCTTCCCGC CTCTGAGGAGCTTCAAGCCAACA TAGCACCCAGCAGCAAGAGCACCAG AGGCCACACTAGTGTGTCTGATC CGGCGGAACAGCCGCCCTGGGCTGC AGTGACTTCTACCCGGGAGCTGT CTGGTGAAAGACTACTTCCCCGAGC GACAGTGGCCTGGAAGGCAGATG CCGTGACCGTGTCCTGGAACTCTGG GCAGCCCCGTCAAGGCGGGAGTG CGCCCTGACCAGCGGAGTGCATACC GAGACCACCAAACCCTCCAAACA TTCCCCGCCGTGCTGCAGAGCAGCG GAGCAACAACAAGTACGCGGCCA GCCTGTACAGCCTGAGCAGCGTGGT GCAGCTACCTGAGCCTGACGCCC GACAGTGCCCAGCAGCAGCCTGGGA GAGCAGTGGAAGTCCCACAGAAG ACCCAGACCTACATCTGCAACGTGA CTACAGCTGCCAGGTCACGCATG ACCACAAGCCCAGCAACACCAAGGT AAGGGAGCACCGTGGAGAAGAC GGACAAGAAGGTGGAACCCAAGAG AGTGGCCCCTGCAGAATGCTCT CTGCGACAAGACCCACACCTGTCCC (SEQ ID NO: 11) CCCTGCCCTGCCCCTGAACTGCTGG GCGGACCCAGCGTGTTCCTGTTCCCC CCAAAGCCCAAGGACACCCTGATGA TCAGCCGGACCCCCGAAGTGACCTG CGTGGTGGTGGACGTGTCCCACGAG GACCCAGAAGTGAAGTTTAATTGGT ACGTGGACGGCGTGGAAGTGCATAA CGCCAAGACCAAGCCCAGAGAGGA ACAGTACAACAGCACCTACCGGGTG GTGTCCGTGCTGACCGTGCTGCACC AGGACTGGCTGAACGGCAAAGAGTA CAAGTGCAAGGTCTCCAACAAGGCC CTGCCTGCCCCCATCGAGAAAACCA TCAGCAAGGCCAAGGGCCAGCCCCG CGAGCCTCAGGTGTACACACTGCCC CCCAGCCGGGATGAGCTGACCAAGA ACCAGGTGTCCCTGACCTGTCTGGT GAAAGGCTTCTACCCCAGCGATATC GCCGTGGAATGGGAGAGCAACGGCC AGCCCGAGAACAATTACAAGACCAC CCCCCCTGTGCTGGACAGCGACGGC TCATTCTTCCTGTACTCCAAGCTGAC CGTGGACAAGAGCCGGTGGCAGCAG GGCAACGTGTTCAGCTGCAGCGTGA TGCACGAGGCCCTGCACAATCACTA CACCCAGAAGTCCCTGAGCCTGAGC CCCGGC (SEQ ID NO: 12) TPP 2015 GCACAAGACATCCAGATGACCCA GAAGTTCAATTGTTAGAGTCTGGTG GTCTCCAGGCACCCTGTCTTTGTC GCGGTCTTGTTCAGCCTGGTGGTTCT TCCAGGGGAAAGAGCCACCCTCT TTACGTCTTTCTTGCGCTGCTTCCGG CCTGCAGGGCCAGTCAGAGTGTT ATTCACTTTCTCTAAGTACAAGATGG AGCAGCAGCTACTTAGCCTGGTA ATTGGGTTCGCCAAGCTCCTGGTAA CCAGCAGAAACCTGGCCAGGCTC AGGTTTGGAGTGGGTTTCTCGTATCG CCAGGCTCCTCATCTATGGTGCAT GTCCTTCTGGTGGCAAGACTATGTAT CCAGCAGGGCCACTGGCATCCCA GCTGACTCCGTTAAAGGTCGCTTCA GACAGGTTCAGTGGCAGTGGGTC CTATCTCTAGAGACAACTCTAAGAA TGGGACAGACTTCACTCTCACCAT TACTCTCTACTTGCAGATGAACAGCT CAGCAGACGGAGCCTGAAGATTT TAAGGGCTGAGGACACGGCCGTGTA TGCAGTGTATTACTGTCAGCAGTA TTACTGTGCGAGAGAGAAAGCGTCG TGGTAGCTCAACGTTCGGCCAAG GATCTATCGGGGACTTATTCTGAGG GGACCAAGGTGGAAATCAGACGA CCCTTGACTACTGGGGCCAGGGAAC ACTGTGGCTGCAATCTGTCTTCAT CCTGGTCACCGTCTCAAGCGCCTCC CTTCCCGCCATCTGATGAGCAGTT ACCAAGGGCCCATCGGTCTTCCCGC GAAATCTGGAACTGCCTCTGTTGT TAGCACCCAGCAGCAAGAGCACCAG GTGCCTGCTGAATAACTTCTATCC CGGCGGAACAGCCGCCCTGGGCTGC CAGAGAGGCCAAAGTACAGTGGA CTGGTGAAAGACTACTTCCCCGAGC AGGTGGATAACGCCCTCCAATCG CCGTGACCGTGTCCTGGAACTCTGG GGTAACTCCCAGGAGAGTGTCAC CGCCCTGACCAGCGGAGTGCATACC AGAGCAGGACAGCAAGGACAGC TTCCCCGCCGTGCTGCAGAGCAGCG ACCTACAGCCTCAGCAGCACCCT GCCTGTACAGCCTGAGCAGCGTGGT GACGCTGAGCAAAGCAGACTACG GACAGTGCCCAGCAGCAGCCTGGGA AGAAACACAAAGTCTACGCCTGC ACCCAGACCTACATCTGCAACGTGA GAAGTCACCCATCAGGGCCTGAG ACCACAAGCCCAGCAACACCAAGGT CTCGCCCGTCACAAAGAGCTTCA GGACAAGAAGGTGGAACCCAAGAG ACAGGGGAGAGTGT (SEQ ID NO: CTGCGACAAGACCCACACCTGTCCC 13) CCCTGCCCTGCCCCTGAACTGCTGG GCGGACCCAGCGTGTTCCTGTTCCCC CCAAAGCCCAAGGACACCCTGATGA TCAGCCGGACCCCCGAAGTGACCTG CGTGGTGGTGGACGTGTCCCACGAG GACCCAGAAGTGAAGTTTAATTGGT ACGTGGACGGCGTGGAAGTGCATAA CGCCAAGACCAAGCCCAGAGAGGA ACAGTACAACAGCACCTACCGGGTG GTGTCCGTGCTGACCGTGCTGCACC AGGACTGGCTGAACGGCAAAGAGTA CAAGTGCAAGGTCTCCAACAAGGCC CTGCCTGCCCCCATCGAGAAAACCA TCAGCAAGGCCAAGGGCCAGCCCCG CGAGCCTCAGGTGTACACACTGCCC CCCAGCCGGGATGAGCTGACCAAGA ACCAGGTGTCCCTGACCTGTCTGGT GAAAGGCTTCTACCCCAGCGATATC GCCGTGGAATGGGAGAGCAACGGCC AGCCCGAGAACAATTACAAGACCAC CCCCCCTGTGCTGGACAGCGACGGC TCATTCTTCCTGTACTCCAAGCTGAC CGTGGACAAGAGCCGGTGGCAGCAG GGCAACGTGTTCAGCTGCAGCGTGA TGCACGAGGCCCTGCACAATCACTA CACCCAGAAGTCCCTGAGCCTGAGC CCCGGC (SEQ ID NO: 14) TPP 2016 GCACAAGACATCCAGATGACCCA GAAGTTCAATTGTTAGAGTCTGGTG GTCTCCAGCCACCCTGTCTGTGTC GCGGTCTTGTTCAGCCTGGTGGTTCT TCCAGGGGAAAGAGCCACCCTCT TTACGTCTTTCTTGCGCTGCTTCCGG CCTGCAGGGCCAGTCAGAATATT ATTCACTTTCTCTAAGTACCGTATGG AATAGAAACTTGGCCTGGTACCA ATTGGGTTCGCCAAGCTCCTGGTAA GCAGAAGCCTGGCCGGGCTCCCA AGGTTTGGAGTGGGTTTCTCGTATCG GACTCCTCATCCATACCGCATCCA GTCCTTCTGGTGGCAAGACTACTTAT CTAGGGCCCCTGGTGTCCCAGTC GCTGACTCCGTTAAAGGTCGCTTCA AGGATCACTGGCAGTGGGTCTGG CTATCTCTAGAGACAACTCTAAGAA AACAGAGTTCACTCTCACCATCA TACTCTCTACTTGCAGATGAACAGCT GCAGCCTGGAACCTGAAGATTTT TAAGGGCTGAGGACACGGCCGTGTA GCAGTGTATTTCTGTCAGCAGTAT TTACTGTGCGAGAGAGAAAACGTCG GCTAGCCCACCTCGGACGTTCGG GATCTATCGGGGAGTTATTCTGAGG CCAAGGGACCAAGGTGGAAATCA CCCTTGACTACTGGGGCCAGGGAAC AGCGAACTGTGGCTGCACCATCT CCTGGTCACCGTCTCAAGCGCCTCC GTCTTCATCTTCCCGCCATCTGAT ACCAAGGGCCCATCGGTCTTCCCGC GAGCAGTTGAAATCTGGAACTGC TAGCACCCAGCAGCAAGAGCACCAG CTCTGTTGTGTGCCTGCTGAATAA CGGCGGAACAGCCGCCCTGGGCTGC CTTCTATCCCAGAGAGGCCAAAG CTGGTGAAAGACTACTTCCCCGAGC TACAGTGGAAGGTGGATAACGCC CCGTGACCGTGTCCTGGAACTCTGG CTCCAATCGGGTAACTCCCAGGA CGCCCTGACCAGCGGAGTGCATACC GAGTGTCACAGAGCAGGACAGCA TTCCCCGCCGTGCTGCAGAGCAGCG AGGACAGCACCTACAGCCTCAGC GCCTGTACAGCCTGAGCAGCGTGGT AGCACCCTGACGCTGAGCAAAGC GACAGTGCCCAGCAGCAGCCTGGGA AGACTACGAGAAACACAAAGTCT ACCCAGACCTACATCTGCAACGTGA ACGCCTGCGAAGTCACCCATCAG ACCACAAGCCCAGCAACACCAAGGT GGCCTGAGCTCGCCCGTCACAAA GGACAAGAAGGTGGAACCCAAGAG GAGC TTCAACAGGGGAGAGTGT CTGCGACAAGACCCACACCTGTCCC (SEQ ID NO: 15) CCCTGCCCTGCCCCTGAACTGCTGG GCGGACCCAGCGTGTTCCTGTTCCCC CCAAAGCCCAAGGACACCCTGATGA TCAGCCGGACCCCCGAAGTGACCTG CGTGGTGGTGGACGTGTCCCACGAG GACCCAGAAGTGAAGTTTAATTGGT ACGTGGACGGCGTGGAAGTGCATAA CGCCAAGACCAAGCCCAGAGAGGA ACAGTACAACAGCACCTACCGGGTG GTGTCCGTGCTGACCGTGCTGCACC AGGACTGGCTGAACGGCAAAGAGTA CAAGTGCAAGGTCTCCAACAAGGCC CTGCCTGCCCCCATCGAGAAAACCA TCAGCAAGGCCAAGGGCCAGCCCCG CGAGCCTCAGGTGTACACACTGCCC CCCAGCCGGGATGAGCTGACCAAGA ACCAGGTGTCCCTGACCTGTCTGGT GAAAGGCTTCTACCCCAGCGATATC GCCGTGGAATGGGAGAGCAACGGCC AGCCCGAGAACAATTACAAGACCAC CCCCCCTGTGCTGGACAGCGACGGC TCATTCTTCCTGTACTCCAAGCTGAC CGTGGACAAGAGCCGGTGGCAGCAG GGCAACGTGTTCAGCTGCAGCGTGA TGCACGAGGCCCTGCACAATCACTA CACCCAGAAGTCCCTGAGCCTGAGC CCCGGC (SEQ ID NO: 16) TPP 2019 GCACAAGACATCCAGATGACCCA GAAGTTCAATTGTTAGAGTCTGGTG GTCTCCAGCCACCCTGTCTTTGTC GCGGTCTTGTTCAGCCTGGTGGTTCT TCCAGGGGAAAGAGCCACCCTCT TTACGTCTTTCTTGCGCTGCTTCCGG CCTGCAGGGCCAGTCAGCGTGTT ATTCACTTTCTCTCGTTACGCTATGT AGCAGCAGCTACTTAACCTGGTA ATTGGGTTCGCCAAGCTCCTGGTAA CCAGCAGAAACCTGGCCAGGCTC AGGTTTGGAGTGGGTTTCTCGTATCT CCAGGCTCCTCATCTATGGTGCAT CTCCTTCTGGTGGCAAGACTCATTAT CCAGCAGGGCCACTGGCATCCCA GCTGACTCCGTTAAAGGTCGCTTCA GACAGGTTCAGTGGCAGTGGGTC CTATCTCTAGAGACAACTCTAAGAA TGGGACAGACTTCACTCTCACCAT TACTCTCTACTTGCAGATGAACAGCT CAGCAGACTGGAGCCTGAAGATT TAAGGGCTGAGGACACGGCCGTGTA TTGCAGTTTATTACTGTCAGCAGT TTACTGTGCGAGACTGTCTCAAACT ATGATAGTACGCCTCCGCTCACCT GGTTATTACCCTCACTACCACTACTA TCGGCGGAGGGACCAAGGTGGAG CGGTATGGACGTCTGGGGCCAAGGG ATCAAACGAACTGTGGCTGCACC ACCACGGTCACCGTCTCAAGCGCCT ATCTGTCTTCATCTTCCCGCCATC CCACCAAGGGCCCATCGGTCTTCCC TGATGAGCAGTTGAAATCTGGAA GCTAGCACCCAGCAGCAAGAGCACC CTGCCTCTGTTGTGTGCCTGCTGA AGCGGCGGAACAGCCGCCCTGGGCT ATAACTTCTATCCCAGAGAGGCC GCCTGGTGAAAGACTACTTCCCCGA AAAGTACAGTGGAAGGTGGATAA GCCCGTGACCGTGTCCTGGAACTCT CGCCCTCCAATCGGGTAACTCCC GGCGCCCTGACCAGCGGAGTGCATA AGGAGAGTGTCACAGAGCAGGAC CCTTCCCCGCCGTGCTGCAGAGCAG AGCAAGGACAGCACCTACAGCCT CGGCCTGTACAGCCTGAGCAGCGTG CAGCAGCACCCTGACGCTGAGCA GTGACAGTGCCCAGCAGCAGCCTGG AAGCAGACTACGAGAAACACAAA GAACCCAGACCTACATCTGCAACGT GTCTACGCCTGCGAAGTCACCCA GAACCACAAGCCCAGCAACACCAAG TCAGGGCCTGAGCTCGCCCGTCA GTGGACAAGAAGGTGGAACCCAAG CAAAGAGCTTCAACAGGGGAGAG AGCTGCGACAAGACCCACACCTGTC TGT (SEQ ID NO: 17) CCCCCTGCCCTGCCCCTGAACTGCTG GGCGGACCCAGCGTGTTCCTGTTCC CCCCAAAGCCCAAGGACACCCTGAT GATCAGCCGGACCCCCGAAGTGACC TGCGTGGTGGTGGACGTGTCCCACG AGGACCCAGAAGTGAAGTTTAATTG GTACGTGGACGGCGTGGAAGTGCAT AACGCCAAGACCAAGCCCAGAGAG GAACAGTACAACAGCACCTACCGGG TGGTGTCCGTGCTGACCGTGCTGCA CCAGGACTGGCTGAACGGCAAAGAG TACAAGTGCAAGGTCTCCAACAAGG CCCTGCCTGCCCCCATCGAGAAAAC CATCAGCAAGGCCAAGGGCCAGCCC CGCGAGCCTCAGGTGTACACACTGC CCCCCAGCCGGGATGAGCTGACCAA GAACCAGGTGTCCCTGACCTGTCTG GTGAAAGGCTTCTACCCCAGCGATA TCGCCGTGGAATGGGAGAGCAACGG CCAGCCCGAGAACAATTACAAGACC ACCCCCCCTGTGCTGGACAGCGACG GCTCATTCTTCCTGTACTCCAAGCTG ACCGTGGACAAGAGCCGGTGGCAGC AGGGCAACGTGTTCAGCTGCAGCGT GATGCACGAGGCCCTGCACAATCAC TACACCCAGAAGTCCCTGAGCCTGA GCCCCGGC (SEQ ID NO: 18)
[0079] In some embodiments, isolated nucleic acid molecules encode an antibody that binds to ATβH and inhibits anticoagulant activity but has minimal binding to ATα, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8.
[0080] In some embodiments, isolated nucleic acid molecules encode an antibody that binds to ATβH and inhibits anticoagulant activity but has minimal binding to ATα, wherein the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7.
[0081] In other embodiments, isolated nucleic acid molecules encode an antibody that binds to ATβ and inhibits anticoagulant activity of ATβ, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8 or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, and one or more amino acid modifications in the heavy chain variable region or light chain variable region.
[0082] Further, also provided are vectors comprising the isolated nucleic acid molecules encoding any of the monoclonal antibodies described above and host cells comprising such vectors.
Methods of Preparing Antibodies to ATβH
[0083] The monoclonal antibody can be produced recombinantly by expressing a nucleotide sequence encoding the variable regions of the monoclonal antibody according to one of the present embodiments in a host cell. With the aid of an expression vector, a nucleic acid containing the nucleotide sequence can be transfected and expressed in a host cell suitable for the production. Accordingly, an exemplary method for producing a monoclonal antibody that binds with human ATβH can comprise: (a) transfecting a nucleic acid molecule encoding a monoclonal antibody into a host cell; (b) culturing the host cell so to express the monoclonal antibody in the host cell, and (c) optionally isolating and purifying the produced monoclonal antibody, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody.
[0084] In one example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" refers to an antibody gene that is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, alternatively, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector.
[0085] Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain encoding genes, the recombinant expression vectors carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter.
[0086] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
[0087] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" encompasses a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, including mammalian host cells, is typical because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Examples of mammalian host cells for expressing the recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr-CH0 cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, HKB11 cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods, such as ultrafiltration, size exclusion chromatography, ion exchange chromatography and centrifugation.
Use of Partial Antibody Sequences to Express Intact Antibodies
[0088] Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. It is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962).
[0089] Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. For this reason, it is necessary to use the corresponding germline leader sequence for expression constructs. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons. The nucleotide sequences of heavy and light chain transcripts are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and light chain sequences can differ from the natural sequences. For example: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and restriction sites are engineered upstream or downstream of the translation initiation sites. For both the heavy and light chain variable regions, the optimized coding, and corresponding non-coding, strand sequences are broken down into 30-50 nucleotide sections at approximately the midpoint of the corresponding non-coding oligonucleotide. For each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150-400 nucleotides.
[0090] Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It can also be desirable to include an overlapping fragment of the heavy or light chain constant region in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs. The reconstructed heavy and light chain variable regions are then combined with cloned promoter, translation initiation, constant region, 3' untranslated, polyadenylation, and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains. In another aspect, the structural features of a human anti-ATβH antibody are used to create structurally related human anti-ATβH antibodies that retain the function of binding to ATP. For example, one or more CDRs of the specifically identified heavy and light chain regions of the monoclonal antibodies can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, human anti-ATβH antibodies.
Pharmaceutical Compositions
[0091] Also provided are pharmaceutical compositions comprising therapeutically effective amounts of anti-ATβH monoclonal antibody and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a substance that can be added to the active ingredient to help formulate or stabilize the preparation and causes no significant adverse toxicological effects to the patient. Examples of such carriers are well known to those skilled in the art and include water, sugars such as maltose or sucrose, albumin, salts such as sodium chloride, etc. Other carriers are described for example in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will contain a therapeutically effective amount of at least one monoclonal antibody.
[0092] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. The composition is in some embodiments formulated for parenteral injection. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, the composition of the carrier includes isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride.
[0093] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Pharmaceutical Uses
[0094] The monoclonal antibody can be used for therapeutic purposes for treating genetic and acquired deficiencies or defects in coagulation. For example, the monoclonal antibodies in the embodiments described above can be used to block the interaction of ATβH with its substrate, which can include Factor Xa or Factor IIa. The monoclonal antibodies have therapeutic use in the treatment of disorders of hemostasis such as thrombocytopenia, platelet disorders and bleeding disorders (e.g., hemophilia A, hemophilia B and hemophilia C). Such disorders can be treated by administering a therapeutically effective amount of the anti-ATβH monoclonal antibody to a patient in need thereof. The monoclonal antibodies also have therapeutic use in the treatment of uncontrolled bleeds in indications such as trauma and hemorrhagic stroke. Thus, also provided is a method for shortening the bleeding time comprising administering a therapeutically effective amount of an anti-ATβH monoclonal antibody to a patient in need thereof.
[0095] In another embodiment, the anti-ATβH antibody can be useful as an antidote for AT treated patients, including for example wherein AT is used for the treatment of sepsis or bleeding disorder.
[0096] The antibodies can be used as monotherapy or in combination with other therapies to address a hemostatic disorder. For example, co-administration of one or more antibodies with a clotting factor such as Factor VIIa, Factor VIII or Factor IX is believed useful for treating hemophilia. In at least some embodiments, a method for treating genetic and acquired deficiencies or defects in coagulation comprises administering: (a) a first amount of a monoclonal antibody that binds to human tissue factor pathway inhibitor; and (b) a second amount of Factor VIII or Factor IX, wherein said first and second amounts together are effective for treating said deficiencies or defects. In at least some embodiments, a method for treating genetic and acquired deficiencies or defects in coagulation comprises administering: (a) a first amount of a monoclonal antibody that binds to human tissue factor pathway inhibitor; and (b) a second amount of factor VIII or Factor IX, wherein said first and second amounts together are effective for treating said deficiencies or defects, and further wherein Factor VII is not co-administered. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of the combination of a monoclonal antibody and Factor VIII or Factor IX, wherein the composition does not contain Factor VII. "Factor VII" includes Factor VII and Factor VIIa. These combination therapies are likely to reduce the necessary infusion frequency of the clotting factor. By co-administration or combination therapy is meant administration of the two therapeutic drugs each formulated separately or formulated together in one composition, and, when formulated separately, administered either at approximately the same time or at different times, but over the same therapeutic period.
[0097] In some embodiments, one or more antibodies described herein can be used in combination to address a hemostatic disorder. For example, co-administration of two or more of the antibodies described herein is believed useful for treating hemophilia or other hemostatic disorder.
[0098] The pharmaceutical compositions can be parenterally administered to subjects suffering from hemophilia A or B at a dosage and frequency that can vary with the severity of the bleeding episode or, in the case of prophylactic therapy, can vary with the severity of the patient's clotting deficiency.
[0099] The compositions can be administered to patients in need as a bolus or by continuous infusion. For example, a bolus administration of an antibody as a Fab fragment can be in an amount from about 0.0025 to about 100 mg/kg body weight, about 0.025 to about 0.25 mg/kg, about 0.010 to about 0.10 mg/kg or about 0.10 to about 0.50 mg/kg. For continuous infusion, an inventive antibody present as an Fab fragment can be administered at about 0.001 to about 100 mg/kg body weight/minute, about 0.0125 to about 1.25 mg/kg/min, about 0.010 to about 0.75 mg/kg/min, about 0.010 to about 1.0 mg/kg/min, or about 0.10 to about 0.50 mg/kg/min for a period of about 1-24 hours, about 1-12 hours, about 2-12 hours, about 6-12 hours, about 2-8 hours, or about 1-2 hours. For administration of an inventive antibody present as a full-length antibody (with full constant regions), dosage amounts can be about 1-10 mg/kg body weight, about 2-8 mg/kg, or about 5-6 mg/kg. Such full-length antibodies would typically be administered by infusion extending for a period of thirty minutes to three hours. The frequency of the administration would depend upon the severity of the condition. Frequency could range from three times per week to once every two weeks to six months.
[0100] Additionally, the compositions can be administered to patients via subcutaneous injection. For example, a dose of about 10 to about 100 mg anti-ATβH antibody can be administered to patients via subcutaneous injection weekly, biweekly or monthly. As used herein, "therapeutically effective amount" means an amount of an anti-ATβH monoclonal antibody or of a combination of such antibody and Factor VIII or Factor IX that is needed to effectively increase the clotting time in vivo or otherwise cause a measurable benefit in vivo to a patient in need thereof. The precise amount will depend upon numerous factors, including the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one skilled in the art.
[0101] Aspects of the present disclosure may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
Example 1
Human and Rabbit ATα and ATβ Purification
[0102] ATα and ATβ were purified from human and rabbit plasma by affinity chromatography on heparin-sepharose according to methods previously described (Carlson and Atencio 1982; Peterson and Blackburn 1985) at Enzyme Research laboratory (South Bend, Ind.). Briefly, the supernatant from a dextran sulphate/calcium chloride precipitation was applied to a heparin-sepharose affinity column (Pharmacia). ATα and ATβ were separated with a NaCl gradient: ATα and ATβ were eluted at 0.8 M and 1.3 M NaCl, respectively. Anion-exchange chromatography (HiTrap-Q, Pharmacia) was employed for further purification of ATβ. Purity and glycan profile of ATα and ATβ were evaluated by protein SDS-PAGE and LC-MS.
Example 2
Determination of the Number and Position of Glycans on ATα and ATβ by Mass-Spectrometry Analysis
[0103] Due to the distinct number of glycans, ATα and ATβ were differentiated based on their mass by Agilent 6520 LC-MS system which is equipped with duo-ESI (or nano ChipCube) source, MassHunter acquisition software and qualitative analysis software including Bioconfirm. Glycosylation sites were determined by a bottom-up method in which proteins are digested by trypsin and Arg-c followed by target MSMS to identify the glycosylated and nono-glycosylated peptide sequences. Data was collected in two experiments: Fragmentor voltage 175v and 430v.
Example 3
AT Antigen Biotinylation
[0104] Human and rabbit ATα and ATβ were labeled with biotins on the surface lysine residues by NHS-biotin. For lysine biotinylation, proteins were first desalted into PBS/Ca++ buffer (Life Technologies Corporation, Carlsbad, Calif.) to remove any amines that might be inhibitory to the biotinylation reaction. Concentrations of desalted proteins were determined by OD280 on the NanoDrop. Protein were then incubated for 1 hour at room temperature (RT) with Sulfo-NHS-Biotin (Pierce Thermo Scientific, Rockford, Ill.) at the 1:5 and/or 1:3 molar ratio of AT:NHS-biotin (i.e. biotin in excess). Free biotin was removed by overnight dialysis into PBS/Ca++ buffer. The amount of biotin in the biotinylated proteins was quantified using Biotin Quantitation Kit (Pierce Thermo Scientific, Rockford, Ill.). Biotinylated ATα and ATβ were analyzed by SDS-PAGE, and biotinylation was confirmed by Western blot analysis using streptavidin-HRP (Pierce Thermo Scientific, Rockford, Ill.) as probe. The functional activities of biotinylated AT were evaluated by FXa inhibition assay. By comparison of the biotinylated ATα and ATβ with unbiotinylated ATα and ATβ, only slight reductions in AT inhibition activity were observed after biotinylation, indicating the biotinylated ATβ and ATα prepared in this way would be representative and could be used in panning for ATβH binders as selective anti-coagulant blockers.
Example 4
Human Monoclonal Antibody Discovery by Phage Display and Panning
[0105] A four-arm panning strategy was designed to discover Fabs specifically against ATβH from a human Fab library (Dyax Fab310). The library was first depleted with biotinylated heparin/Fondaparinux-bound ATα and biotinylated ATα and then was panned against heparin/Fondaparinux-bound ATβ and biotinylated ATβ on streptavidin beads, respectively. For each round of panning, the heparin-bound ATα (ATαH) was included in the binding buffer as a competitor. To keep hATβ in active conformation (heparin bound form), heparin was added to the wash buffer in all three rounds of panning. After panning, pooled clones were screened for hATβ and hATβH specific binding and counter-screened for hATα by ELISA. These clones were also examined for differential binding to rabbit ATβ over rabbit ATα. Clones showing differential binding to both hATβH and rATβH over hATα and rATα were further subject to FXa--deinhibition assay with hATβ spike-in. Positive hits (Fabs) were reformatted into IgG1, expressed in HEK293 cells and purified by protein-A column. These purified IgG1s were extensively tested in AT-depleted human plasma and hemA patient plasma for TGA assay (Thrombin Generation Assay) and dPT (diluted Prothrombin Time) assay to measure the clotting time.
Example 5
ELISA (Enzyme-Linked Immunosorbent Assay)
[0106] 2 ug/ml biotinlyated AT antigens in PBS were coated on Streptavidin Microplates (Greiner, 781997) with or without heparin (50 ug/ml, heparin-Natrium-5000, Apotheke, Fa. Ratiopharm). After overnight antigen coating at 4°, plates were washed with PBST+/- heparin and blocked with 5% milk in PBST+/- heparin at 37° for one hour. After removal of blocking buffer, 20 ug/ml Fab or 4 ug/ml IgG in blocking buffer (5% milk in PBST+/- heparin) was then added to the plates and plates were incubated at room temperature for 1 hour. Plates were then washed three times. Anti-human IgG POD (Sigma, A0170) in blocking buffer was added to plates and plates were incubated at room temperature for 30 minutes. Amplex red (In vitrogen, Cat#A22170) was used for detection at 1:1000 together with H2O2. After 30 min incubation, plates were read at Ex535, Em 590 in a fluorescent plate reader.
Example 6
FXa De-Inhibition Assay--AT with Heparin
[0107] Heparin was incubated with ATβ or ATα to form stable ATH complexes. Antibody was then added to the ATβH or ATαH complexes. In the meantime, 10 μl of 200 ng/ml FXa (HTI) and 20 μl of 50 μg/ml Fluophen FXa fluorogenic substrate (Hyphen Biomed) were mixed in a separate plate. The antibody-ATH mixture was added to the FXa/substrate solution quickly and fluorescent kinetic measurement was started immediately at Ex360 nm and Em465 nm. All necessary dilutions is made in 100 mM NaCl, 20 mM Tris, 2.5 mM CaCl2, 0,1% BSA, 0,1% PEG8000.
Example 7
Thrombin Generation Assay (TGA) in FVIII Deficiency Human Plasma
[0108] A 1:2 serial dilution of ATβH antibody was made in HemA human plasma starting from 1 uM of final concentration to 0.015 uM. Heparin was added in each antibody solution at a final concentration of 50 nM. An 80 ul of the antibody-heparin-plasma mixture was then added to each well containing 20 ul of reconstitute PPP reagent or calibrator in a 96 well TGA plate. The plate was placed in the TGA instrument and the machine automatically dispensed 20 ul of FluCa (Fluo substrate+CaCl2) into each well. The reaction was allowed to run 60 min. Plasma alone was used as the negative control.
Example 8
Thrombin Generation Assay (TGA) in AT-Depleted Human Plasma with Spiked-in ATα and ATβ Respectively
[0109] Antibodies were added to human AT-deficient plasma spiked with 15 nm of ATα or ATβ. Heparin was then pipetted into each reaction at a final concentration of 50 nM. 80 ul of plasma samples containing ATH-specific antibody, heparin and ATα or ATβ were added into wells of a 96 well TGA plate with 20 ul of PPP reagent or calibrator. Plates were placed in the TGA instrument, and then 20 ul of FluCa (Fluo substrate+CaCl2) was dispensed into each well. Reactions were allowed to continue for 60 min.
Example 9
Diluted Prothrombin Time Assay (dPT) in Human hemA Plasma and AT Deficient Plasma
[0110] A serial dilution of anti-ATβH hmAbs was made in hemA plasma starting at 250 nM with 0.1 U/mL of heparin. The mixture of antibody, plasma and heparin was incubated at room temperature for 20-30 min. Then 50 uL of this mixture was added to a 50 uL of diluted Innovin (1/2000) (Dade Behring), incubated for 4 min at 37° C., followed by adding 50 uL of 25 mM CaCl2 (HemSil). dPT test program was set on ACL Top coagulometer with acquisition time of 360 seconds. For dTP in AT deficient plasma, AT-DP was spiked in with either AT or AT at a final concentration of 0.2 uM with 0.1 U/ml of heparin. Anti-ATβH mAbs was added to AT-DP/heparin/AT or AT-DP/heparin/AT mixtures at a final concentration of 0.25 uM and incubated at room temperature for 20-30 min. For each reaction, a 50 uL of plasma/antibody/heparin mixture was added to 50 uL of diluted Innovin (1/4000), incubate 4 min at 37° C., followed by adding 50 uL of 25 mM CaCl2 (HemSil) as above.
Example 10
Antibody Purification
[0111] Pre-washed protein A agarose beads were incubated with antibody in binding buffer (volume ratio: 1:1) with rotation overnight at 4° C. Beads were then packed into a column and washed with 1×PBS until O.D.280<0.05. Residual solution was drained. Antibodies were eluted with elution buffer and collected into tubes containing neutralizing buffer. Eluted fractions were dialyzed against 1×PBS overnight at 4° C. with at least twice buffer changes. IgG concentration was measured at 280 nm by nanodrop. The antibody purity was examined by either ELISA, SDS-PAGE or SSC.
Example 11
Antibody Binding Affinity Study by Biacore
[0112] Antibody affinity measurement was performed on a Biacore T100 or T200 processing unit. Anti-human Fc antibody or streptavidin was immobilized on a CM5 chip. hATβH or biotinylated hmAb antibodies were injected and captured on the chip. ATβ or ATα at different concentration with/without heparin were injected. Only AT and ATH bound to the antibodies generate binding constants. The binding results were reported as Equilibrium Dissociation Constants (KD) in nanoMoles. When AT/heparin complex was analyzed, heparin at 1 uM is included in the running buffer.
[0113] The foregoing disclosure and examples are not intended to narrow the scope of the claims in any way. It should be understood that various modifications and changes can be made, and equivalents can be substituted, to the foregoing embodiments and teachings without departing from the true spirit and scope of the claims appended hereto. The specification and examples are, accordingly, to be regarded in an illustrative sense rather than in a restrictive sense. Furthermore, the disclosure of all articles, books, patent applications, patents, and other material referred to herein are incorporated herein by reference in their entireties.
Sequence CWU
1
1
501215PRTArtificial SequenceTPP2009, Light Chain Variable Region 1Ala Gln
Ser Val Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly 1 5
10 15 Gln Thr Val Arg Ile Thr Cys
Gln Gly Asp Ser Leu Arg Ser Tyr Tyr 20 25
30 Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Val Leu Val Ile 35 40 45
Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60 Ser Ser Ser
Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala 65
70 75 80 Glu Asp Glu Ala Asp Tyr Tyr
Cys Asn Ser Arg Asp Ser Ser Gly Asn 85
90 95 His Leu 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 Gly Ser Pro Val Lys Ala 145 150
155 160 Gly Val Glu Thr Thr Lys 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 Ala Glu Cys Ser
210 215 2125PRTArtificial SequenceTPP2009, Heavy
Chain Variable Region 2Glu 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 Ala Tyr
20 25 30 Arg Met Gly
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Tyr Ser Ser Gly Gly
Arg Thr Arg 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 Glu Lys
Ala Ser Asp Leu Ser Gly Ser Phe Ser Glu Ala Leu 100
105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125
3216PRTArtificial SequenceTPP2015, Light Chain Variable Region 3Ala Gln
Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5
10 15 Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser 20 25
30 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg 35 40 45
Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
50 55 60 Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65
70 75 80 Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser 85
90 95 Ser Arg Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Arg 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 Gln Gly Leu Ser Ser Pro Val Thr 195
200 205 Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 4125PRTArtificial SequenceTPP2015,
Heavy Chain Variable Region 4Glu 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 Lys Tyr
20 25 30 Lys Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Gly Pro Ser Gly
Gly Lys Thr Met 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 Glu
Lys Ala Ser Asp Leu Ser Gly Thr Tyr Ser Glu Ala Leu 100
105 110 Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125
5216PRTArtificial SequenceTPP2016, Light Chain Variable Region 5Ala Gln
Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 1 5
10 15 Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Asn Ile Asn 20 25
30 Arg Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg
Ala Pro Arg Leu 35 40 45
Leu Ile His Thr Ala Ser Thr Arg Ala Pro Gly Val Pro Val Arg Ile
50 55 60 Thr Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65
70 75 80 Glu Pro Glu Asp Phe Ala Val
Tyr Phe Cys Gln Gln Tyr Ala Ser Pro 85
90 95 Pro Arg 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 Gln Gly Leu Ser Ser Pro Val Thr 195
200 205 Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 6125PRTArtificial SequenceTPP2016,
Heavy Chain Variable Region 6Glu 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 Lys Tyr
20 25 30 Arg Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Gly Pro Ser Gly
Gly Lys Thr Thr 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 Glu
Lys Thr Ser Asp Leu Ser Gly Ser Tyr Ser Glu Ala Leu 100
105 110 Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125
7218PRTArtificial SequenceTPP2019, Light Chain Variable Region 7Ala Gln
Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 1 5
10 15 Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Arg Val Ser 20 25
30 Ser Ser Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg 35 40 45
Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
50 55 60 Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65
70 75 80 Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Asp Ser 85
90 95 Thr Pro Pro Leu Thr Phe Gly Gly 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 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 8126PRTArtificial
SequenceTPP2019, Heavy Chain Variable Region 8Glu 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 Arg Tyr 20 25
30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser
Arg Ile Ser Pro Ser Gly Gly Lys Thr His 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 Leu Ser Gln Thr Gly Tyr Tyr Pro His Tyr His Tyr Tyr Gly
100 105 110 Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 125 9217PRTArtificial SequenceConsensus
Sequence; Light Chain anti-ATbetaH mAbs 9Ala Gln Asp Ile Gln Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser 1 5
10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Asn Ile Ser 20 25
30 Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg
Leu 35 40 45 Leu
Ile His Thr Ala Ser Thr Arg Ala Pro Gly Ile Pro Val Arg Ile 50
55 60 Ser Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70
75 80 Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln
Gln Tyr Asp Ser Ser 85 90
95 Pro Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105 110 Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115
120 125 Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135
140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly 145 150 155
160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175 Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180
185 190 Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val 195 200
205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 10213PRTArtificial SequenceConsensus Sequence; Heavy
Chain anti-ATbetaH mAbs 10Glu 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 Lys
Tyr 20 25 30 Arg
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Gly Pro Ser
Gly Gly Lys Thr Thr 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
Glu Lys Thr Ser Asp Leu Ser Gly Ser Tyr Ser Glu Ala Leu 100
105 110 Asp 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 210 11645DNAArtificial
SequenceTPP2009 Light Chain_V region 11gcacagagcg tcttgactca ggaccctgct
gtgtctgtgg ccttgggaca gacagtcagg 60atcacatgcc aaggagacag cctcagaagc
tattatgcaa gctggtacca gcagaagcca 120ggacaggccc ctgtacttgt catctatggt
aaaaacaacc ggccctcagg gatcccagac 180cgattctctg gctccagctc aggaaacaca
gcttccttga ccatcactgg ggctcaggcg 240gaagatgagg ctgactatta ctgtaactcc
cgggacagca gtggtaacca tctggtattc 300ggcggaggga ccaagctgac cgtcctaggt
cagcccaagg ctgccccctc ggtcactctg 360ttcccgccct cctctgagga gcttcaagcc
aacaaggcca cactagtgtg tctgatcagt 420gacttctacc cgggagctgt gacagtggcc
tggaaggcag atggcagccc cgtcaaggcg 480ggagtggaga ccaccaaacc ctccaaacag
agcaacaaca agtacgcggc cagcagctac 540ctgagcctga cgcccgagca gtggaagtcc
cacagaagct acagctgcca ggtcacgcat 600gaagggagca ccgtggagaa gacagtggcc
cctgcagaat gctct 645121362DNAArtificial
SequenceTPP2009 Heavy chain 12gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gcttaccgta
tgggttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctcgt atctattctt
ctggtggccg tactcgttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac acggccgtgt
attactgtgc gagagagaaa 300gcgtcggatc tatcggggag tttttctgag gcccttgact
actggggcca gggaaccctg 360gtcaccgtct caagcgcctc caccaagggc ccatcggtct
tcccgctagc acccagcagc 420aagagcacca gcggcggaac agccgccctg ggctgcctgg
tgaaagacta cttccccgag 480cccgtgaccg tgtcctggaa ctctggcgcc ctgaccagcg
gagtgcatac cttccccgcc 540gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg
tgacagtgcc cagcagcagc 600ctgggaaccc agacctacat ctgcaacgtg aaccacaagc
ccagcaacac caaggtggac 660aagaaggtgg aacccaagag ctgcgacaag acccacacct
gtcccccctg ccctgcccct 720gaactgctgg gcggacccag cgtgttcctg ttccccccaa
agcccaagga caccctgatg 780atcagccgga cccccgaagt gacctgcgtg gtggtggacg
tgtcccacga ggacccagaa 840gtgaagttta attggtacgt ggacggcgtg gaagtgcata
acgccaagac caagcccaga 900gaggaacagt acaacagcac ctaccgggtg gtgtccgtgc
tgaccgtgct gcaccaggac 960tggctgaacg gcaaagagta caagtgcaag gtctccaaca
aggccctgcc tgcccccatc 1020gagaaaacca tcagcaaggc caagggccag ccccgcgagc
ctcaggtgta cacactgccc 1080cccagccggg atgagctgac caagaaccag gtgtccctga
cctgtctggt gaaaggcttc 1140taccccagcg atatcgccgt ggaatgggag agcaacggcc
agcccgagaa caattacaag 1200accacccccc ctgtgctgga cagcgacggc tcattcttcc
tgtactccaa gctgaccgtg 1260gacaagagcc ggtggcagca gggcaacgtg ttcagctgca
gcgtgatgca cgaggccctg 1320cacaatcact acacccagaa gtccctgagc ctgagccccg
gc 136213642DNAArtificial SequenceTPP2015 Light
Chain_V region 13gcacaagaca tccagatgac ccagtctcca ggcaccctgt ctttgtctcc
aggggaaaga 60gccaccctct cctgcagggc cagtcagagt gttagcagca gctacttagc
ctggtaccag 120cagaaacctg gccaggctcc caggctcctc atctatggtg catccagcag
ggccactggc 180atcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac
catcagcaga 240cggagcctga agattttgca gtgtattact gtcagcagta tggtagctca
acgttcggcc 300aagggaccaa ggtggaaatc agacgaactg tggctgcaat ctgtcttcat
cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt
642141362DNAArtificial SequenceTPP2015 Heavy Chain
14gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct aagtacaaga tggattgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctcgt atcggtcctt ctggtggcaa gactatgtat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac acggccgtgt attactgtgc gagagagaaa
300gcgtcggatc tatcggggac ttattctgag gcccttgact actggggcca gggaaccctg
360gtcaccgtct caagcgcctc caccaagggc ccatcggtct tcccgctagc acccagcagc
420aagagcacca gcggcggaac agccgccctg ggctgcctgg tgaaagacta cttccccgag
480cccgtgaccg tgtcctggaa ctctggcgcc ctgaccagcg gagtgcatac cttccccgcc
540gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgacagtgcc cagcagcagc
600ctgggaaccc agacctacat ctgcaacgtg aaccacaagc ccagcaacac caaggtggac
660aagaaggtgg aacccaagag ctgcgacaag acccacacct gtcccccctg ccctgcccct
720gaactgctgg gcggacccag cgtgttcctg ttccccccaa agcccaagga caccctgatg
780atcagccgga cccccgaagt gacctgcgtg gtggtggacg tgtcccacga ggacccagaa
840gtgaagttta attggtacgt ggacggcgtg gaagtgcata acgccaagac caagcccaga
900gaggaacagt acaacagcac ctaccgggtg gtgtccgtgc tgaccgtgct gcaccaggac
960tggctgaacg gcaaagagta caagtgcaag gtctccaaca aggccctgcc tgcccccatc
1020gagaaaacca tcagcaaggc caagggccag ccccgcgagc ctcaggtgta cacactgccc
1080cccagccggg atgagctgac caagaaccag gtgtccctga cctgtctggt gaaaggcttc
1140taccccagcg atatcgccgt ggaatgggag agcaacggcc agcccgagaa caattacaag
1200accacccccc ctgtgctgga cagcgacggc tcattcttcc tgtactccaa gctgaccgtg
1260gacaagagcc ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg
1320cacaatcact acacccagaa gtccctgagc ctgagccccg gc
136215648DNAArtificial SequenceTPP2016 Light Chain_V region 15gcacaagaca
tccagatgac ccagtctcca gccaccctgt ctgtgtctcc aggggaaaga 60gccaccctct
cctgcagggc cagtcagaat attaatagaa acttggcctg gtaccagcag 120aagcctggcc
gggctcccag actcctcatc cataccgcat ccactagggc ccctggtgtc 180ccagtcagga
tcactggcag tgggtctgga acagagttca ctctcaccat cagcagcctg 240gaacctgaag
attttgcagt gtatttctgt cagcagtatg ctagcccacc tcggacgttc 300ggccaaggga
ccaaggtgga aatcaagcga actgtggctg 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 600cagggcctga
gctcgcccgt cacaaagagc ttcaacaggg gagagtgt
648161362DNAArtificial SequenceTPP2016 Heavy Chain 16gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg
cttccggatt cactttctct aagtaccgta tggattgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttctcgt atcggtcctt ctggtggcaa gactacttat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga
acagcttaag ggctgaggac acggccgtgt attactgtgc gagagagaaa 300acgtcggatc
tatcggggag ttattctgag gcccttgact actggggcca gggaaccctg 360gtcaccgtct
caagcgcctc caccaagggc ccatcggtct tcccgctagc acccagcagc 420aagagcacca
gcggcggaac agccgccctg ggctgcctgg tgaaagacta cttccccgag 480cccgtgaccg
tgtcctggaa ctctggcgcc ctgaccagcg gagtgcatac cttccccgcc 540gtgctgcaga
gcagcggcct gtacagcctg agcagcgtgg tgacagtgcc cagcagcagc 600ctgggaaccc
agacctacat ctgcaacgtg aaccacaagc ccagcaacac caaggtggac 660aagaaggtgg
aacccaagag ctgcgacaag acccacacct gtcccccctg ccctgcccct 720gaactgctgg
gcggacccag cgtgttcctg ttccccccaa agcccaagga caccctgatg 780atcagccgga
cccccgaagt gacctgcgtg gtggtggacg tgtcccacga ggacccagaa 840gtgaagttta
attggtacgt ggacggcgtg gaagtgcata acgccaagac caagcccaga 900gaggaacagt
acaacagcac ctaccgggtg gtgtccgtgc tgaccgtgct gcaccaggac 960tggctgaacg
gcaaagagta caagtgcaag gtctccaaca aggccctgcc tgcccccatc 1020gagaaaacca
tcagcaaggc caagggccag ccccgcgagc ctcaggtgta cacactgccc 1080cccagccggg
atgagctgac caagaaccag gtgtccctga cctgtctggt gaaaggcttc 1140taccccagcg
atatcgccgt ggaatgggag agcaacggcc agcccgagaa caattacaag 1200accacccccc
ctgtgctgga cagcgacggc tcattcttcc tgtactccaa gctgaccgtg 1260gacaagagcc
ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg 1320cacaatcact
acacccagaa gtccctgagc ctgagccccg gc
136217654DNAArtificial SequenceTPP2019 Light Chain_V region 17gcacaagaca
tccagatgac ccagtctcca gccaccctgt ctttgtctcc aggggaaaga 60gccaccctct
cctgcagggc cagtcagcgt gttagcagca gctacttaac ctggtaccag 120cagaaacctg
gccaggctcc caggctcctc atctatggtg catccagcag ggccactggc 180atcccagaca
ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240ctggagcctg
aagattttgc agtttattac tgtcagcagt atgatagtac gcctccgctc 300accttcggcg
gagggaccaa ggtggagatc aaacgaactg 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
654181365DNAArtificial SequenceTPP2019 Heavy chain 18gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg
cttccggatt cactttctct cgttacgcta tgtattgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttctcgt atctctcctt ctggtggcaa gactcattat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga
acagcttaag ggctgaggac acggccgtgt attactgtgc gagactgtct 300caaactggtt
attaccctca ctaccactac tacggtatgg acgtctgggg ccaagggacc 360acggtcaccg
tctcaagcgc ctccaccaag ggcccatcgg tcttcccgct agcacccagc 420agcaagagca
ccagcggcgg aacagccgcc ctgggctgcc tggtgaaaga ctacttcccc 480gagcccgtga
ccgtgtcctg gaactctggc gccctgacca gcggagtgca taccttcccc 540gccgtgctgc
agagcagcgg cctgtacagc ctgagcagcg tggtgacagt gcccagcagc 600agcctgggaa
cccagaccta catctgcaac gtgaaccaca agcccagcaa caccaaggtg 660gacaagaagg
tggaacccaa gagctgcgac aagacccaca cctgtccccc ctgccctgcc 720cctgaactgc
tgggcggacc cagcgtgttc ctgttccccc caaagcccaa ggacaccctg 780atgatcagcc
ggacccccga agtgacctgc gtggtggtgg acgtgtccca cgaggaccca 840gaagtgaagt
ttaattggta cgtggacggc gtggaagtgc ataacgccaa gaccaagccc 900agagaggaac
agtacaacag cacctaccgg gtggtgtccg tgctgaccgt gctgcaccag 960gactggctga
acggcaaaga gtacaagtgc aaggtctcca acaaggccct gcctgccccc 1020atcgagaaaa
ccatcagcaa ggccaagggc cagccccgcg agcctcaggt gtacacactg 1080ccccccagcc
gggatgagct gaccaagaac caggtgtccc tgacctgtct ggtgaaaggc 1140ttctacccca
gcgatatcgc cgtggaatgg gagagcaacg gccagcccga gaacaattac 1200aagaccaccc
cccctgtgct ggacagcgac ggctcattct tcctgtactc caagctgacc 1260gtggacaaga
gccggtggca gcagggcaac gtgttcagct gcagcgtgat gcacgaggcc 1320ctgcacaatc
actacaccca gaagtccctg agcctgagcc ccggc
13651911PRTArtificial SequenceTPP2009 LCDR1 19Gln Gly Asp Ser Leu Arg Ser
Tyr Tyr Ala Ser 1 5 10
2012PRTArtificial SequenceTPP2015 LCDR1 20Arg Ala Ser Gln Ser Val Ser Ser
Ser Tyr Leu Ala 1 5 10
2111PRTArtificial SequenceTPP2016 LCDR1 21Arg Ala Ser Gln Asn Ile Asn Arg
Asn Leu Ala 1 5 10 2212PRTArtificial
SequenceTPP2019 LCDR1 22Arg Ala Ser Gln Arg Val Ser Ser Ser Tyr Leu Thr 1
5 10 237PRTArtificial
SequenceTPP2009 LCDR2 23Gly Lys Asn Asn Arg Pro Ser 1 5
247PRTArtificial SequenceTPP2015 LCDR2 24Gly Ala Ser Ser Arg Ala
Thr 1 5 257PRTArtificial SequenceTPP2016 LCDR2
25Thr Ala Ser Thr Arg Ala Pro 1 5
267PRTArtificial SequenceTPP2019 LCDR2 26Gly Ala Ser Ser Arg Ala Thr 1
5 2711PRTArtificial SequenceTPP2009 LCDR3 27Asn Ser
Arg Asp Ser Ser Gly Asn His Leu Val 1 5
10 288PRTArtificial SequenceTPP2015 LCDR3 28Gln Gln Tyr Gly Ser Ser
Arg Thr 1 5 299PRTArtificial SequenceTPP2016
LCDR3 29Gln Gln Tyr Ala Ser Pro Pro Arg Thr 1 5
3010PRTArtificial SequenceTPP2019 LCDR3 30Gln Gln Tyr Asp Ser Thr
Pro Pro Leu Thr 1 5 10 315PRTArtificial
SequenceTPP2009 HCDR1 31Ala Tyr Arg Met Gly 1 5
325PRTArtificial SequenceTPP2015 HCDR1 32Lys Tyr Lys Met Asp 1
5 335PRTArtificial SequenceTPP2016 HCDR1 33Lys Tyr Arg Met Asp 1
5 345PRTArtificial SequenceTPP2019 HCDR1 34Arg Tyr Ala Met
Tyr 1 5 3517PRTArtificial SequenceTPP2009 HCDR2 35Arg Ile
Tyr Ser Ser Gly Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 3617PRTArtificial
SequenceTPP2015 HCDR2 36Arg Ile Gly Pro Ser Gly Gly Lys Thr Met Tyr Ala
Asp Ser Val Lys 1 5 10
15 Gly 3717PRTArtificial SequenceTPP2016 HCDR2 37Arg Ile Gly Pro Ser
Gly Gly Lys Thr Thr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 3817PRTArtificial SequenceTPP2019
HCDR2 38Arg Ile Ser Pro Ser Gly Gly Lys Thr His Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
3918PRTArtificial SequenceTPP2009 HCDR3 39Ala Arg Glu Lys Ala Ser Asp Leu
Ser Gly Ser Phe Ser Glu Ala Leu 1 5 10
15 Asp Tyr 4018PRTArtificial SequenceTPP2015 HCDR3
40Ala Arg Glu Lys Ala Ser Asp Leu Ser Gly Thr Tyr Ser Glu Ala Leu 1
5 10 15 Asp Tyr
4118PRTArtificial SequenceTPP2016 HCDR3 41Ala Arg Glu Lys Thr Ser Asp Leu
Ser Gly Ser Tyr Ser Glu Ala Leu 1 5 10
15 Asp Tyr 4219PRTArtificial SequenceTPP2019 HCDR3
42Ala Arg Leu Ser Gln Thr Gly Tyr Tyr Pro His Tyr His Tyr Tyr Gly 1
5 10 15 Met Asp Val
43215PRTArtificial SequenceTPP2009, hIgG, Light_Chain 43Ala Gln Ser Val
Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly 1 5
10 15 Gln Thr Val Arg Ile Thr Cys Gln Gly
Asp Ser Leu Arg Ser Tyr Tyr 20 25
30 Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu
Val Ile 35 40 45
Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly 50
55 60 Ser Ser Ser Gly Asn
Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala 65 70
75 80 Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser
Arg Asp Ser Ser Gly Asn 85 90
95 His Leu 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 Gly
Ser Pro Val Lys Ala 145 150 155
160 Gly Val Glu Thr Thr Lys 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 Ala Glu Cys Ser 210
215 44454PRTArtificial SequenceTPP2009, hIgG, Heavy_Chain 44Glu 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 Ala Tyr 20 25
30 Arg Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45
Ser Arg Ile Tyr Ser Ser Gly Gly Arg Thr Arg 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 Glu Lys Ala Ser Asp Leu Ser Gly Ser
Phe Ser Glu Ala Leu 100 105
110 Asp 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 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 Ser Arg Asp Glu Leu
Thr Lys 355 360 365
Asn Gln Val Ser Leu Thr 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
450 45216PRTArtificial SequenceTPP-2015, hIgG,
Light_Chain 45Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser 1 5 10 15 Pro
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
20 25 30 Ser Ser Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 35
40 45 Leu Leu Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg 50 55
60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg 65 70 75
80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
85 90 95 Ser Arg Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Arg 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 Gln Gly Leu Ser Ser Pro Val
Thr 195 200 205 Lys
Ser Phe Asn Arg Gly Glu Cys 210 215
46453PRTArtificial SequenceTPP-2015, hIgG, Heavy_Chain 46Glu 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 Lys Tyr 20 25
30 Lys Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45
Ser Arg Ile Gly Pro Ser Gly Gly Lys Thr Met 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 Glu Lys Ala Ser Asp Leu Ser Gly Thr Tyr Ser Glu Ala
Leu 100 105 110 Asp
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 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 Ser Arg Asp Glu Leu Thr Lys
355 360 365 Asn Gln Val Ser
Leu Thr 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 Gly 450
47216PRTArtificial SequenceTPP-2016, hIgG, Light_Chain, Kappa
47Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 1
5 10 15 Pro Gly Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Asn 20
25 30 Arg Asn Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Arg Ala Pro Arg Leu 35 40
45 Leu Ile His Thr Ala Ser Thr Arg Ala Pro Gly Val Pro Val
Arg Ile 50 55 60
Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65
70 75 80 Glu Pro Glu Asp Phe
Ala Val Tyr Phe Cys Gln Gln Tyr Ala Ser Pro 85
90 95 Pro Arg 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 Gln Gly Leu Ser Ser Pro Val Thr 195
200 205 Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 48454PRTArtificial SequenceTPP-2016,
hIgG, Heavy_Chain 48Glu 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 Lys Tyr
20 25 30 Arg Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Gly Pro Ser Gly Gly Lys
Thr Thr 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 Glu Lys
Thr Ser Asp Leu Ser Gly Ser Tyr Ser Glu Ala Leu 100
105 110 Asp 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 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 Ser Arg Asp Glu Leu Thr Lys 355 360
365 Asn Gln Val Ser Leu Thr 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 450 49218PRTArtificial
SequenceTPP-2019, hIgG, Light_Chain, Kappa 49Ala Gln Asp Ile Gln Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser 1 5
10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Arg Val Ser 20 25
30 Ser Ser Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg 35 40 45 Leu
Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg 50
55 60 Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70
75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Asp Ser 85 90
95 Thr Pro Pro Leu Thr Phe Gly Gly 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 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 50455PRTArtificial SequenceTPP-2019, hIgG,
Heavy_Chain 50Glu 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 Arg Tyr
20 25 30 Ala Met Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Arg Ile Ser Pro Ser Gly Gly Lys
Thr His 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 Leu Ser
Gln Thr Gly Tyr Tyr Pro His Tyr His Tyr Tyr Gly 100
105 110 Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser 115 120
125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr 130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145
150 155 160 Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165
170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser 180 185
190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile 195 200 205 Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210
215 220 Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230
235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 245 250
255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270 Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275
280 285 Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295
300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 305 310 315
320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335 Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 340
345 350 Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr 355 360
365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser 370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385
390 395 400 Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405
410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe 420 425
430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 435 440 445
Ser Leu Ser Leu Ser Pro Gly 450 455
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