Methods of Generating Bioactive Peptide-bearing Antibodies and Compositions Comprising the Same

Abstract

In one aspect, the invention provides a method of making a bioactive peptide-bearing antibody, or fragment thereof, comprising (a) engrafting the amino acid sequence of at least one bioactive peptide of interest into (i) at least one of CDR-H1, CDR-H2 or CDR-H3 of a heavy chain variable region comprising one or more chicken framework regions and/or (ii) at least one of CDR-L1, CDR-L2 or CDR-L3 of the light chain variable region comprising one or more chicken framework regions, and (b) determining whether the antibody has substantially the same biological activity as the bioactive peptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Application Ser. No. 61/962,289, filed Mar. 15, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for generating bioactive peptide-bearing antibodies and fragments thereof, such as antibodies comprising bioactive peptides for inhibiting complement activation.

STATEMENT REGARDING SEQUENCE LISTING

[0003] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is MP_--1_--0164_US2_Sequence_Listing_--20140312_ST25. The text file is 198 KB, was created on Mar. 11, 2014; and is being submitted via EFS-Web with the filing of the specification.

BACKGROUND

[0004] The complement system provides an early acting mechanism to initiate, amplify and orchestrate the immune response to microbial infection and other acute insults (M. K. Liszewski and J. P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W. E. Paul, Raven Press, Ltd., New York) in humans and other vertebrates. While complement activation provides a valuable first-line defense against potential pathogens, the activities of complement that promote a protective immune response can also represent a potential threat to the host (K. R. Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994; B. P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, the C3 and C5 proteolytic products recruit and activate neutrophils. While indispensable for host defense, activated neutrophils are indiscriminate in their release of destructive enzymes and may cause organ damage. In addition, complement activation may cause the deposition of lytic complement components on nearby host cells as well as on microbial targets, resulting in host cell lysis.

[0005] The complement system has also been implicated in the pathogenesis of numerous acute and chronic disease states, including: myocardial infarction, stroke, acute respiratory distress syndrome, reperfusion injury, septic shock, capillary leakage following thermal burns, post cardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, age-related macular degeneration, paroxysmal nocturnal hemoglobinuria, and Alzheimer's disease. In almost all of these conditions, complement is not the cause but is one of several factors involved in pathogenesis. Nevertheless, complement activation may be a major pathological mechanism and represents an effective point for clinical control in many of these disease states.

[0006] The growing recognition of the importance of complement-mediated tissue injury in a variety of disease states underscores the need for effective complement inhibitory drugs. To date, Eculizumab (Soliris®), an antibody against C5, is the only complement-targeting drug that has been approved for human use. Yet, C5 is one of several effector molecules located "downstream" in the complement system, and blockade of C5 does not inhibit activation of the complement system. Therefore, an inhibitor of the initiation steps of complement activation would have many significant advantages over a "downstream" complement inhibitor.

[0007] Three distinct pathways of complement activation have been defined. The classical pathway is activated upon binding of particular antibody isotypes to a pathogen or host antigen. The lectin pathway is activated upon binding of pattern recognition lectins, such as mannan-binding lectin (MBL), CL-11, or ficolins L, M, or H to complex microbial or host macromolecules such as polysaccharides. Finally, the alternative pathway serves to amplify the signals generated by the classical and lectin pathways. A family of serine proteases is integral to the initial activation steps of all three pathways. C1r and C1s form the enzymatic components of the C1 complex that is assembled by complement-activating antibodies. In addition, there are three MBL-associated serine proteases (MASPs) that initiate and/or propagate the protease cascades of the lectin and alternative pathways (Yongqing et al., Biochim. Biophys. Acta 1824:253, 2012).

[0008] MASP-1, MASP-2 and MASP-3 share identical domain organizations with those of C1r and C1s, the enzymatic components of the C1 complex (Sim, R. B., et al., Biochem. Soc. Trans. 28:545, 2000). These domains include an N-terminal C1r/C1s/sea urchin VEGF/bone morphogenic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complement control protein domains, and a serine protease domain. As in the C1 proteases, activation of the MASP proteases occurs through cleavage of an Arg-Ile bond adjacent to the serine protease domain, which splits the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain.

[0009] The generation of specific peptide inhibitors of MASP-1 and MASP-2, termed SGMI-1 and SGMI-2, respectively, is described in Heja et al., J Biol Chem 287:20290 (2012) and Heja et al., PNAS 109:10498 (2012), each of which is hereby incorporated herein by reference. SGMI-1 and SGMI-2 are each 36 amino acid peptides which were selected from a phage library of variants of the Schistocerca gregaria protease inhibitor 2 in which six of the eight positions of the protease binding loop were fully randomized. Mechanistically, both SGMI-1 and SGMI-2 block the lectin pathway of complement activation without affecting the classical or alternative pathways (Heja et al., 2012. Proc. Natl. Acad. Sci. 109:10498). However, peptides such as SGMI-1 and SGMI-2 have limited potential for use in therapeutic applications because of the short half-life of peptides in serum.

SUMMARY

[0010] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0011] In one aspect, the invention provides a method of making a bioactive peptide-bearing antibody, or fragment thereof, comprising (a) engrafting the amino acid sequence of at least one bioactive peptide of interest into (i) at least one of CDR-H1, CDR-H2 or CDR-H3 of a heavy chain variable region comprising one or more chicken framework regions and/or (ii) at least one of CDR-L1, CDR-L2 or CDR-L3 of a light chain variable region comprising one or more chicken framework regions, and (b) determining whether the antibody has substantially the same biological activity as the bioactive peptide.

[0012] In another aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, comprising one or more bioactive peptide amino acid sequence(s), wherein at least one bioactive peptide amino acid sequence is engrafted into at least one of: (i) a light chain variable region comprising one or more chicken framework regions and/or (ii) a heavy chain variable region comprising one or more chicken framework regions. In some embodiments, a bioactive peptide amino acid sequence is engrafted into at least one of CDR-H1, CDR-H2 or CDR-H3 of a heavy chain variable region comprising one or more chicken framework regions. In some embodiments, the bioactive peptide amino acid sequence is engrafted into at least one of CDR-L1, CDR-L2 or CDR-L3 of a light chain variable region comprising one or more chicken framework regions.

[0013] In another aspect, the invention provides a method of making a bioactive peptide-bearing antibody, comprising (a) fusing the amino acid sequence of at least one bioactive peptide of interest onto: (i) an amino terminal region of at least one of: a light chain variable region comprising one or more chicken framework regions and/or a heavy chain variable region comprising one or more chicken framework regions, and/or (ii) a carboxy terminal region of at least one of: a light chain constant region and/or a heavy chain constant region; and (b) determining whether the antibody has substantially the same biological activity as the bioactive peptide.

[0014] In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, comprising a bioactive peptide amino acid sequence, wherein the bioactive peptide amino acid sequence is fused to at least one of (i) the amino terminal region of at least one of: a light chain variable region comprising one or more chicken framework regions and/or a heavy chain variable region comprising one or more chicken framework regions; or (ii) the carboxy terminal region of at least one of: a light chain constant region and/or a heavy chain constant region, wherein the antibody has substantially the same biological activity as the bioactive peptide.

[0015] In another aspect, the invention provides an isolated polypeptide comprising: (i) a region comprising an SGMI core sequence, the SGMI core sequence comprising an amino acid sequence according to: X₁CTX₂X₃X₄CX₅Q (SEQ ID NO:5), wherein: X₁ is F or V, X₂ is R or K, X₃ is K or L, X₄ is L or W, and X₅ is Y or N; and (ii) a region comprising human IgG1 Fc, wherein the polypeptide inhibits the activity of at least one of MASP-1 or MASP-2.

[0016] In another aspect, the invention provides pharmaceutical compositions comprising the bioactive peptide-bearing antibodies, fragments thereof, and polypeptides, as disclosed herein.

[0017] In another aspect, the invention provides a method of inhibiting lectin pathway complement activation in a mammalian subject comprising administering a composition comprising a bioactive peptide-bearing antibody, or fragment thereof, or polypeptide, as disclosed herein, in an amount sufficient to inhibit lectin pathway complement activation in said mammalian subject.

DESCRIPTION OF THE DRAWINGS

[0018] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0019] FIG. 1 is a bar graph showing the percent C5b-C9 formation in the presence of positive serum, negative serum, isotype control, SGMI-1Fc or SGMI-2Fc, demonstrating that both SGMI-1Fc and SGMI-2Fc inhibit the activation of the lectin pathway;

[0020] FIG. 2 graphically illustrates the level of C3b deposition for 1% normal serum plus isotype control, SGMI-1Fc or SGMI-2Fc over a concentration range of 0.15 nM to 1000 nM, demonstrating that both SGMI-1Fc and SGMI-2Fc inhibited C3b deposition from normal serum in mannan-coated ELISA wells;

[0021] FIG. 3 illustrates an exemplary parental (DTLacO) variable heavy chain polypeptide sequence compared to a variable heavy chain polypeptide sequence comprising a bioactive peptide amino acid sequence engrafted within complementarity determining region-3 (CDR-3);

[0022] FIG. 4 shows an alignment of the amino acid sequences of exemplary variable heavy chain polypeptides comprising the bioactive peptide SGMI-1, and variants thereof, engrafted within CDR-3, including optional linkers at the C-terminus and/or N-terminus of the bioactive peptide;

[0023] FIG. 5 illustrates an exemplary parental (DTLacO) variable light chain polypeptide sequence compared to a variable light chain polypeptide sequence comprising a bioactive peptide engrafted within CDR-1;

[0024] FIG. 6 shows an alignment of the amino acid sequences of exemplary variable light chain polypeptides comprising the bioactive peptide SGMI-1 or SGMI-2, and variants thereof, engrafted within CDR-1, including optional linkers at the C-terminus and/or N-terminus of the bioactive peptide.

[0025] FIG. 7A graphically illustrates the inhibitory activity of various representative chimeric chicken/human mAbs containing SGMI-1 engrafted into CDR-H3 on C5b-C9 deposition;

[0026] FIG. 7B graphically illustrates the inhibitory activity of additional various representative chimeric chicken/human mAbs containing SGMI-1 engrafted into CDR-3 on C5b-C9 deposition;

[0027] FIG. 8A graphically illustrates the inhibitory activity of various representative chimeric chicken/human mAbs containing SGMI-1 engrafted into CDR-H3 on complement C3b deposition activity in a dose-response manner;

[0028] FIG. 8B graphically illustrates the inhibitory activity of additional various representative chimeric chicken/human mAbs containing SGMI-1 engrafted into CDR-H3 on complement C3b deposition activity in a dose-response manner;

[0029] FIG. 9A graphically illustrates the inhibitory activity of a chimeric chicken/human mAb comprising SGMI-2 engrafted within CDR-L1 (Ab-SGMI-2L-Igλ) and a combination of SGMI-1 engrafted within CDR-H3 and SGMI-2 engrafted within CDR-L1 (Ab-SGMI-1-L1-IgG1/SGMI-2L-Igλ), demonstrating that the chimeric combination SGMI-1-SGMI-2 mAb (Ab-SBMI-1-L1-IgG1/SGMI-2L-Igλ) inhibits C5b-C9 deposition;

[0030] FIG. 9B graphically illustrates the inhibitory activity of a chimeric chicken/human mAb comprising a combination of SGMI-1 engrafted within CDR-H3 and SGMI-2 engrafted within CDR-L1 (Ab-SGMI-1-L1-IgG1/SGMI-2L-Igλ), demonstrating that the chimeric combination SGMI-1-SGMI-2 mAb (Ab-SGMI-1-L1-IgG 1/SGMI-2L-Igλ) inhibits C5b-C9 deposition;

[0031] FIG. 10 illustrates a chimeric chicken/human antibody comprising bioactive peptides fused to the N-terminus of the heavy chain variable region (A); and/or the N-terminus of the light chain variable region (B); and/or the C-terminus of the heavy chain constant region (C); and/or the C-terminus of the light chain constant region (D);

[0032] FIG. 11 graphically illustrates the inhibitory activity of chimeric chicken/human antibodies comprising bioactive SGMI-1 or SGMI-2 peptides fused to the N- or C-terminus of the heavy or light chain, demonstrating that all of the peptide-mAb fusions inhibit C5b-C9 deposition.

DESCRIPTION OF THE SEQUENCE LISTING

[0033] SEQ ID NO:1 human MASP-1 cDNA;

[0034] SEQ ID NO:2 human MASP-1 protein (with leader sequence);

[0035] SEQ ID NO:3 human MASP-2 cDNA;

[0036] SEQ ID NO:4 human MASP-2 protein (with leader sequence);

[0037] SEQ ID NO:5: SGMI peptide core sequence;

[0038] SEQ ID NO:6 SGMI-1L peptide (full length);

[0039] SEQ ID NO:7 SGMI-1M peptide (medium truncated version);

[0040] SEQ ID NO:8 SGMI-1S peptide (short truncated version);

[0041] SEQ ID NO:9 SGMI-2L peptide (full length);

[0042] SEQ ID NO:10 SGMI-2M peptide (medium truncated version);

[0043] SEQ ID NO:11 SGMI-2S peptide (short truncated version);

[0044] SEQ ID NO:12 human IgG1-Fc polypeptide;

[0045] SEQ ID NO:13 peptide linker #1 (12aa);

[0046] SEQ ID NO:14: peptide linker #2 (10aa);

[0047] SEQ ID NO:15: nucleic acid encoding polypeptide fusion comprising the human IL-2-signal sequence, SGMI-1L, linker#1, and human IgG1-Fc;

[0048] SEQ ID NO:16: mature polypeptide fusion comprising SGMI-1L, linker#1 and human IgG1-Fc (SGMI-1Fc);

[0049] SEQ ID NO:17: nucleic acid encoding polypeptide fusion comprising the human IL-2-signal sequence, SGMI-2L, linker#1 and human IgG1-Fc;

[0050] SEQ ID NO:18: mature polypeptide fusion comprising SGMI-2L, linker#1 and human IgG1-Fc (SGMI-2Fc);

[0051] SEQ ID NO:19: SGMI-1 forward primer;

[0052] SEQ ID NO:20: SGMI-1 reverse primer;

[0053] SEQ ID NO:21: SGMI-2 forward primer;

[0054] SEQ ID NO:22: SGMI-2 reverse primer;

[0055] SEQ ID NO:23: parent DTLacO (clone #1) chicken heavy chain variable region (DTLacO_VH);

[0056] SEQ ID NO:24: conserved FR-1 region from chicken heavy chain variable region;

[0057] SEQ ID NO:25: conserved FR-2 region from chicken heavy chain variable region;

[0058] SEQ ID NO:26: conserved FR-3 region from chicken heavy chain variable region;

[0059] SEQ ID NO:27: conserved FR-3 flanking region adjacent to CDR-H3 from chicken heavy chain variable region;

[0060] SEQ ID NO:28: conserved FR-4 region from chicken heavy chain variable region;

[0061] SEQ ID NO:29: conserved FR-4 flanking region adjacent to CDR-H3 from chicken heavy chain variable region;

[0062] SEQ ID NO:30: Parent DTLacO (clone #1) chicken light chain variable region (DTLacO_VL);

[0063] SEQ ID NO:31: conserved FR-1 region from chicken light chain variable region;

[0064] SEQ ID NO:32: conserved FR-1 flanking region adjacent to CDR-L1 from chicken light chain variable region;

[0065] SEQ ID NO:33: conserved FR-2 region from chicken light chain variable region;

[0066] SEQ ID NO:34: conserved FR-2 flanking region adjacent to CDR-L1 from chicken light chain variable region;

[0067] SEQ ID NO:35: conserved FR-3 region from chicken light chain variable region;

[0068] SEQ ID NO:36: conserved FR-4 region from chicken light chain variable;

[0069] SEQ ID NO:37-46: peptide linkers

[0070] SEQ ID NO:47: human IgG1 constant region (CH1-hinge-CH2-CH3);

[0071] SEQ ID NO:48: human lambda light chain constant region;

[0072] SEQ ID NO:49: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1L-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1L-IgG1);

[0073] SEQ ID NO:50: mature polypeptide comprising the SGMI-1L-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1L-IgG1);

[0074] SEQ ID NO:51: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1M-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1M-IgG1);

[0075] SEQ ID NO:52: mature polypeptide comprising the SGMI-1M-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1M-IgG1);

[0076] SEQ ID NO:53: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1S-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1S-IgG1);

[0077] SEQ ID NO:54: mature polypeptide comprising the SGMI-1S-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1S-IgG1);

[0078] SEQ ID NO:55: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L1-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L1-IgG1);

[0079] SEQ ID NO:56: mature polypeptide comprising the SGMI-1-L1-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L1-IgG1);

[0080] SEQ ID NO:57: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L2-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L2-IgG1);

[0081] SEQ ID NO:58: mature polypeptide comprising the SGMI-1-L2-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L2-IgG1);

[0082] SEQ ID NO:59: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L3-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L3-IgG1);

[0083] SEQ ID NO:60: mature polypeptide comprising the SGMI-1-L3-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L3-IgG1);

[0084] SEQ ID NO:61: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L4-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L4-IgG1);

[0085] SEQ ID NO:62: mature polypeptide comprising the SGMI-1-L4-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L4-IgG1);

[0086] SEQ ID NO:63: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L5-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L5-IgG1);

[0087] SEQ ID NO:64: mature polypeptide comprising the SGMI-1-L5-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L5-IgG1);

[0088] SEQ ID NO:65: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L6-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L6-IgG1);

[0089] SEQ ID NO:66: mature polypeptide comprising the SGMI-1-L6-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L6-IgG1);

[0090] SEQ ID NO:67: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L7-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L7-IgG1);

[0091] SEQ ID NO:68: mature polypeptide comprising the SGMI-1-L7-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L7-IgG1);

[0092] SEQ ID NO:69: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L8-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L8-IgG1);

[0093] SEQ ID NO:70: mature polypeptide comprising the SGMI-1-L8-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L8-IgG1);

[0094] SEQ ID NO:71: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L9-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L9-IgG1);

[0095] SEQ ID NO:72: mature polypeptide comprising the SGMI-1-L9-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L9-IgG1);

[0096] SEQ ID NO:73: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L10-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L10-IgG1);

[0097] SEQ ID NO:74: mature polypeptide comprising the SGMI-1-L10-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L10-IgG1);

[0098] SEQ ID NO:75: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L11-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L11-IgG1);

[0099] SEQ ID NO:76: mature polypeptide comprising the SGMI-1-L1'-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L11-IgG1);

[0100] SEQ ID NO:77: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-L12-bearing chicken VH sequence and the human IgG1 constant region (pcDNA3-SGMI-1-L12-IgG1);

[0101] SEQ ID NO:78: mature polypeptide comprising the SGMI-1-L12-bearing chicken VH region and the human IgG1 constant region (Ab-SGMI-1-L12-IgG1);

[0102] SEQ ID NO:79: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the SGMI-2L-bearing chicken VL sequence and the human Igλ constant region (pcDNA3-SGMI-2L-Igλ);

[0103] SEQ ID NO:80: mature polypeptide comprising the SGMI-2L-bearing chicken VL region and the human Igλ constant region (Ab-SGMI-2L-Igλ);

[0104] SEQ ID NO:81: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the SGMI-2M-bearing chicken VL sequence and the human Igλ constant region (pcDNA3-SGMI-2M-Igλ);

[0105] SEQ ID NO:82: mature polypeptide comprising the SGMI-2M-bearing chicken VL region and the human Igλ constant region (Ab-SGMI-2M-Igλ);

[0106] SEQ ID NO:83: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the SGMI-2S-bearing chicken VL sequence and the human Igλ constant region (pcDNA3-SGMI-2S-Igλ);

[0107] SEQ ID NO:84: mature polypeptide comprising the SGMI-2S-bearing chicken VL region and the human Igλ constant region (Ab-SGMI-2S-Igλ);

[0108] SEQ ID NO:85: polynucleotide encoding the polypeptide comprising the SGMI-1L-bearing chicken VL region and the human Igλ constant region (pcDNA3-SGMI-1L-Igλ);

[0109] SEQ ID NO:86: mature polypeptide comprising the SGMI-1L-bearing chicken VL region and the human Igλ constant region (Ab-SGMI-1L-Igλ);

[0110] SEQ ID NO:87: polynucleotide encoding a polypeptide comprising the SGMI-1M-bearing chicken VL region and the human Igλ constant region (pcDNA3-SGMI-1M-Igλ);

[0111] SEQ ID NO:88: mature polypeptide comprising the SGMI-1M-bearing chicken VL region and the human Igλ constant region (Ab-SGMI-1M-Igλ);

[0112] SEQ ID NO:89: polynucleotide encoding a polypeptide comprising the SGMI-1S-bearing chicken VL region and the human Igλ constant region (pcDNA3-SGMI-1S-Igλ);

[0113] SEQ ID NO:90: mature polypeptide comprising the SGMI-1S-bearing chicken VL region and the human Igλ constant region (Ab-SGMI-1S-Igλ);

[0114] SEQ ID NO:91: DTLacO chicken (clone #2) heavy chain variable region (DTLacO VH);

[0115] SEQ ID NO:92: DTLacO chicken (clone#2) light chain variable region (DTLacO VL);

[0116] SEQ ID NO:93: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-1-fused chicken VH sequence, and the human IgG1 constant region (pcDNA3-IgG1-S10);

[0117] SEQ ID NO:94: mature polypeptide comprising the SGMI-1-fused chicken VH region and the human IgG 1 constant region (Ab-IgG1-S10);

[0118] SEQ ID NO:95: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the SGMI-2-fused chicken VH sequence, and the human IgG1 constant region (pcDNA3-IgG1-S20);

[0119] SEQ ID NO:96: mature polypeptide comprising the SGMI-2-fused chicken VH region and the human IgG1 constant region (Ab-IgG1-S20);

[0120] SEQ ID NO:97: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the chicken VH sequence, and the SGMI-1-fused human IgG1 constant region (pcDNA3-IgG1-S01);

[0121] SEQ ID NO:98: mature polypeptide comprising the chicken VH region and the SGMI-1-fused human IgG1 constant region (Ab-IgG1-S01);

[0122] SEQ ID NO:99: polynucleotide encoding the polypeptide comprising the human VH signal sequence, the chicken VH sequence, and the SGMI-2-fused human IgG1 constant region (pcDNA3-IgG1-S02);

[0123] SEQ ID NO:100: mature polypeptide comprising the chicken VH region and the SGMI-2-fused human IgG1 constant region (Ab-IgG1-S02);

[0124] SEQ ID NO:101: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the SGMI-1-fused VL sequence and the human Igλ constant region (pcDNA3-Igλ-S10);

[0125] SEQ ID NO:102: mature polypeptide comprising the SGMI-1-fused chicken VL region and the human Igλ constant region (Ab-Igλ-S10);

[0126] SEQ ID NO:103: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the SGMI-2-fused VL sequence and the human Igλ constant region (pcDNA3-Igλ-S20);

[0127] SEQ ID NO:104: mature polypeptide comprising the SGMI-2-fused chicken VL region and the human Igλ constant region (Ab-Igλ-520);

[0128] SEQ ID NO:105: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the chicken VL sequence, and the SGMI-1-fused human Igλ constant region (pcDNA3-Igλ-S01);

[0129] SEQ ID NO: 106: mature polypeptide comprising the chicken VL region, and the SGMI-1-fused human Igλ constant region (Ab-Igλ-S01;

[0130] SEQ ID NO:107: polynucleotide encoding the polypeptide comprising the human VL signal sequence, the chicken VL sequence, and the SGMI-2-fused human Igλ constant region (pcDNA3-Igλ-S02); and

[0131] SEQ ID NO 108: mature polypeptide comprising the chicken VL region, and the SGMI-2-fused human Igλ constant region (Ab-Igλ-S02);

DETAILED DESCRIPTION

I. Definitions

[0132] Unless specifically defined herein, all terms used herein have the same meaning as would be understood by those of ordinary skill in the art of the present invention. The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention.

[0133] As used herein, an "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site, located in the variable region (also referred to herein as the variable domain) of the immunoglobulin molecule. In some embodiments, the antibody as disclosed herein comprises a variable region comprising chicken framework regions and further comprising a bioactive peptide amino acid sequence engrafted into a CDR region. In some embodiments, the antibody as disclosed herein comprises a variable region comprising chicken framework regions and further comprises a bioactive peptide fused to the amino and/or carboxy terminal region of the light and/or heavy chain. As used herein, the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as a single variable region antibody (dAb), or other known antibody fragments such as Fab, Fab', F(ab')₂, Fv and the like, single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity. "Diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also a particular form of antibody contemplated herein. Minibodies comprising a scFv joined to a CH3 domain are also included herein (S. Hu et al, Cancer Res., 56, 3055-3061, 1996). See e.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); PCT/US92/09965; WO94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996; S. Hu et al, Cancer Res., 56, 3055-3061, 1996. Nanobodies and maxibodies are also contemplated (see, e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO06/079372, WO/2010037402).

[0134] As used herein, the term "engrafted into a CDR region" refers to introducing a bioactive peptide sequence into at least one CDR region of a variable region of a heavy or light chain comprising chicken framework regions (FR1, FR2, FR3 and FR4) parental generic heavy or light chain, wherein the flanking framework regions remain intact, and wherein either the entire native CDR sequence is replaced with the bioactive peptide, or at least one amino acid, at least two, at least three, at least four, at least five, or more, up to all the amino acid residues of the native CDR sequence are retained as linker sequences flanking the bioactive peptide in the heavy or light chain variable region comprising the engrafted bioactive peptide.

[0135] As used herein, the term `fused onto a light or heavy chain" refers to fusing a bioactive peptide sequence at the amino terminal region or at the carboxy terminal region of a heavy chain or light chain of an antibody comprising chicken framework regions.

[0136] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chains that binds to the antigen of interest, including a polypeptide fragment that contains at least one bioactive peptide engrafted into a CDR, or a bioactive peptide fused to a light chain or heavy chain, wherein the polypeptide fragment binds to a target of the bioactive peptide, such as MASP-1 or MASP-2. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set forth herein, wherein the antibodies bind a target of a bioactive peptide of interest, such as MASP-1 or MASP-2. An antigen-binding fragment of the herein described MASP-1 or MASP-2-specific antibodies is capable of binding to MASP-1 or MASP-2. In certain embodiments, binding of an antigen-binding fragment prevents or inhibits binding of a target of a bioactive peptide of interest (e.g., a GPCR ligand to its receptor), interrupting the biological response resulting from ligand binding to the receptor. In certain embodiments, the antigen-binding fragment binds specifically to and/or inhibits or modulates the biological activity of a target of a bioactive peptide of interest. In certain embodiments, the antigen-binding fragment binds specifically to and/or inhibits or modulates the biological activity of human MASP-1 and/or human MASP-2.

[0137] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, including a target molecule or a portion of a molecule capable of being bound by a bioactive peptide of interest, and/or additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

[0138] As used herein, an "epitope" refers to the site on a protein (e.g., a target of a bioactive peptide, such as MASP-1 or MASP-2 protein) that is bound by an antibody. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residue(s), including linear and non-linear epitopes. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind a protein target when it preferentially recognizes its target protein in a complex mixture of proteins and/or macromolecules. An antibody is said to specifically bind a target protein (also referred to as a target antigen) when the equilibrium dissociation constant is less than or equal to 10^-6 M, or less than or equal to 10^-7 M, or less than or equal to 10^-8 M. In some embodiments, the equilibrium dissociation constant may be less than or equal to 10^-9 M or less than or equal to 10^-10 M.

[0139] The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab')₂ fragment which comprises both antigen-binding sites. An Fv fragment for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V_H::V_L heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. See e.g., Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0140] In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (III et al., Prot. Eng. 10: 949-57 (1997); minibodies (Martin et al., EMBO J. 13: 5305-9 (1994); diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins (Traunecker et al., EMBO J. 10: 3655-59 (1991) and Traunecker et al. Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity. In still other embodiments, bispecific or chimeric antibodies may be made that encompass the engrafted bioactive peptides and/or bioactive peptide fusions of the present disclosure. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies, while bispecific antibodies may be generated that bind specifically to the target of a first bioactive peptide through one binding domain and to a target of a second bioactive peptide through a second binding domain. In another embodiment, bi-specific and/or tri-specific antibodies may be generated that bind to the target of the parent antibody through one binding domain and to a target of the first and/or second bioactive peptide through a second and/or third binding domain introduced by the presence of the bioactive peptide. These antibodies may be produced through recombinant molecular biological techniques or may be physically conjugated together.

[0141] A single chain Fv (scFv) polypeptide is a covalently linked V_H::V_L heterodimer which is expressed from a gene fusion including V_H- and V_L-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated--but chemically separated--light and heavy polypeptide chains from an antibody variable (V) region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al. A dAb fragment of an antibody consists of a VH domain (Ward, E. S. et al., Nature 341, 544-546 (1989)).

[0142] In certain embodiments, an antibody as herein disclosed (e.g., a MASP-1 or MASP-2-specific antibody) is in the form of a diabody. Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).

[0143] Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Diabodies and scFv can be constructed without an Fc region, using only variable regions, potentially reducing the effects of anti-idiotypic reaction.

[0144] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).

[0145] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This proprietary antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies do not activate the complement system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one area on the UniBody® that can bind to cognate antigens (e.g., disease targets) and the UniBody® therefore binds univalently to only one site on target cells. For certain cancer cell surface antigens, this univalent binding may not stimulate the cancer cells to grow as may be seen using bivalent antibodies having the same antigen specificity, and hence UniBody® technology may afford treatment options for some types of cancer that may be refractory to treatment with conventional antibodies. The UniBody® is about half the size of a regular IgG4 antibody. This small size can be a great benefit when treating some forms of cancer, allowing for better distribution of the molecule over larger solid tumors and potentially increasing efficacy.

[0146] In certain embodiments, the antibodies of the present disclosure may take the form of a nanobody. Nanobodies are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g. U.S. Pat. No. 6,765,087), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see e.g. U.S. Pat. No. 6,838,254). The production process is scalable and multi-kilogram quantities of nanobodies have been produced. Nanobodies may be formulated as a ready-to-use solution having a long shelf life. The Nanoclone method (see eg. WO 06/079372) is a proprietary method for generating Nanobodies against a desired target, based on automated high-throughput selection of B-cells.

[0147] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

[0148] As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 70-90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical" structures--regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

[0149] As used herein, the term "chicken framework region or a variant thereof" refers to the FR regions of a chicken antibody, and conserved variants thereof, for example as disclosed herein and further described in Wu et al., J. Immunol. 188:322-333 (2012), hereby incorporated herein by reference.

[0150] The structures and locations of immunoglobulin variable regions may be determined by reference to Kabat, E. A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (immuno.bme.nwu.edu).

[0151] A "monoclonal antibody" refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')₂, Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody."

[0152] "Humanized" antibodies refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species (e.g., a chicken) and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable regions fused onto constant domains or only the CDRs grafted (including CDRs comprising engrafted bioactive peptide sequences) onto appropriate framework regions in the variable regions. Epitope binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A. F. et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327).

[0153] In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, in one embodiment, a chimeric antibody is comprised of an antigen-binding fragment of an antibody comprising a bioactive peptide sequence engrafted into a CDR of a variable region operably linked or otherwise fused to a heterologous Fc portion of a different antibody, or fused to the N- or C-terminus of the heavy or light chain. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be comprised of CH₂ and CH3 domains from one or more of the different Ig classes. As noted above with regard to humanized antibodies, the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may comprise an entire variable region (VL, VH or both).

[0154] In certain embodiments, an antibody comprising an engrafted bioactive peptide sequence comprises one or more of the CDRs of the antibodies described herein. In this regard, it has been shown in some cases that the transfer of only the VH-CDR3 of an antibody can be done while still retaining desired specific binding (Barbas et al., PNAS (1995) 92: 2529-2533). See also, McLane et al., PNAS (1995) 92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

[0155] As used herein, the term "MASP-2-dependent complement activation" comprises MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca⁺+) leading to the formation of the C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n.

[0156] As used herein, the term "MASP-1-dependent complement activation" comprises MASP-1 dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca⁺+) leading to the formation of the C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n.

[0157] As used herein, the term "lectin pathway" refers to complement activation that occurs via the specific binding of serum and non-serum carbohydrate-binding proteins including mannan-binding lectin (MBL), CL-11 and the ficolins (H-ficolin, M-ficolin, or L-ficolin).

[0158] As used herein, the term "MASP-2 inhibitory antibody" refers to any MASP-2 antibody, or MASP-2 binding fragment thereof, that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation. MASP-2 inhibitory antibodies useful in the method of the invention may reduce MASP-2-dependent complement activation by greater than 20%, such as greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%.

[0159] As used herein, the term "MASP-1 inhibitory antibody" refers to any MASP-1 antibody, or MASP-1 binding fragment thereof, that binds to or directly interacts with MASP-1 and effectively inhibits MASP-1-dependent complement activation. MASP-1 inhibitory antibodies useful in the method of the invention may reduce MASP-1-dependent complement activation by greater than 20%, such as greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%.

[0160] As used herein, the term "MASP-2 blocking antibody" refers to MASP-2 inhibitory antibodies that reduce MASP-2-dependent complement activation by greater than 90%, such as greater than 95%, or greater than 98% (i.e., resulting in MASP-2 complement activation of only 10%, such as only 9%, or only 8%, or only 7%, or only 6%, such as only 5% or less, or only 4%, or only 3% or only 2% or only 1%).

[0161] As used herein, the term "MASP-1 blocking antibody" refers to MASP-1 inhibitory antibodies that reduce MASP-1-dependent complement activation by greater than 90%, such as greater than 95%, or greater than 98% (i.e., resulting in MASP-1 complement activation of only 10%, such as only 9%, or only 8%, or only 7%, or only 6%, such as only 5% or less, or only 4%, or only 3% or only 2% or only 1%).

[0162] As used herein, the term "variant" antibody sequence refers to a molecule which differs in amino acid sequence from a "parent" or reference antibody amino acid sequence by virtue of addition, deletion, and/or substitution of one or more amino acid residue(s) in the parent antibody sequence. In one embodiment, a variant antibody sequence refers to a molecule which contains one or more framework regions that are identical to the parent framework domains, except for a combined total of 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 amino acid substitutions within the framework regions of the heavy chain variable region, and/or up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions with said framework regions of the light chain variable region. In some embodiments, the amino acid substitutions are conservative sequence modifications. In some embodiments, the variant framework region(s) of the variable light chain and/or the variable heavy chain comprise or consist of an amino acid sequence having at least 85% identity, such as least 86%, or at least 87%, or at least 88% or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94% or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% or 100% identity with at least one or more of the chicken framework regions VL-FR1, VL-FR2, VL-FR3 and VL-FR4 amino acid sequences set forth in SEQ ID NO:s 31, 33, 35 and 36, respectively; or with at least one or more of the chicken framework regions. VH-FR-1, VH-FR2, VH-FR3 and VH-FR4 amino acid sequences set forth in SEQ ID NO:s 24, 25, 26, and 28, respectively.

[0163] As used herein, the term "parent chicken antibody" refers to an antibody which is encoded by an amino acid sequence used for the preparation of the variant comprising a bioactive peptide engrafted into or onto at least one of the variable region of the heavy or light chain. The parent antibody has a chicken framework region and, if present, typically has human antibody constant region(s).

[0164] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V).

[0165] In the broadest sense, the naturally occurring amino acids can be divided into groups based upon the chemical characteristic of the side chain of the respective amino acids. By "hydrophobic" amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By "hydrophilic" amino acid is meant either Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subclassed as follows. By "uncharged hydrophilic" amino acid is meant either Ser, Thr, Asn or Gln. By "acidic" amino acid is meant either Glu or Asp. By "basic" amino acid is meant either Lys, Arg or His.

[0166] As used herein the term "conservative amino acid substitution" is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

[0167] As used herein, the term "isolated antibody" refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0168] As used herein, an "isolated nucleic acid molecule" is a nucleic acid molecule (e.g., a polynucleotide) that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.

[0169] As used herein, a "nucleic acid molecule construct" is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.

[0170] As used herein, an "expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

[0171] As used herein, the terms "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.

[0172] As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; reference to "an antibody" includes a plurality of such antibodies and reference to "a framework region" includes reference to one or more framework regions and equivalents thereof known to those skilled in the art, and so forth.

[0173] As used herein, "a subject" includes all mammals, including without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

[0174] As used herein, the term "bioactive peptide" refers to a peptide having a biological activity.

[0175] The term "peptide" as used herein refers to a plurality of amino acids joined together in a linear chain via peptide bonds, including a dipeptide, tripeptide, oligopeptide and polypeptide. The term oligopeptide is typically used to describe peptides having from at least 2 to about 50 or more (e.g., from 2 amino acids to 60 amino acids in length, such as from about 5 to about 50 amino acids, such as from about 5 to about 40, or from about 5 to about 30 amino acids in length). Peptides larger than 60 amino acids are referred to herein as polypeptides or proteins.

[0176] As used herein, the term "bioactive" or "bioactivity" as used herein includes, but is not limited to, any type of interaction with another biomolecule, such as a protein, glycoprotein, carbohydrate, for example an oligosaccharide or polysaccharide, nucleotide, polynucleotide, fatty acid, hormone, enzyme, cofactor or the like, whether the interactions involve covalent or noncovalent binding. Bioactivity further includes interactions of any type with other cellular components or constituents including salts, ions, metals, nutrients, foreign or exogenous agents present in a cell such as viruses, phage and the like, for example binding, sequestration or transport-related interactions. Bioactivity of a peptide can be detected, for example, by observing phenotypic effects in a host cell in which it is expressed, or by performing an in vitro assay for a particular bioactivity, such as affinity binding to a target molecule, alteration of an enzymatic activity, or the like. Examples of bioactive peptides include antimicrobial peptides and peptide drugs. Antimicrobial peptides are peptides that adversely affect a microbe such as a bacterium, virus, protozoan, or the like. Antimicrobial peptides include, for example, inhibitory peptides that slow the growth of a microbe, microbiocidal peptides that are effective to kill a microbe (e.g., bacteriocidal and virocidal peptide drugs, sterilants, and disinfectants), and peptides effective to interfere with microbial reproduction, host toxicity, or the like. Peptide drugs for therapeutic use in humans or other animals include, for example, antimicrobial peptides that are not prohibitively toxic to the patient, and peptides designed to elicit, speed up, slow down, or prevent various metabolic processes in the host such as insulin, oxytocin, calcitonin, gastrin, somatostatin, anticancer peptides, and the like.

[0177] As used herein, the term "wherein the isolated antibody has substantially the same biological activity as the unmodified bioactive peptide" refers to wherein the isolated antibody comprising the bioactive peptide sequence has at least 70%, or at least 80%, or at least 85%, or at least 90% or at least 95%, or at least 98%, or at least 99% of the biological activity as compared to the original, unmodified form of the corresponding bioactive peptide.

[0178] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0179] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, N.Y.); or other relevant Current Protocol publications and other like references. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0180] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

II. Overview

[0181] Bioactive peptides are peptides (i.e., from 2 to 60 amino acid residues in length, such as from about 5 to about 50 amino acids, such as from about 5 to about 40 amino acids in length, such as from about 5 to about 30 amino acids in length, or such as a peptide having a length of no more than 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 amino acid residues) that elicit a biological activity. For example, the bioactive peptides SGMI-1 (set forth as SEQ ID NO:6) and SGMI-2 (set forth as SEQ ID NO:9) are each 36 amino acid residues in length and are highly specific inhibitors of MASP-1 and MASP-2, respectively. However, as peptide they have limited potential for use in biological studies and therapeutic applications. For example, peptide instability within the biological system of interest often occurs, as evidenced by the unwanted degradation of potential peptide drugs by proteases and/or peptidases in the host cells.

[0182] In order to engineer bioactive peptides, such as SGMI-1 and SGMI-2, for use as therapeutic agents, the inventors have generated bioactive peptide-bearing antibodies and fragments thereof by engrafting amino acid sequences encoding bioactive peptides into or fused onto three distinct scaffolds: (1) fused onto the amino terminus of human IgG1 Fc region to create an Fc-fusion protein, as described in Example 2; (2) engrafted into various complementarity-determining regions (CDR) of a chimeric chicken (variable regions)--human (IgG1 and Igλ constant regions) antibody, as described in Example 3; and (3) fused onto the amino or carboxy termini of the heavy and/or light chains of an antibody, as described in Example 4. Using the methods described herein, the inventors have produced bioactive peptide-bearing antibodies and fragments thereof which surprisingly have substantially the same biological activity of the bioactive peptide when measured in vitro, with the advantages of increased stability for use as a therapeutic agent in a living subject.

III. Bioactive Peptide-Bearing Antibodies

[0183] In accordance with the foregoing, in one aspect, the invention provides a method of making a bioactive peptide-bearing antibody, the method comprising (a) engrafting the amino acid sequence of at least one bioactive peptide of interest into (i) at least one of CDR-H1, CDR-H2 or CDR-H3 of a heavy chain variable region comprising chicken framework regions and/or (ii) at least one of CDR-L1, CDR-L2 or CDR-L3 of the light chain variable region comprising chicken framework regions, and (b) determining whether the antibody has substantially the same biological activity as the bioactive peptide.

[0184] The method in accordance with this aspect of the invention may be used to generate a bioactive peptide-bearing antibody, wherein the antibody comprises the amino acid sequence of any bioactive peptide of interest. Bioactive peptides have been isolated from a variety of systems, exhibit a wide range of actions, and have been utilized as therapeutic agents in the field of medicine and as diagnostic tools in both basic and applied research. The mode of action of bioactive peptides has been found to be due to the interaction of the bioactive peptide with a specific protein target. The bioactive peptide acts by binding to and either activating or inactivating its protein target with extremely high specificities. Binding constants of bioactive peptides for their protein targets typically have been determined to be in the nanomolar (nM) range with binding constants as potent as picomolar range having been reported.

[0185] The methods of this aspect of the invention may be used to generate an antibody comprising an amino acid sequence with any bioactive peptide. Exemplary bioactive peptides for use in the methods of the invention include (i) bioactive peptides that inhibit medically-important proteases, (ii) neuropeptides (iii) bioactive peptides that inhibit or activate neuropeptide activity, (iii) peptide hormones, (iv) bioactive peptides that inhibit or activate peptide hormone activity, (v) peptides that are ligands for Class A GPCRs, (vi) bioactive peptides that inhibit or activate Class A GPCRs, (vii) Class B GPCR ligands, and (viii) bioactive peptides that inhibit or activate Class B GPCRs.

[0186] For example, medically-important proteases that are inhibited by bioactive peptides include, but are not limited to: Gamma-secretase, PAR-1, PAR-2, PAR-3, Cathepsin, Incretin, Dipeptidyl peptidase IV, Angiotensin-converting enzyme, Calpain, Caspase-3, Carboxypeptidase, Thrombin, and proteases in the clotting cascade and complement pathways. Examples of complement pathway serine protease inhibitors (e.g., MASP-1, MASP-2 inhibitors), include the bioactive peptide inhibitors SGMI-1 and SGMI-2.

[0187] Examples of neuropeptides include, but are not limited to: N-Acetylaspartylglutamic acid, agouti-related peptide, alpha-endorphin, Big dynorphin, Bombesin, Bombesin-like peptides, Carbetocin, Cocaine-and-amphetamine regulated transcript (CART), Cholecystokinin, Corazonin, Corticotropin-like intermediate peptide, Cortistatin, Demoxytocin, Dynorphin A, Dynorphin B, Eledoisin, Encephalin, Galanin, Galanin-like peptide, Galmic, Galnon, Gamma-endorphin, Ghrelin, Hemopressin, Kisspeptin, Neurokinin B, Neuromedin B, Neuromedin N, Neuromedin S, Neuromedin U, Neuromedin S, Neuromedin Y, Neuropeptide Y, Neurotensin, Nociceptin, Opiorphin, Orexin, Orexin-A, Oxytocin, Physalaemin, Preprotachykinin, Proctolin, Proenkephalin, Proopiomelanocortin, Protein episteme, Relaxin-3, RVD-Pa, Somatostatin, Substance P, TAC1, Tacchykinin peptides, TRH, Vasopressin, Vasotocin, VIP, and VGF.

[0188] Examples of peptide hormones include, but are not limited to: Activin and inhibin, Adiponectin, Adipose-derived hormones, Adrenocorticotropic hormone, Afamelanotide, Agouti gene, Agouti signaling peptide, Allatostatin, Amylin, Amylin family, Angiotensin, Atrial natriuretic peptide, Big gastrin, Bovine somatotropin, Bradykinin, Brain-derived neurotrophic factor, Calcitonin, cholecystokinin, Ciliary neurotrophic factor, CJC-1293, CJC-1295, Corticotropin-releasing hormone, Cosyntropin, Crustacean neurohormone family, Endothelian, Enteroglucagon, FGF15, GFG15/19, Follicle-stimulating hormone, Gastrin, Gastroinhibitory peptide, Ghrelin, Glucagon, Glucagon-like peptide-1, Gonadotropin, Gonadotropin-preparations, Gonadotropin-releasing hormone, Granulocyte-colony-stimulating factor, Growth hormone, Growth-hormone-releasing hormone, Hepcidin, Human chorionic gonadotropin, Human placental lactogen, Incretin, Insulin, Insulin analog, Insulin aspart, Insulin degludec, Insulin glargine, Insulin lispro, Insulin-like growth factor, Insulin-like growth factor-1, Insulin-like growth factor-2, Leptin, Liraglutide, Little gastrin I, Luteinizing hormone, Melanocortin, Melanocyte-stimulating hormone, Alpha-Melanocyte-stimulating hormone, Melanotan II, Minigastrin, N-terminal prohormone of brain natriuretic peptide, Nerve growth factor, Neurotrophin-3, Neurotrophin-4, NPH insulin, Obestatin, Orexin, Osteocalcin, Pancreatic hormone, Parathyroid hormone, Peptide hormone, Peptide YY, Plasma renin activity, Pramlintide, Preprohormone, Prolactin, Relaxin, Relaxin family peptide hormone, Renin, Salcatonin, Secretin, Secretin family peptide hormone, Sincalide, Teleost leptins, Temporin, Tesamorelin, Thyroid-stimulating hormone, Thyrotropin-releasing hormone, Urocortin, Urocortin II, Urocortin III, Vasoactive intestinal peptide, and Vitellogenin.

[0189] Examples of Class B GPCR ligands include, but are not limited to: VIP (28aa), PACAP (38aa), and CRF1 (41aa).

[0190] Tables 1 and 2 list representative bioactive peptides suitable for use in the methods of the invention.

TABLE-US-00001 TABLE 1 Representative Bioactive Peptides Utilized in Medicine Size (amino acid Name Isolated from residues) Therapeutic Use Angiotensin II Human Plasma 8 Vasoconstrictor Bradykinin Human Plasma 9 Vasodilator Caerulein From Skin 10 Choleretic Agent Calcitonin Human Parathyroid Gland 32 Calcium Regulator Cholecystokinin Porcine Intestine 33 Choleretic Agent Corticotropin Porcine Pituitary Gland 39 Hormone Eledoisin Octopod Venom 11 Hypotensive Agent Gastrin Porcine Stomach 17 Gastric Activator Glucagon Porcine Pancreas 29 Antidiabetic Agent Gramicidin D Bacillus brevis 11 Antibacterial Agent Insulin Canine Pancreas Antidiabetic Agent Insulin A 21 Insulin B 30 Kallidin Human Plasma 10 Vasodilator Luteinizing Bovine Hypothalamus 10 Hormone Stimulator Hormone Releasing Factor Melittin Bee Venom 26 Antirheumatic Agent Oxytocin Bovine Pituitary Gland 9 Oxytocic Agent Secretin Canine Intestine 27 Hormone Sermorelin Human Pancreas 29 Hormone Stimulator Somatostatin Bovine Hypothalamus 14 Hormone Inhibitor Vasopressin Bovine Pituitary Gland 9 Antidiuretic Agent

TABLE-US-00002 TABLE 2 Representative Bioactive Peptides Utilized in Applied Research Size (amino acid Name Isolated from residues) Biological activity Atrial Natriuretic Rat Atria 28 Natriuretic Agent Peptide Peptide Bombesin Frog Skin 14 Gastric Activator Conantokin G Snail Venom 17 Neurotransmitter Conotoxin G1 Snail Venom 13 Neuromuscular Inhibitor Defensin HNP-1 Human Neutrophils 30 Antimicrobial Agent Delta Sleep-Inducing Rabbit Brain 9 Neurological Affector Peptide Dermaseptin Frog Skin 34 Antimicrobial Agent Dynorphin Porcine Brain 17 Neurotransmitter EETI II Ecballium elaterium 29 Protease Inhibitor seeds Endorphin Human Brain 30 Neurotransmitter Enkephalin Human Brain 5 Neurotransmitter Histatin 5 Human Saliva 24 Antibacterial Agent Mastoparan Vespid Wasps 14 Mast Cell Degranulator Magainin 1 Frog Skin 23 Antimicrobial Agent Melanocyte Porcine Pituitary 13 Hormone Stimulator Motilin Canine Intestine 22 Gastric Activator Neurotensin Bovine Brain 13 Neurotransmitter Physalaemin Frog Skin 11 Hypotensive Agent Substance P Horse Intestine 11 Vasodilator Vasoactive Intestinal Porcine Intestine 28 Hormone Peptide

[0191] In accordance with the methods of this aspect of the invention, an amino acid sequence of a bioactive peptide of interest is engrafted into at least one CDR region of a variable region of a heavy chain comprising one or more chicken framework regions (VH-FR1, VH-FR2, VH-FR3, VH-FR4), or is engrafted into at least one CDR region of a variable region of a light chain comprising one or more chicken framework regions (VL-FR1, VL-FR2, VL-FR3,VL-FR4), such as a heavy chain or light chain variable region from a parental chicken generic antibody, as described in Example 3 and illustrated in FIGS. 3-6. The bioactive peptide is engrafted into a CDR such that the flanking framework regions adjacent the CDR in the variable heavy or light chain remain intact. In some embodiments, the entire native CDR sequence of the generic parental antibody is removed and replaced with the bioactive peptide sequence.

[0192] As shown in FIGS. 3-6, in some embodiments, at least one peptide linker sequence (typically from 1 amino acid residue to 20 amino acid residues in length) is included between the CDR-engrafted bioactive peptide amino acid sequence and one or both of the chicken framework region(s) adjacent the bioactive peptide-bearing antibody. The peptide linker may be any flexible linker sequence, such a sequence shown in TABLE 4. In some embodiments, as illustrated in FIGS. 4 and 6, native CDR amino acid residues from the parental antibody are used to form a linker on one or both flanking regions of the bioactive peptide adjacent the framework regions. In some embodiments, at least one amino acid, or at least two, at least three, at least four, at least five, or more, up to all the amino acid residues of the native CDR sequence are retained as linker sequences flanking the bioactive peptide in the heavy or light chain variable region comprising the engrafted bioactive peptide.

[0193] In some embodiments, the bioactive peptide sequence is engrafted into a heavy chain variable region of an antibody, wherein the heavy chain variable region comprises a region having general formula (I):

N--X--B--Y--C (I)

wherein:

[0194] N is an amino terminal region of the heavy chain variable region,

[0195] X is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids;

[0196] B is a bioactive peptide amino acid sequence and consists of an amino acid sequence of no more than 60, or consists of no more than 50 amino acid residues to a minimum of at least 3 amino acid residues;

[0197] Y is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; and

[0198] C is a carboxy terminal region of the heavy chain variable region, and

[0199] wherein at least one of the following applies:

[0200] N comprises FR-1, set forth as SEQ ID NO:24, or a variant thereof, and C comprises FR-2, set forth as SEQ ID NO:25, or a variant thereof; or

[0201] N comprises FR-2, set forth as SEQ ID NO:25, or a variant thereof, and C comprises FR-3, set forth as SEQ ID NO:26, or a variant thereof; or

[0202] N comprises FR-3, set forth as SEQ ID NO:26, or a variant thereof, or flanking SEQ ID NO:27, and C comprises FR-4, set forth as SEQ ID NO:28, or a variant thereof, or flanking SEQ ID NO:29.

[0203] In one embodiment, a bioactive peptide sequence is engrafted into a heavy chain variable region of an antibody, wherein N comprises FR-3, set forth as SEQ ID NO:26, or a variant thereof, or flanking SEQ ID NO:27, and C comprises FR-4, set forth as SEQ ID NO:28, or a variant thereof, or flanking SEQ ID NO:29.

[0204] In one embodiment, the heavy chain comprising one or more chicken framework regions (VH-FR1, VH-FR2, VH-FR3, VH-FR4) and at least one bioactive peptide engrafted into a CDR further comprises the human IgG1 constant region, set forth as SEQ ID O:47, or a variant thereof.

[0205] In some embodiments, the bioactive peptide sequence is engrafted into a light chain variable region of an antibody, wherein the light chain variable region comprises a region having general formula (II):

N--X--B--Y--C (II)

wherein:

[0206] N is an amino terminal region of the light chain variable region,

[0207] X is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids;

[0208] B is a bioactive peptide amino acid sequence and consists of an amino acid sequence of no more than 60, or consists of no more than 50 amino acid residues to a minimum of at least 3 amino acid residues;

[0209] Y is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; and

[0210] C is a carboxy terminal region of the light chain variable region, and

[0211] wherein at least one of the following applies:

[0212] N comprises FR-1, set forth as SEQ ID NO:31, or a variant thereof, or flanking SEQ ID NO:32, and C comprises FR-2, set forth as SEQ ID NO:33, or a variant thereof, or flanking SEQ ID NO:34; or

[0213] N comprises FR-2, set forth as SEQ ID NO:33, or a variant thereof, and C comprises FR-3, set forth as SEQ ID NO:35, or a variant thereof; or

[0214] N comprises FR-3, set forth as SEQ ID NO:35, or a variant thereof, and C comprises FR-4, set forth as SEQ ID NO:36, or a variant thereof.

[0215] In one embodiment, a bioactive peptide is engrafted into a light chain variable region of an antibody, wherein N comprises FR-1, set forth as SEQ ID NO:31, or a variant thereof, or flanking SEQ ID NO:32, and C comprises FR-2, set forth as SEQ ID NO:33, or a variant thereof, or flanking SEQ ID NO:34.

[0216] In one embodiment, the light chain comprising one or more chicken framework regions (VL-FR1, VL-FR2, VL-FR3, VL-FR4) and at least one bioactive peptide engrafted into a CDR further comprises the human lambda light chain, set forth as SEQ ID NO:48, or a variant thereof.

[0217] In one embodiment, the methods according to this aspect of the invention comprise engrafting a bioactive peptide comprising an SGMI core amino acid sequence into at least one of the heavy chain variable region and/or light chain variable region comprising chicken framework regions, wherein the SGMI core amino acid sequence comprises:

TABLE-US-00003 (SEQ ID NO: 5) X₁CTX₂X₃X₄CX₅Q



[0218] wherein:

[0219] X₁ is F or V,

[0220] X₂ is R or K,

[0221] X₃ is K or L,

[0222] X₄ is L or W, and

[0223] X₅ is Y or N; and

[0224] wherein the bioactive peptide inhibits the activity of at least one of MASP-1 or MASP-2.

[0225] In one embodiment, the method comprises engrafting a bioactive peptide selected from the group consisting of SEQ ID NO:6 to SEQ ID NO:11.

[0226] In one embodiment, the method comprises engrafting a bioactive peptide that inhibits the activity of MASP-1, wherein the bioactive peptide is at least one of SEQ ID NO: 6 to 8.

[0227] In one embodiment, the method comprises engrafting a bioactive peptide that inhibits the activity of MASP-2, wherein the bioactive peptide is at least one of SEQ ID NO: 9 to 11.

[0228] In another aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, comprising one or more bioactive peptide amino acid sequence(s), wherein at least one of the bioactive peptide amino acid sequence is engrafted into at least one of: (i) a light chain variable region comprising chicken framework regions and/or (ii) a heavy chain variable region comprising chicken framework regions. In some embodiments, the bioactive peptide amino acid sequence is engrafted into at least one of CDR-H1, CDR-H2 or CDR-H3 of the heavy chain variable region. In some embodiments, the bioactive peptide amino acid sequence is engrafted into at least one of CDR-L1, CDR-L2 or CDR-L3 of the light chain variable region. Various embodiments of the isolated antibodies or antigen-binding fragments thereof comprising the one or more bioactive peptide amino acid sequences engrafted into one or more CDR regions of a heavy and/or light chain are generated according to the methods as described herein.

[0229] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a bioactive peptide amino acid sequence comprising an SGMI core sequence set forth as SEQ ID NO:5. In one embodiment, the isolated antibody or fragment thereof comprises a bioactive peptide sequence engrafted into a CDR, wherein the bioactive peptide sequence comprises or consists of at least one of SEQ ID NO:6 to SEQ ID NO:11. In one embodiment, the isolated antibody or antigen binding fragment thereof comprises at least one of SEQ ID NO:50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or SEQ ID NO:90, or a variant thereof having at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 95%, or at least 98% identity to SEQ ID NO:50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or SEQ ID NO:90. In another embodiment, a nucleic acid molecule is provided that encodes the isolated antibody or antigen fragment thereof, the nucleic acid molecule comprising at least one of SEQ ID NO:49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or SEQ ID NO:89, or a variant thereof having at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 95%, or at least 98% identity to SEQ ID NO:49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 or SEQ ID NO:89.

[0230] In another aspect, the invention provides a method of making a bioactive peptide-bearing antibody, comprising (a) fusing the amino acid sequence of at least one bioactive peptide of interest onto: (i) an amino terminal region of at least one of: a light chain variable region comprising chicken framework regions and/or a heavy chain variable region comprising chicken framework regions, and/or (ii) a carboxy terminal region of at least one of: a light chain constant region and/or a heavy chain constant region; and (b) determining whether the antibody has substantially the same biological activity as the bioactive peptide.

[0231] The methods of this aspect of the invention may be carried out with any bioactive peptide of interest, such as the exemplary bioactive peptides described herein. FIG. 9 is a schematic diagram illustrating the various embodiments of bioactive-peptide bearing antibodies that may be generated using the methods of this aspect of the invention, as further described in Example 4.

[0232] In some embodiments, the method according to this aspect of the invention comprises fusing the amino acid sequence of a bioactive peptide of interest to the amino terminal region of at least one of a light chain variable region comprising chicken framework regions and/or a heavy chain variable region comprising chicken framework regions.

[0233] In some embodiments, the method according to this aspect of the invention comprises fusing the amino acid sequence of a bioactive peptide of interest to the carboxy terminal region of at least one of: a light chain constant region and/or a heavy chain constant region.

[0234] As shown in FIG. 9, in some embodiments, at least one peptide linker sequence (typically from 1 amino acid residue to 20 amino acid residues) is included between the bioactive peptide sequence and the amino terminus of the light or heavy chain region, or between the bioactive peptide sequence and the carboxy terminus of the light or heavy constant region.

[0235] In some embodiments, a bioactive peptide of interest is fused to the amino terminus of a heavy chain variable region comprising the VH-FR1, VH-FR-2, VH-FR-3 and VH-FR-4 amino acid sequences set forth as SEQ ID NO:24, 25, 26 and 28, respectively, or variants thereof. In some embodiments, the heavy chain further comprises a human IgG1 constant region, for example, as set forth as SEQ ID NO:47, or a variant thereof.

[0236] In some embodiments, a bioactive peptide of interest is fused to the carboxy terminus of a heavy chain constant region, wherein the heavy chain further comprises a variable region comprising the VH-FR1, VH-FR-2, VH-FR-3 and VH-FR-4 amino acid sequences set forth as SEQ ID NO:24, 25, 26 and 28, respectively, or variants thereof.

[0237] In some embodiments, a bioactive peptide of interest is fused to the amino terminus of a light chain variable region comprising the VL-FR1, VL-FR2, VL-FR3, VL-FR4 amino acid sequences set forth as SEQ ID NO:31, 33, 35 and 36, respectively, or variants thereof. In some embodiments, the light chain further comprises a human lambda light chain constant region, for example, as set forth as SEQ ID NO:48.

[0238] In one embodiment, the methods according to this aspect of the invention comprise fusing a bioactive peptide comprising an SGMI core amino acid sequence onto at least one of a heavy and/or light chain comprising chicken framework regions, wherein the SGMI core amino acid sequence comprises:

TABLE-US-00004 (SEQ ID NO: 5) X₁CTX₂X₃X₄CX₅Q



[0239] wherein:

[0240] X₁ is F or V,

[0241] X₂ is R or K,

[0242] X₃ is K or L,

[0243] X₄ is L or W, and

[0244] X₅ is Y or N; and

[0245] wherein the bioactive peptide inhibits the activity of at least one of MASP-1 or MASP-2.

[0246] In one embodiment, the method comprises fusing a bioactive peptide selected from the group consisting of SEQ ID NO:6 to SEQ ID NO:11.

[0247] In one embodiment, the method comprises fusing a bioactive peptide that inhibits the activity of MASP-1, wherein the bioactive peptide is at least one of SEQ ID NO: 6 to 8.

[0248] In one embodiment, the method comprises fusing a bioactive peptide that inhibits the activity of MASP-2, wherein the bioactive peptide is at least one of SEQ ID NO:9 to 11.

[0249] In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, comprising one or more bioactive peptide amino acid sequence(s), wherein at least one bioactive peptide amino acid sequence is fused to at least one of (i) the amino terminal region of at least one of: a light chain variable region comprising chicken framework regions and/or a heavy chain variable region comprising chicken framework regions; or (ii) the carboxy terminal region of at least one of: a light chain constant region and/or a heavy chain constant region, wherein the antibody has substantially the same biological activity as the bioactive peptide. Various embodiments of the isolated antibodies or fragments thereof comprising the one or more bioactive peptide amino acids fused to the amino terminal region of a light or heavy chain variable region, or fused to the carboxy terminal region of a light chain constant region or a heavy chain constant region are generated according to the methods as described herein.

[0250] In one embodiment, the isolated antibody or antigen binding fragment thereof comprises a bioactive peptide amino acid sequence comprising an SGMI core sequence set forth as SEQ ID NO:5. In one embodiment, the isolated antibody or fragment thereof comprises a bioactive peptide fused onto the amino terminal region of a light or heavy chain variable region, or fused to the carboxy terminal region of a light chain constant region or a heavy chain constant region, wherein the bioactive peptide sequence comprises or consists of at least one of SEQ ID NO:6 to SEQ ID NO:11. In one embodiment, the isolated antibody or antigen binding fragment thereof comprises at least one of SEQ ID NO:94, 96, 98, 100, 102, 104, 106, or SEQ ID NO:108, or a variant thereof having at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 95%, or at least 98% identity to SEQ ID NO:94, 96, 98, 100, 102, 104, 106, or SEQ ID NO:108. In another embodiment, a nucleic acid molecule is provided that encodes the isolated antibody or antigen fragment thereof, the nucleic acid molecule comprising at least one of SEQ ID NO:93, 95, 97, 99, 101, 103, 105 or SEQ ID NO:107, or a variant thereof having at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 95%, or at least 98% identity to SEQ ID NO:93, 95, 97, 99, 101, 103, 105 or SEQ ID NO:107.

[0251] In another aspect, the invention provides an isolated polypeptide comprising: (i) a region comprising an SGMI core sequence, the SGMI core sequence comprising an amino acid sequence according to: X₁CTX₂X₃X₄CX₅Q (SEQ ID NO:5), wherein: X₁ is F or V, X₂ is R or K, X₃ is K or L, X₄ is L or W, and X₅ is Y or N; and (ii) a region comprising human IgG1 Fc, wherein the polypeptide inhibits the activity of at least one of MASP-1 or MASP-2.

[0252] In one embodiment, the region comprising the human IgG1 Fc region is located at the amino terminus of the region comprising the SGMI core sequence. In another embodiment, the region comprising the human IgG1 Fc region is located at the carboxy terminus of the region comprising the SGMI core sequence.

[0253] In one embodiment, the region comprising the IgG1 Fc comprises or consists of SEQ ID NO:12, or a variant thereof.

[0254] In one embodiment, the region comprising the SGMI core sequence comprises or consists of at least one of SEQ ID NO:6 to SEQ ID NO:11. In one embodiment, the region comprising human IgG1 Fc is fused directly to at least one of SEQ ID NO:6 to SEQ ID NO:11. In one embodiment, the polypeptide further comprises a linker region of from 1 amino acid residue to 20 amino acid residues, wherein the linker region is included between the region comprising the SGMI core sequence and the region comprising human IgG1 Fc. In one embodiment, the linker sequence comprises at least one of SEQ ID NO:13 or SEQ ID NO:14. In one embodiment, the polypeptide comprises at least one of SEQ ID NO:16 or SEQ ID NO:18, or a variant thereof having at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 95%, or at least 98% identity to SEQ ID NO:16 or SEQ ID NO:18.

[0255] In another embodiment, a nucleic acid molecule is provided that encodes the polypeptide, the nucleic acid molecule comprising at least one of SEQ ID NO:15 or SEQ ID NO:17, or a variant thereof having at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 95%, or at least 98% identity to SEQ ID NO:15 or SEQ ID NO:17.

[0256] Methods for Producing Antibodies

[0257] The antibodies and polypeptides of the invention can be produced by standard recombinant genetic engineering methods, which are well known to those of skill in the art of molecular biology and immunology.

[0258] For recombinant production of a fusion polypeptide of the invention, DNA sequences encoding the polypeptide components of a fusion polypeptide (e.g., a bioactive peptide sequence and a heavy chain or light chain polypeptide sequence of interest) may be assembled using conventional methodologies. In one example, the components may be assembled separately and ligated into an appropriate expression vector. For example, the 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase.

[0259] For recombinant production of a bioactive peptide sequence engrafted into a CDR region of a heavy chain variable region or a light chain variable region, the nucleic acid components may be assembled and ligated into an appropriate expression vector, with or without a peptide linker, such that the nucleic acid sequence encoding the bioactive peptide sequence is in phase with the nucleic acid sequence encoding the adjacent framework regions of the variable light chain or variable heavy chain.

[0260] As described herein, a peptide linker sequence may be employed to separate a bioactive peptide sequence from a heterologous polypeptide sequence by some defined distance, for example a distance sufficient to ensure that the advantages of the invention are achieved, e.g., biological activity of the bioactive peptide engrafted into a CDR region, or fused onto an amino or carboxy terminal region of a heavy or light chain polypeptide. Such a peptide linker sequence may be incorporated into the bioactive peptide-bearing antibodies using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based, for example, on the factors such as: (1) their ability to adopt a flexible extended conformation; and (2) their inability to adopt a secondary structure that could interfere with the activity of the bioactive peptide sequence. Illustrative peptide linker sequences, for example, may contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed herein as well as those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 20 amino acids in length, for example.

[0261] The invention further includes nucleic acid molecules encoding the polypeptides of the invention as described herein. A vector that contains such a nucleic acid is also included. When the method is performed in a host cell, the host cell is first transformed or transfected with an exogenous nucleic acid encoding the stabilized polypeptide, then the polypeptides and antibodies are expressed and recovered. The host cells can be prokaryotic, such as bacteria, or eukaryotic, as described further herein.

[0262] In many embodiments, the nucleic acids encoding a subject monoclonal antibody are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded antibody.

[0263] In some embodiments, the invention provides a cell comprising a nucleic acid molecule encoding an antibody or polypeptide of the invention.

[0264] In some embodiments, the invention provides an expression cassette comprising a nucleic acid molecule encoding an antibody or polypeptide of the invention.

[0265] In some embodiments, the invention provides a method of producing an antibody or polypeptide of the invention comprising culturing a cell comprising a nucleic acid molecule encoding an antibody of the invention.

[0266] According to certain related embodiments there is provided a recombinant host cell which comprises one or more constructs as described herein; a nucleic acid encoding any antibody, CDR, VH or VL domain, or antigen-binding fragment thereof; and a method of production of the encoded product, which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, an antibody or antigen-binding fragment thereof, may be isolated and/or purified using any suitable technique, and then used as desired.

[0267] For example, any cell suitable for expression of expression cassettes may be used as a host cell, for example, yeast, insect, plant, etc., cells. In many embodiments, a mammalian host cell line that does not ordinarily produce antibodies is used, examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CV1 cells transformed by SV40 (COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, (1980); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1). Additional cell lines will become apparent to those of ordinary skill in the art. A wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.

[0268] Methods of introducing nucleic acids into cells are well known in the art. Suitable methods include electroporation, particle gun technology, calcium phosphate precipitation, cationic lipid nucleic acid delivery, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3d ed., Wiley & Sons, 1995. In some embodiments, lipofectamine and calcium mediated gene transfer technologies are used.

[0269] After the subject nucleic acids have been introduced into a cell, the cell is typically incubated, normally at 37° C., sometimes under selection, for a suitable time to allow for the expression of the antibody. In most embodiments, the antibody is typically secreted into the supernatant of the media in which the cell is growing in.

[0270] In mammalian host cells, a number of viral-based expression systems may be utilized to express a subject antibody. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0271] For long-term, high-yield production of recombinant antibodies, stable expression may be used. For example, cell lines, which stably express the antibody molecule, may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and grow to form foci which in turn can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

[0272] Once an antibody molecule of the invention has been produced, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In many embodiments, antibodies are secreted from the cell into culture medium and harvested from the culture medium. For example, a nucleic acid sequence encoding a signal peptide may be included adjacent the coding region of the antibody or fragment. Such a signal peptide may be incorporated adjacent to the 5' end of the amino acid sequences set forth herein for the subject antibodies in order to facilitate production of the subject antibodies.

[0273] In one embodiment, the antibodies according to certain embodiments of the present invention may be generated using an in vitro system based on the DT40 chicken B cell lymphoma line. The DT40 chicken B cell lymphoma line has been used for antibody evolution ex vivo (Cumbers, S. J. et al. Nat Biotechnol 20:1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23:731-735 (2005).). DT40 cells command enormous potential V region sequence diversity, as they can access two distinct physiological pathways for diversification, gene conversion and somatic hypermutation, which create templated and nontemplated mutations, respectively (Maizels, N., Immunoglobulin gene diversification. Ann. Rev. Genet. 39:23-46 (2005)). However, the utility of DT40 cells for antibody evolution has been limited in practice because as in other transformed B cell lines--diversification occurs at less than 1% the physiological rate. Diversification can be accelerated several-fold by disabling the homologous recombination pathway (Cumbers et al., supra), but cells thus engineered lose the ability to carry out efficient gene targeting. Diversification can also be accelerated by treatment of cells with the histone deacetylase inhibitor, trichostatin A (Seo et al., supra), but resulting mutations are exclusively templated, which limits potential diversity and may not produce antibodies of required affinity or specificity.

[0274] The DT40 cells used herein to generate antibodies are modified to accelerate the rate of immunoglobulin (Ig) gene diversification without sacrificing the capacity for further genetic modification or the potential for both gene conversion and somatic hypermutation to contribute to mutagenesis. This was accomplished by putting Ig gene diversification under control of the potent E. coli lactose operator/repressor regulatory network. Multimers consisting of approximately 100 polymerized repeats of the potent E. coli lactose operator (PolyLacO) were inserted upstream of the rearranged and expressed Igλ and IgH genes by homologous gene targeting. Regulatory factors fused to lactose repressor protein (Lad) can then be tethered to the LacO regulatory elements to regulate diversification, taking advantage of the high affinity (KD=10^-14 M) of lactose repressor for operator DNA. DT40 PolyLacO-λ_R cells, in which PolyLacO was integrated only at Igλ, exhibited a 5-fold increase in Ig gene diversification rate relative to the parental DT40 cells prior to any engineering (Cummings, W. J. et al. PLoS Biol 5, e246 (2007)). Diversification was further elevated in cells engineered to carry PolyLacO targeted to both the Igλ and the IgH genes ("DTLacO").

[0275] Pharmaceutical Compositions

[0276] In another aspect, the invention provides pharmaceutical compositions comprising the bioactive peptide-bearing antibodies and fragments thereof, as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the invention provides compositions comprising bioactive peptide-bearing antibodies and fragments thereof capable of inhibiting activation of the lectin complement pathway. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the bioactive peptide-bearing antibody (and any other therapeutic agents combined therewith). Exemplary pharmaceutically acceptable carriers for polypeptides are described in U.S. Pat. No. 5,211,657 to Yamada. The bioactive peptide-bearing antibodies and polypeptides may be formulated into preparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. The invention also contemplates local administration of the compositions by coating medical devices and the like.

[0277] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0278] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0019369 A1. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.

[0279] For intra-articular or intravenous delivery, the bioactive peptide-bearing antibodies or polypeptides may be carried in above-described liquid or gel carriers that are injectable, above-described sustained-release delivery vehicles that are injectable, or a hyaluronic acid or hyaluronic acid derivative.

[0280] For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriately sterile delivery systems (e.g., liquids; gels, suspensions, etc.) can be used to administer the present invention.

[0281] The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

[0282] To achieve high concentrations of the subject antibodies for local delivery, the antibodies may be formulated as a suspension of particulates or crystals in solution for subsequent injection, such as for intramuscular injection of a depot.

[0283] Therapeutic Methods:

[0284] In another aspect, the invention provides methods of inhibiting lectin pathway complement activation in a mammalian subject, such as a human subject, comprising administering a composition comprising a bioactive peptide-bearing antibody or polypeptide as disclosed herein to said human subject, wherein the bioactive peptide inhibits activation of the lectin complement pathway. As described herein, the bioactive peptides SGMI-1 and SGMI-2 block the lectin pathway of complement activation without affecting the classical or alternative pathways (Heja et al., 2012. Proc. Natl. Acad. Sci. 109:10498). As described in U.S. Pat. No. 7,919,094, co-pending U.S. patent application Ser. No. 13/083,441, and co-pending U.S. patent application Ser. No. 12/905,972 (each of which is assigned to Omeros Corporation, the assignee of the instant application), each of which is hereby incorporated by reference, MASP-2 dependent lectin complement activation has been implicated as contributing to the pathogenesis of numerous acute and chronic disease states, including MASP-2-dependent complement mediated vascular condition, an ischemia reperfusion injury, atherosclerosis, inflammatory gastrointestinal disorder, a pulmonary condition, an extracorporeal reperfusion procedure, a musculoskeletal condition, a renal condition, a skin condition, organ or tissue transplant, nervous system disorder or injury, a blood disorder, a urogenital condition, diabetes, chemotherapy or radiation therapy, malignancy, an endocrine disorder, a coagulation disorder, a thrombotic microangiopathy, or an ophthalmologic condition. Therefore, the lectin pathway inhibitory antibodies of the present invention may be used to treat the above-referenced diseases and conditions.

[0285] In one embodiment, the composition is formulated to specifically inhibit MASP-1 or MASP-2 activity. In one embodiment, the composition is formulated to inhibit MASP-1 activity. In one embodiment, the composition is formulated to inhibit MASP-2 activity.

[0286] In one embodiment, the composition is formulated for systemic delivery, such as, by intra-arterial, intravenous, intracranial, intramuscular, inhalational, nasal or subcutaneous administration.

[0287] As used herein, the terms "systemic delivery" and "systemic administration" are intended to include but are not limited to oral and parenteral routes including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively result in dispersal of the delivered antibody to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the present compositions include intravenous, intramuscular, subcutaneous, and inhalational. It will be appreciated that the exact systemic administration route for selected agents utilized in particular compositions of the present invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of administration.

[0288] The bioactive peptide-bearing antibodies and polypeptides can be delivered into a subject in need thereof by any suitable means. Methods of delivery include administration by oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), inhalation (such as via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual routes of administration, and can be formulated in dosage forms appropriate for each route of administration.

[0289] The compositions of the present invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. For example, compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. These factors will include the extent of progress of the condition being treated, the patient's age, sex and weight, and other clinical factors. The dosage for each individual agent will vary as a function of the particular antibody that is included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delivery vehicle). In addition, the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).

[0290] Therapeutic efficacy of MASP-2 and MASP-1 inhibitory compositions and methods of the present invention in a given subject, and appropriate dosages, can be determined in accordance with complement assays well known to those of skill in the art. Complement generates numerous specific products. During the last decade, sensitive and specific assays have been developed and are available commercially for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize antibodies that react with new antigens (neoantigens) exposed on the fragment, but not on the native proteins from which they are formed, making these assays very simple and specific. Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their `desArg` fragments, which are the major forms found in the circulation. Unprocessed fragments and C5a.sub.desArg are rapidly cleared by binding to cell surface receptors and are hence present in very low concentrations, whereas C3a.sub.desArg does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of the fluid-phase product of membrane attack pathway activation, sC5b-9, provides evidence that complement is being activated to completion. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of these two pathways has generated the activation products.

[0291] The inhibition of lectin-dependent complement activation is characterized by at least one of the following changes in a component of the complement system that occurs as a result of administration of an anti-MASP-2 antibody in accordance with the present invention: the inhibition of the generation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a and/or C5b-9 (MAC), the reduction of C4 cleavage and C4b deposition, or the reduction of C3 cleavage and C3b deposition.

[0292] Articles of Manufacture

[0293] In another aspect, the present invention provides an article of manufacture containing a bioactive peptide-bearing antibody, or antigen binding fragment thereof, or polypeptide as described herein in a unit dosage form suitable for therapeutic administration to a human subject, such as, for example, a unit dosage in the range of 1 mg to 5000 mg, such as from 1 mg to 2000 mg, such as from 1 mg to 1000 mg, such as 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg, or 1000 mg. In some embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the bioactive peptide-bearing antibody or antigen binding fragment thereof or polypeptide of the invention. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for administering the antibody composition to the patient. Articles of manufacture and kits comprising combinatorial therapies described herein are also contemplated.

[0294] The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

Example 1

Overview of the Strategy for Generating Inhibitory MASP Polypeptides by Engrafting Bioactive Peptides into or onto Antibodies, or Fragments Thereof

[0295] Rationale:

[0296] The generation of specific inhibitors of MASP-1 and MASP-2, termed SGMI-1 and SGMI-2, respectively, is described in Heja et al., J Biol Chem 287:20290 (2012) and Heja et al., PNAS 109:10498 (2012), each of which is hereby incorporated herein by reference. SGMI-1 and SGMI-2 are each 36 amino acid peptides which were selected from a phage library of variants of the Schistocerca gregaria protease inhibitor 2 in which six of the eight positions of the protease binding loop were fully randomized. Subsequent in vitro evolution yielded mono-specific inhibitors with single digit nM K_I values (Heja et al., J. Biol. Chem. 287:20290, 2012). Structural studies revealed that the optimized protease binding loop forms the primary binding site that defines the specificity of the two inhibitors. The amino acid sequences of the extended secondary and internal binding regions are common to the two inhibitors and contribute to the contact interface (Heja et al., 2012. J. Biol. Chem. 287:20290). Mechanistically, both SGMI-1 and SGMI-2 block the lectin pathway of complement activation without affecting the classical or alternative pathways (Heja et al., 2012. Proc. Natl. Acad. Sci. 109:10498).

[0297] The amino acid sequences of the SGMI-1 and SGMI-2 inhibitors are set forth below:

TABLE-US-00005 SGMI-1-full-length: (SEQ ID NO: 6) LEVTCEPGTTFKDKCNTCRCGSDGKSAFCTRKLCYQ SGMI-1-medium: (SEQ ID NO: 7) TCEPGTTFKDKCNTCRCGSDGKSAFCTRKLCYQ SGMI-1-short: (SEQ ID NO: 8) TCRCGSDGKSAFCTRKLCYQ SGMI-2-full-length: (SEQ ID NO: 9) LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ SGMI-2-medium: (SEQ ID NO: 10) TCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ SGMI-2-short: (SEQ ID NO: 11) ................TCRCGSDGKSAVCTKLWCNQ

[0298] The above SGMI sequences share a core SGMI sequence (underlined), which is set forth below as SEQ ID NO:5:

TABLE-US-00006 (SEQ ID NO: 5) X₁CTX₂X₃X₄CX₅Q

[0299] wherein:

[0300] X₁ is F or V,

[0301] X₂ is R or K,

[0302] X₃ is K or L,

[0303] X₄ is L or W, and

[0304] X₅ is Y or N

[0305] The bioactive peptides derived from SGMI-1 (set forth as SEQ ID NOs:6-8) and SGMI-2 (set forth as SEQ ID NO:9-11) are highly specific inhibitors of MASP-1 and MASP-2, respectively. However, as peptides they have limited potential for use in biological studies and therapeutic applications. To address these limitations, we engrafted these bioactive peptide amino acid sequences (i.e., amino acid sequences encoding the bioactive peptides) into three distinct scaffolds: (1) onto the amino terminus of human IgG1 Fc region to create an Fc-fusion protein, as described in Example 2; (2) into selected CDRs of a chimeric chicken (variable regions)--human (IgG1 and Igλ constant regions) antibody, as described in Example 3; and (3) onto the amino or carboxy termini of the heavy and/or light chains of an antibody, as described in Example 4.

[0306] As described herein, introduction of a bioactive peptide sequence into an antibody scaffold results in a product with the bioactivity of the bioactive peptide and with improved therapeutic properties, such as a longer half-life and antibody effector functions.

Example 2

[0307] This Example describes the generation of recombinant SGMI-Fc fusion proteins and demonstrates that these fusion proteins are able to inhibit the lectin pathway.

[0308] Methods:

[0309] To express the SGMI-IgG1 Fc fusion proteins, polynucleotides encoding the SGMI-1 (SEQ ID NO:6) and SGMI-2 (SEQ ID NO:9) peptides were synthesized (DNA 2.0) and inserted into the expression vector pFUSE-hIgG1-Fc2 (InvivoGen) between nucleotide sequences encoding the IL-2 signal sequence and the human IgG1 Fc region (SEQ ID NO:12). In some embodiments, an optional flexible polypeptide linker (e.g., SEQ ID NO:13 or SEQ ID NO:14) was included between the SGMI peptide and the IgG1 Fc region.

[0310] Exemplary Flexible Polypeptide Linker Sequences:

TABLE-US-00007 (SEQ ID NO: 13) GTGGGSGSSSRS (SEQ ID NO: 14) GTGGGSGSSS

[0311] It is noted that in another embodiment, the invention encompasses an alternative version of the SGMI-IgG1 Fc fusion proteins containing the IgG1 Fc region fused to the amino terminus of the SGMI peptides. It is further noted that in further embodiments, the invention encompasses alternative versions of the SGMI-IgG1 Fc fusion proteins comprising a bioactive peptide amino acid sequence comprising the core SGMI sequence (SEQ ID NO:5), and having a length of from at least 9 amino acid residues to 36 amino acid residues, including various truncated versions of SGMI-1 or SGMI-2 bioactive peptides (e.g., SGMI peptides comprising the core sequence of SEQ ID NO:5, such as any of SEQ ID NO:6 to SEQ ID NO:11).

[0312] The resulting constructs are described as follows:

[0313] A polynucleotide encoding the polypeptide fusion comprising the human IL-2 signal sequence, SGMI-1, linker and human IgG1-Fc (pFUSE-SGMI-1Fc), is set forth as SEQ ID NO:15, which encodes the mature polypeptide fusion comprising SGMI-1 (underlined), linker region (italicized) and human IgG1-Fc (together referred to as "SGMI-1Fc"), which is set forth as SEQ ID NO:16.

TABLE-US-00008 SEQ ID NO: 16 LEVTCEPGTTFKDKCNTCRCGSDGKSAFCTRKLCYQGTGGGS GSSSRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK

[0314] A polynucleotide encoding the polypeptide fusion comprising the human IL-2 signal sequence, SGMI-2, linker and human IgG1-Fc (pFUSE-SGMI-2Fc), is set forth as SEQ ID NO:17, which encodes the mature polypeptide fusion comprising SGMI-2 (underlined), linker region (italicized) and human IgG1-Fc (together referred to as "SGMI-2Fc"), which is set forth as SEQ ID NO:18:

TABLE-US-00009 SEQ ID NO: 18 LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQGTGGGS GSSSRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK

[0315] Production of Recombinant Proteins:

[0316] Freestyle 293-F or Expi293F cells (Invitrogen) were transiently transfected according to the supplier's protocol with one of the two expression plasmids (pFUSE-SGMI-1Fc (SEQ ID NO:15) and pFUSE-SGMI-2Fc (SEQ ID NO:17). After four days of incubation at 37° C., the culture media were harvested. The Fc-fusion proteins were purified by Protein A affinity chromatography.

[0317] Assays Measuring Activation of the Lectin Pathway.

[0318] The Wieslab® Complement System Screen (Euro Diagnostic, Malmo, Sweden), MBL assay measures C5b-C9 deposition in conditions that isolated the lectin pathway. The assay was carried out according to the manufacturer's instructions with the Fc fusion proteins being tested at final concentrations of 400 nM.

[0319] FIG. 1 is a bar graph showing the inhibitory activity of the SGMI-1Fc (SEQ ID NO:16) or SGMI-2Fc (SEQ ID NO:18) fusion proteins in comparison to the positive and negative sera provided with the assay kit, as well as an isotype control antibody. As shown in FIG. 1, both SGMI-1Fc and SGMI-2Fc inhibit the activation of the lectin pathway, whereas the isotype control antibody does not.

[0320] The SGMI-1Fc and SGMI-2Fc fusion proteins were also tested for the ability to inhibit deposition of C3b from 1% serum on a mannan-coated 96-well plate, which is another measure of lectin pathway activity. SGMI-1Fc and SGMI-2Fc were pre-incubated with 1% normal human serum for one hour on ice before addition to wells coated with mannan (2 μg/well). C3b deposition was measured by ELISA as described in Schwaeble et al. PNAS 108:7523, 2011.

[0321] FIG. 2 graphically illustrates the level of C3b deposition for 1% normal human serum plus isotype control, SGMI-1Fc or SGMI-2Fc over a concentration range of 0.15 to 1000 nM. As shown in FIG. 2, both SGMI-1Fc and SGMI-2Fc inhibited C3b deposition from normal serum in mannan-coated ELISA wells, with IC50 values of approximately 27 nM and 300 nM, respectively.

[0322] These results demonstrate that the MASP-1 and MASP-2 inhibitory functions of the SGMI peptides are retained in the SGMI-1Fc and SGMI-2Fc fusion proteins.

Example 3

[0323] This Example describes the generation of chimeric chicken (V region)/human constant region) antibodies comprising a bioactive peptide amino acid sequence (e.g., SGMI-1 or SGMI-2) engrafted into at least one CDR region of a heavy chain variable region and/or at least one CDR region of a light chain variable region (e.g., CDR-H3 and/or CDR-L1).

[0324] Background/Rationale:

[0325] A modified DT40 cell line, DTLacO, that permits reversible induction of diversification of a particular polypeptide, is described in WO2009029315 and US2010093033, each of which is hereby incorporated herein by reference. DT40 is a chicken B cell line that is known to constitutively mutate its heavy and light chain immunoglobulin (Ig) genes in culture. Like other B cells, this constitutive mutagenesis targets mutations to the V region of Ig genes, and thus, the CDRs of the expressed antibody molecules. Constitutive mutagenesis in DT40 cells takes place by gene conversion using as donor sequences an array of non-functional V gene segments (pseudo-V genes; ψV) situated upstream of each functional V region. Deletion of the ψV region was previously shown to cause a switch in the mechanism of diversification from gene conversion to somatic hypermutation, the mechanism commonly observed in human B cells. The DT40 chicken B cell lymphoma line has been shown to be a promising starting point for antibody evolution ex vivo (Cumbers, S. J. et al. Nat Biotechnol 20, 1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23, 731-735 (2005)). DT40 cells proliferate robustly in culture, with an 8-10 hour doubling time (compared to 20-24 hr for human B cell lines), and they support very efficient homologous gene targeting (Buerstedde, J. M. et al. Embo J 9, 921-927 (1990)). DT40 cells command enormous potential V region sequence diversity given that they can access two distinct physiological pathways for diversification, gene conversion and somatic hypermutation, which create templated and nontemplated mutations, respectively (Maizels, N. Annu Rev Genet. 39, 23-46 (2005)). Diversified heavy and light chain immunoglobulins (Igs) are expressed in the form of a cell-surface displayed IgM. Surface IgM has a bivalent form, structurally similar to an IgG molecule. Cells that display IgM with specificity for a particular antigen can be isolated by binding either immobilized soluble or membrane displayed versions of the antigen. However, utility of DT40 cells for antibody evolution has been limited in practice because--as in other transformed B cell lines--diversification occurs at less than 1% the physiological rate.

[0326] In the system used in this example, as described in WO2009029315 and US2010093033, the DT40 cells were engineered to accelerate the rate of Ig gene diversification without sacrificing the capacity for further genetic modification or the potential for both gene conversion and somatic hypermutation to contribute to mutagenesis. Two key modifications to DT40 were made to increase the rate of diversification and, consequently, the complexity of binding specificities in the library of cells (Yabuki et al., PLoS One 7:e36032, 2012). First, Ig gene diversification was put under the control of the potent E. coli lactose operator/repressor regulatory network. Multimers consisting of approximately 100 polymerized repeats of the potent E. coli lactose operator (PolyLacO) were inserted upstream of the rearranged and expressed Igλ and IgH genes by homologous gene targeting. Regulatory factors fused to lactose repressor protein (Lad) can then be tethered to the LacO regulatory elements to regulate diversification, taking advantage of the high affinity (k_D=10^-14 M) of lactose repressor for operator DNA. DT40 PolyLacO-λ_R cells, in which PolyLacO was integrated only at Igλ, exhibited a 5-fold increase in Ig gene diversification rate relative to the parental DT40 cells prior to any engineering (Cummings, W. J. et al. PLoS Biol 5, e246 (2007)). Diversification was further elevated in cells engineered to carry PolyLacO targeted to both the Igλ and the IgH genes ("DTLacO"). DTLacO cells were demonstrated to have diversification rates 2.5- to 9.2-fold elevated relative to the 2.8% characteristic of the parental DT40 PolyLacO-λ_R LacI-HP1 line. Thus, targeting PolyLacO elements to both the heavy and light chain genes accelerated diversification 21.7-fold relative to the DT40 parental cell line. Tethering regulatory factors to the Ig loci not only alters the frequency of mutagenesis, but also can change the pathway of mutagenesis creating a larger collection of unique sequence changes (Cummings et al. 2007; Cummings et al. 2008). Second, a diverse collection of sequence starting points for the tethered factor-accelerated Ig gene diversification was generated. These diverse sequence starting points were added to DTLacO by targeting rearranged Ig heavy-chain variable regions, isolated from a two month old chick, to the heavy chain locus. The addition of these heavy chain variable regions created a repertoire of 10⁷ new starting points for antibody diversification. Building these new starting points into the DTLacO cell line permits the identification of clones that bind a particular target, and then enable rapid affinity maturation by the tethered factors. Following affinity maturation, a full-length, recombinant chimeric IgG is made by cloning the matured, rearranged heavy- and light-chain variable sequences (VH and Vλ consisting of chicken framework regions and the CDRs) into expression vectors containing human IgG1 and lambda constant regions. These recombinant mAbs are suitable for in vitro and in vivo applications, and they serve as the starting point for humanization.

[0327] Through the use of the DTLacO system, the inventors have observed large inserts of more than 25 amino acids in CDR-H3 of the chicken heavy (VH) and CDR-L1 of the chicken light (VL) chain variable regions. In contrast, the average CDR-H3 size for mice and humans is much smaller (average size of 9 amino acids and 12 amino acids, respectively). Given the potential of these chicken CDRs to accommodate large blocks of sequence, the inventors tested the capacity of the CDRs to present the bioactive peptides SGMI-1 and SGMI-2 in an active conformation. The value of this strategy is several-fold: (1) the in vivo stability of an antibody is conferred to the SGMI-inhibitors, an important benefit for therapeutic applications; (2) integration of the VH and VL genes carrying a bioactive peptide, (such as the SGMI-1 or -2 sequence) into the DTLacO cell line provides the means for ex vivo mutagenesis and selection of V regions with greater affinity and potency; (3) engrafting a first bioactive peptide (e.g. SGMI-1) into one of the long CDRs and engrafting a second bioactive peptide (e.g. SGMI-2) into another of the long CDRs of an antibody, will create a bi-specific antibody that has two functional activities (e.g., inhibits MASP-1 and MASP-2). While this example describes the invention in the context of engrafting SGMI sequences into the CDR-H3 and/or CDR-L1 of the chicken variable regions and retaining inhibitory activity, it will be understood by one of skill in the art that results here establish a paradigm for the display and delivery of other bio-active peptides within CDRs of the variable light and/or heavy chain of antibodies comprising chicken variable regions.

[0328] Methods:

[0329] To generate the chimeric chicken-human antibodies bearing bioactive peptides (SGMI-1 or SGMI-2) within CDR-H3 and/or CDR-L1, polynucleotides encoding the SGMI-1 and SGMI-2 peptides were inserted by In-Fusion cloning (Clontech primers shown in Table 3) into the pcDNA3 (Invitrogen)-based expression vectors of chicken-human chimeric heavy- and light-chain antibodies, described in WO2009029315 and US2010093033, incorporated herein by reference.

TABLE-US-00010 TABLE 3 PCR primers used for cloning the SGMI-1 and SGMI-2 polynucleotides into chicken V-regions, resulting in SGMI-1L-IgG1 and SGMI-2L-Igλ chimeric antibodies. Primer Sequence SGMI-1 Forward CTACTGCGCCAAACTCGAGGTGACATGTGA (SEQ ID NO: 19) SGMI-1 Reverse CGTGGCCCCATGCCTGGTAGCACAATTTCC (SEQ ID NO: 20) SGMI-2 Forward CGGGGGTGGCAGC TTGGAAGTGACGTGTGA (SEQ ID NO: 21) SGMI-2 Reverse AGCCATAATAGTA CTGGTTACACCAGAGCT (SEQ ID NO: 22)

[0330] 1. SGMI-1 and SGMI-2 Engrafted into the CDR-H3 of a Parental Chicken Heavy Chain Variable Region.

[0331] The DT40 chicken heavy chain variable region was chosen as the starting parental clone for use as a scaffold into which SGMI-1 or SGMI-2 peptide sequences were engrafted into the CDR-H3 region, as shown in FIGS. 3 and 4.

[0332] FIG. 3 illustrates an exemplary parental (DTLacO) variable heavy chain polypeptide sequence compared to a modified version of the variable heavy chain polypeptide sequence comprising a bioactive peptide amino acid sequence engrafted within CDR-H3. As shown in FIG. 3, the chicken heavy chain variable region contains three CDRs (CDR-H1, CDR-H2 and CDR-H3), flanked by four framework regions (FR-1, FR-2, FR-3 and FR-4). The inventors have surprisingly discovered that by engrafting a bioactive peptide (e.g. SGMI-1 or SGMI-2) into a CDR of the heavy chain variable region of the DT40 parental chicken antibody (which provides the antibody scaffold), the biological activity of the bioactive peptide was conferred to the parental antibody comprising the engrafted bioactive peptide sequence.

[0333] The parental chicken antibody provides the framework regions (FR1, FR2, FR3 and FR4) of the heavy and light chains, which are conserved between various clones. Any parental chicken antibody clone may be selected for use as a scaffold. In some embodiments, the parental chicken antibody clone may be selected based on desirable properties, such as stability.

[0334] Exemplary parental chicken heavy chain variable regions are provided below. As shown in FIG. 4, although the native CDR regions vary between parental clones, the Framework regions between the CDRs are conserved in chicken, accordingly, a consensus FR-1, FR-2, FR-3 and FR-4 sequence derived from an alignment of several different parental chicken heavy chain regions is also provided below. As further shown in FIG. 4, in FR-3 there is a conserved cysteine (C) residue at the third position N-terminal to CDR-H3 in the parental clones (corresponding to the cysteine at position 31 in SEQ ID NO:26), which is retained in FR-3 in the constructs containing an engrafted bioactive peptide in CDR-H3. As further shown in FIG. 4, in FR-4 there is a conserved tryptophan (W) residue at the position immediately adjacent to CDR-H3 in the parental clones (corresponding to the tryptophan at position 1 in SEQ ID NO:28), which is retained in FR-4 in the constructs containing an engrafted bioactive peptide in CDR-H3.

TABLE-US-00011 DTLacO parental chicken (clone #1) heavy chain variable region: (DTLacO VH) (SEQ ID NO: 23) AVTLDESGGGLQTPGRALSLVCKASGFTFSSYNMGWVRQAPGKGLE FVAGIDNTGRYTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTG TYYCAKAAGGSGYCGSGAYIDAWGHGTEVIVSS

[0335] The V_H CDRs (31-35 (H1); 50-66 (H2); and 99-114 (H3) are underlined, and the Framework regions (1-30 (FR-1); 36-49 (FR-2); 67-98 (FR-3) and 115-125 (FR-4) are italicized.

TABLE-US-00012 DTLacO parental chicken (clone #2) heavy chain variable region (DTLacO VH) (SEQ ID NO: 91) AVTLDESGGGLQTPGGALSLVCKASGFTFSSNAMGWVRQAPGKGLEW VAGIDDDGSGTRYAPAVKGRATISRDNGQSTLRLQLNNLRAEDTGIY YCTKCAYSSGCDYEGGYIDAWGHGTEVIVSS Conserved FR-1 region from the DTLacO VH is set forth as SEQ ID NO: 24: AVTLDESGGGLQTPGXALSLVCKASGFTFS Where X = R or G Conserved FR-2 region from the DTLacO VH is set forth as SEQ ID NO: 25: WVRQAPGKGLEXVA Where X = F or W Conserved FR-3 region from the DTLacO VH is set forth as SEQ ID NO: 26: RATISRDNGQSTX₁RLQLNNLRAEDTGIYYCX₂K Where: X₁ = V or L, and X₂ = A or T Conserved FR-3 flanking region adjacent to CDR-H3 is set forth as SEQ ID NO: 27: YYCXK where X = A or T Conserved FR-4 region from the DTLacO VH is set forth as SEQ ID NO: 28: WGHGTEVIVSS Conserved FR-4 flanking region adjacent to CDR-H3 is set forth as SEQ ID NO: 29 WGHGT

[0336] As shown in FIG. 4, in some embodiments, a peptide linker was included at the amino terminus of the bioactive peptide, or at the carboxy terminus of the bioactive peptide, or at both locations. The peptide linker may be any flexible linker sequence, such a sequence shown in TABLE 4. In some embodiments, the linker sequence was derived from the native CDR-H3 sequence in the parental clone. As further shown in FIG. 4, in some embodiments, the bioactive peptide sequence replaced all but one of the sixteen original amino acid residues of the native CDR-H3 (see, e.g. SGMI-1L), wherein the remaining one amino acid sequence is included as a linker. In some embodiments, eight of the sixteen original amino acid residues of the native CDR-H3 were retained in either the C-terminal linker (see e.g., SGMI-1L5), and up to fourteen of the original sixteen amino acid residues of the native CDR-H3 were retained in the C-terminal and N-terminal linker regions (see SGMI-L7).

[0337] 2. SGMI-1 and SGMI-2 Engrafted into the CDR-L1 of a Parental Chicken Light Chain Variable Region.

[0338] A DT40 chicken light chain variable region was chosen as the starting parental clone for use as a scaffold into which SGMI-1 or SGMI-2 peptide sequences were engrafted into the CDR-L1 region, as shown in FIGS. 5 and 6.

[0339] FIG. 5 illustrates an exemplary parental (DTLacO) variable light chain polypeptide sequence compared to a variable light chain polypeptide sequence comprising a bioactive peptide amino acid sequence engrafted within CDR-L1. As shown in FIG. 5, the chicken light chain variable region contains three CDRs (CDR-L1, CDR-L2 and CDR-L3), flanked by four framework regions (FR-1, FR-2, FR-3 and FR-4). Similar to the results obtained with CDR-H3 in the variable heavy chain polypeptide, the inventors have discovered that by engrafting a bioactive peptide sequence (e.g. SGMI-1) into CDR-L1 of the variable light chain polypeptide from a parental chicken antibody (which provides the antibody scaffold), the parental antibody comprising the engrafted bioactive peptide sequence is converted into an antibody that comprises biological activity of the bioactive peptide (i.e., inhibition of the lectin pathway was observed with the construct SGMI-IL, data not shown).

[0340] Exemplary parental chicken light chain variable regions are provided below. As shown in FIG. 6, although the native CDR regions vary between parental clones, the Framework regions between the CDRs are conserved in chicken, accordingly, a consensus FR-1, FR-2, FR-3 and FR-4 sequence derived from an alignment of several different parental chicken light chain regions is also provided below. As further shown in FIG. 6, in FR-1 there is a conserved cysteine (C) residue at the position immediately adjacent to CDR-L1 in the parental clones (corresponding to the cysteine at position 23 in SEQ ID NO:31), which is retained in FR-1 in the constructs containing an engrafted bioactive peptide in CDR-L1. As further shown in FIG. 6, in FR-2 there is a conserved tryptophan (W) residue at the position immediately adjacent the CDR-L1 in the parental clones (corresponding to the tryptophan at position 1 in SEQ ID NO:33), which is retained in FR-2 in the constructs containing an engrafted bioactive peptide in CDR-L1.

TABLE-US-00013 DTLacO chicken (clone #1) light chain variable region (DTLacO VL) (SEQ ID NO: 30) ALTQP SVSANPG TVKITCSGDSSYYGWYQQKAPGSAPVT I YDNTNRPS IPSRFSGS SGST TLTITGVRADD AVY CASTDSSSTAFGAGTTLTVL

[0341] The V_L CDRs (21-28 (L1); 45-51 (L2); and 84-92 are underlined and the Framework regions (1-20 (FR-1); 29-44 (FR-2); 52-83 (FR-3) and 93-102 (FR-4) are italicized.

TABLE-US-00014 DTLacO chicken (clone#2) light chain variable region (DTLacO VL) SEQ ID NO: 92) ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVT LIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSAD NSGAAFGAGTTLTVL Conserved FR-1 region from the DTLacO VL is set forth as SEQ ID NO: 31: ALTQPX₁SVSANX₂GX₃TVKITC Where: X₁ = A or S X₂ = L or P X₃ = G or E Conserved FR-1 flanking region adjacent to CDR-L1 is set forth as SEQ ID NO: 32 VKITC Conserved FR-2 region from the DTLacO VL is set forth as SEQ ID NO: 33: WYQQKX₁PGSAPVTX₂IY Where X₁ = A or S, X₂ = V or L Conserved FR-2 flanking region adjacent to CDR-L1 is set forth as SEQ ID NO: 34 WYQQK Conserved FR-3 region from the DTLacO VL is set forth as SEQ ID NO: 35: X₁IPSRFSGSX₂SGSTX₃TLTITGVRADDX₄AVYX₅C Where: X₁ = N or D X₂ = K or L X₃ = A or N X₄ = N or E X₅ = Y or F Conserved FR-4 region from the DTLacO VL is set forth as SEQ ID NO: 36 FGAGTTLTVL

[0342] As shown in FIG. 6, in some embodiments, a peptide linker was included at the amino terminus of the bioactive peptide, or at the carboxy terminus of the bioactive peptide, or at both locations. The peptide linker may be any flexible linker sequence, such as the sequences shown in TABLE 4. In some embodiments, the linker sequence was derived from the native CDR-L1 sequence in the parental clone. As further shown in FIG. 6, in some embodiments, the bioactive peptide replaced five of the thirteen original amino acid residues of the native CDR-L1 (see, e.g. SGMI-2L), retaining a portion of the original CDR-L1 sequence as a peptide linker flanking the bioactive peptide sequence.

[0343] Table 4: Exemplary Peptide Linkers for Engrafting Bioactive Peptides into CDRs:

TABLE-US-00015 38 AAGGSGGSGA 39 YIDA 40 AYIDA 41 GTGGGSGSSSYIDA 42 GSGAYIDA 43 AAGGSGGSGAYIDA 44 SGGGS 45 YYYG 46 GSGA

[0344] In some embodiments, the chicken variable heavy chain region is fused to a human IgG1 constant region, resulting in a chicken/human chimeric antibody. An exemplary human IgG1 constant region is provided below as SEQ ID NO:47.

TABLE-US-00016 human IgG1 Constant Region (CH1-hinge-CH2-CH3): SEQ ID NO: 47 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0345] In some embodiments, the chicken variable light chain region is fused to a human lambda light chain constant region, resulting in a chicken/human chimeric antibody. An exemplary human lambda light chain constant region is provided below as SEQ ID NO:48.

TABLE-US-00017 Human lambda light chain constant region (SEQ ID NO: 48) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTV EKTVAPTECS

[0346] The resulting polynucleotide constructs were designated pcDNA3-SGMI-1L-IgG1, -1M-IgG1, -1S-IgG1, and -1-L1-IgG1 to -1-L12-IgG1 (SEQ ID NOS: 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 and 77) and pcDNA3-SGMI-2L-Igλ, -2M-Igλ, and -2S-Igλ(SEQ ID NOS:79, 81 and 83), while the polypeptides were termed Ab-SGMI-1L-IgG1, -1M-IgG1, -1S-IgG1, and -1-L1-IgG1 to -1-L12-IgG1 (SEQ ID NOS: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 and 78), as shown in FIG. 4, and Ab-SGMI-2L-Igλ-, -2M-Igλ, and -2S-Igλ(SEQ ID NOS: 80, 82 and 84), as show in FIG. 6.

[0347] Freestyle 293-F or Expi293F cells were transiently transfected with combinations of expression plasmids as follows: (a) pcDNA3-SGMI-1-IgG1-1L, (et al.), plus a light chain plasmid encoding the DTLacO VL; (b) pcDNA3-SGMI-2-Igλ-L1 (et. al.), plus a heavy chain plasmid encoding the DTLacO VH; (c) pcDNA3-SGMI-1-IgG1-1L plus pcDNA3-SGMI-2-Igλ-L1. After four days of incubation at 37° C., the culture media were harvested and the SGMI-bearing chimeric antibodies were purified by Protein A affinity chromatography.

[0348] Results:

[0349] Chimeric Chicken/Human Antibodies Comprising SGML-1 Engrafted into CDR-H3

[0350] The Wieslab® Complement System Screen, MBL Pathway, as described in Example 2, was used to measure functionality of the chimeric antibodies. Assays were run in duplicate with the SGMI-1Fc (generated as described in Example 2) as the positive (inhibitory) controls. A matching isotype antibody was included as a negative control.

[0351] FIGS. 7A and 7B graphically illustrate the inhibitory activity of various representative chimeric chicken/human mAbs containing SGMI-1 engrafted into CDR-H3 on MBL complement activity. The data are distributed across two figures because the assays were conducted at different times. As shown in FIGS. 7A and 7B, several of the chimeric mAbs containing SGMI-1 engrafted within the CDR-H3 inhibit C5b-C9 deposition to a degree similar to the positive SGMI-1-Fc fusion protein (e.g., Ab-SGMI-1-L2, -L3, -L4, -L5, -L7, -L9, -L1, -L10, -L11 and -L12). As shown in FIG. 4, these constructs differ only in the nature of the flexible linkers separating the inhibitory peptide from the antibody framework regions. Interestingly, another chimeric mAb with SGMI-1 engrafted into the CDR-H3, referred to as "Ab-SGMI-1L," has no inhibitory activity. As shown in FIG. 4, Ab-SGMI-1L has only a one amino acid residue linker between the bioactive peptide and framework segments.

[0352] The Ab-SGMI-1 antibodies were also assessed for lectin pathway inhibition in an assay of C3b deposition on mannan-coated beads. This assay, which determines degree of activity by flow cytometry, offers greater resolution than the Wieslab® assay. The Lectin Pathway bead assay was carried out as follows: mannan was adsorbed to 7 μM-diameter polystyrene beads (Bangs Laboratories; Fishers, Ind., USA) overnight at 4° C. in carbonate-bicarbonate buffer (pH 9.6). The beads were washed in PBS and exposed to 10% serum, or 10% serum pre-incubated with antibodies or inhibitors. The serum-bead mixture was incubated at room temperature for one hour while agitating. Following the serum incubation, the beads were washed, and C3 deposition on the beads was measured by detection with an anti-C3c rabbit polyclonal antibody (Dako North America; Carpinteria, Calif., USA) and a PE-Cy5 conjugated goat anti-rabbit secondary antibody (Southern Biotech; Birmingham, Ala., USA). Following the staining procedure, the beads were analyzed using a FACS Calibur cytometer. The beads were gated as a uniform population using forward and side scatter, and C3 deposition was apparent as FL3-positive particles (FL-3, or "FL-3 channel" indicates the 3rd or red channel on the cytometer). The Geometric Mean Fluorescence Intensity (MFI) for the population for each experimental condition was plotted relative to the antibody/inhibitor concentration to evaluate lectin pathway inhibition.

[0353] As shown in FIGS. 8A and 8B, all of the antibodies containing SGMI-1 engrafted into CDR-H3 inhibited lectin pathway activity in the bead assay, but with varying degrees of potency. It is noted that the differences between the antibodies are more readily discerned in this bead assay as compared to the Wieslab® assay.

[0354] In summary, these results demonstrate that inhibitory therapeutic polypeptides may be generated by engrafting a bioactive peptide into the CDR-H3 of a chicken antibody scaffold.

[0355] Chimeric Chicken/Human Antibodies Comprising SGMI-2 Engrafted into CDR-L1

[0356] FIG. 9A graphically illustrates that a chimeric chicken/human mAb comprising SGMI-2 engrafted within CDR-L1 (Ab-SGMI-2L-Igλ) exerts little to no inhibitory activity in the Wieslab complement system MBL pathway assay. These results leave room for optimization of the linker elements flanking the bioactive peptide, which significantly impacted the efficacy of the SGMI-1-containing mAbs (as shown in FIGS. 7A and 7B).

[0357] Chimeric Chicken/Human Antibodies Comprising SGMI-1 and SGMI-2

[0358] FIG. 9A also shows the activity of a chimeric chicken/human antibody comprising SGMI-1 and SGMI-2 engrafted into CDR-H3 and CDR-L1, respectively. Ab-SGMI-1-L1-IgG 1/SGMI-2-L-Igλ is nearly as potent as the mAb containing only the SGMI-1 peptide (Ab-SGMI-1-L1-IgG1). This outcome was confirmed using the flow cytometric mannan-coated bead assay, as shown in FIG. 9B. Together, these data demonstrate that the SGMI-1 peptide engrafted into CDR-H3 inhibits the lectin pathway whether or not the SGMI-2 peptide is present engrafted into CDR-L1. Based on the results described herein, further optimization of the SGMI-2 flanking linkers is expected to add MASP-2 inhibitory activity to the antibody already carrying SGMI-1-mediated MASP-1 inhibitory activity.

Example 4

[0359] This Example describes the generation of chimeric antibodies comprising one or more bioactive peptides (e.g. SGMI-1 or SGMI-2) fused onto the amino or carboxy termini of the heavy and light chains of a chimeric chicken/human antibody.

[0360] Rationale:

[0361] As demonstrated in Examples 2 and 3, the inhibitory functions of the SGMI-1 and SGMI-2 peptides (and truncated variants thereof) were preserved in the SGMI-Fc proteins and also, for SGMI-1, when displayed within the CDR regions of a full antibody. In this Example, experiments were carried out to determine whether the SGMI peptides would retain activity when fused to the amino or carboxy termini of antibody heavy or light chains of a chimeric chicken/human antibody.

TABLE-US-00018 TABLE 5 Chimeric chicken/human antibodies with the bioactive peptides SGMI-1 and SGMI-2 fused to the N- or C-termini of the heavy or light chains. Peptide Location on Antibody Antibody HC-N HC-C LC-N LC-C SEQ ID NO: Ab-IgG1-S10 SGMI-1 -- -- -- 94 Ab-IgG1-S20 SGMI-2 -- -- 96 Ab-IgG1-S01 -- SGMI-1 -- -- 98 Ab-IgG1-S02 -- SGMI-2 -- -- 100 Ab-Igλ-S10 -- SGMI-1 -- 102 Ab-Igλ-S20 SGMI-2 104 Ab-Igλ-S01 -- SGMI-1 106 Ab-Igλ-S02 SGMI-2 108 Abbreviations in Table 5: "HC-N" = amino terminus of heavy chain "HC-C" = carboxyl terminus of heavy chain "LC-N" = amino terminus of light chain "LC-C" = carboxyl terminus of light chain

[0362] For the N-terminal fusions shown in TABLE 5, a peptide linker (SEQ ID NO:14) was added between the bioactive peptide and the chicken variable region.

[0363] For the C-terminal fusions shown in TABLE 5, a peptide linker (SEQ ID NO:37) was added between the constant region and the bioactive peptide, and a second peptide "GSGA" was added at the C-terminal end of the fusion polypeptide to protect C-terminal SGMI peptides from degradation. These fusion constructs are illustrated schematically in FIG. 10.

[0364] FIG. 11 illustrates the inhibitory activity of the N- and C-terminal peptides in the Wieslab assay. Compared to the positive and negative controls, all of the fusion mAbs inhibited C5b-9 deposition. All except for one fusion mAb--SGMI-1 fused to the C-terminus of the light chain--exhibited levels of inhibition comparable to those of the control SGMI-1 and SGMI-2 Fc-fusion proteins. Several of these N- and C-terminal peptide-mAb fusions were also tested in the flow cytometric mannan-coated bead assay described in Example 3, with similar results (data not shown). These antibodies m for the development of bi-specific antibodies bearing combinations of SGMI-1 and SGMI-2.

[0365] While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Sequence CWU 1

1

10816476DNAHomo Sapiens 1acacacagag tgatacaaat acctgcttga gcccctcagt tattttctct caagggctga 60agtcagccac acaggataaa ggagggaagg gaaggagcag atcttttcgg taggaagaca 120gattttgttg tcaggttcct gggagtgcaa gagcaagtca aaggagagag agaggagaga 180ggaaaagcca gagggagaga gggggagagg ggatctgttg caggcagggg aaggcgtgac 240ctgaatggag aatgccagcc aattccagag acacacaggg acctcagaac aaagataagg 300catcacggac accacaccgg gcacgagctc acaggcaagt caagctggga ggaccaaggc 360cgggcagccg ggagcaccca aggcaggaaa atgaggtggc tgcttctcta ttatgctctg 420tgcttctccc tgtcaaaggc ttcagcccac accgtggagc taaacaatat gtttggccag 480atccagtcgc ctggttatcc agactcctat cccagtgatt cagaggtgac ttggaatatc 540actgtcccag atgggtttcg gatcaagctt tacttcatgc acttcaactt ggaatcctcc 600tacctttgtg aatatgacta tgtgaaggta gaaactgagg accaggtgct ggcaaccttc 660tgtggcaggg agaccacaga cacagagcag actcccggcc aggaggtggt cctctcccct 720ggctccttca tgtccatcac tttccggtca gatttctcca atgaggagcg tttcacaggc 780tttgatgccc actacatggc tgtggatgtg gacgagtgca aggagaggga ggacgaggag 840ctgtcctgtg accactactg ccacaactac attggcggct actactgctc ctgccgcttc 900ggctacatcc tccacacaga caacaggacc tgccgagtgg agtgcagtga caacctcttc 960actcaaagga ctggggtgat caccagccct gacttcccaa acccttaccc caagagctct 1020gaatgcctgt ataccatcga gctggaggag ggtttcatgg tcaacctgca gtttgaggac 1080atatttgaca ttgaggacca tcctgaggtg ccctgcccct atgactacat caagatcaaa 1140gttggtccaa aagttttggg gcctttctgt ggagagaaag ccccagaacc catcagcacc 1200cagagccaca gtgtcctgat cctgttccat agtgacaact cgggagagaa ccggggctgg 1260aggctctcat acagggctgc aggaaatgag tgcccagagc tacagcctcc tgtccatggg 1320aaaatcgagc cctcccaagc caagtatttc ttcaaagacc aagtgctcgt cagctgtgac 1380acaggctaca aagtgctgaa ggataatgtg gagatggaca cattccagat tgagtgtctg 1440aaggatggga cgtggagtaa caagattccc acctgtaaaa ttgtagactg tagagcccca 1500ggagagctgg aacacgggct gatcaccttc tctacaagga acaacctcac cacatacaag 1560tctgagatca aatactcctg tcaggagccc tattacaaga tgctcaacaa taacacaggt 1620atatatacct gttctgccca aggagtctgg atgaataaag tattggggag aagcctaccc 1680acctgccttc cagtgtgtgg gctccccaag ttctcccgga agctgatggc caggatcttc 1740aatggacgcc cagcccagaa aggcaccact ccctggattg ccatgctgtc acacctgaat 1800gggcagccct tctgcggagg ctcccttcta ggctccagct ggatcgtgac cgccgcacac 1860tgcctccacc agtcactcga tccggaagat ccgaccctac gtgattcaga cttgctcagc 1920ccttctgact tcaaaatcat cctgggcaag cattggaggc tccggtcaga tgaaaatgaa 1980cagcatctcg gcgtcaaaca caccactctc cacccccagt atgatcccaa cacattcgag 2040aatgacgtgg ctctggtgga gctgttggag agcccagtgc tgaatgcctt cgtgatgccc 2100atctgtctgc ctgagggacc ccagcaggaa ggagccatgg tcatcgtcag cggctggggg 2160aagcagttct tgcaaaggtt cccagagacc ctgatggaga ttgaaatccc gattgttgac 2220cacagcacct gccagaaggc ttatgccccg ctgaagaaga aagtgaccag ggacatgatc 2280tgtgctgggg agaaggaagg gggaaaggac gcctgtgcgg gtgactctgg aggccccatg 2340gtgaccctga atagagaaag aggccagtgg tacctggtgg gcactgtgtc ctggggtgat 2400gactgtggga agaaggaccg ctacggagta tactcttaca tccaccacaa caaggactgg 2460atccagaggg tcaccggagt gaggaactga atttggctcc tcagccccag caccaccagc 2520tgtgggcagt cagtagcaga ggacgatcct ccgatgaaag cagccatttc tcctttcctt 2580cctcccatcc cccctccttc ggcctatcca ttactgggca atagagcagg tatcttcacc 2640cccttttcac tctctttaaa gagatggagc aagagagtgg tcagaacaca ggccgaatcc 2700aggctctatc acttactagt ttgcagtgct gggcaggtga cttcatctct tcgaacttca 2760gtttcttcat aagatggaaa tgctatacct tacctacctc gtaaaagtct gatgaggaaa 2820agattaacta atagatgcat agcacttaac agagtgcata gcatacactg ttttcaataa 2880atgcacctta gcagaaggtc gatgtgtcta ccaggcagac gaagctctct tacaaacccc 2940tgcctgggtc ttagcattga tcagtgacac acctctcccc tcaaccttga ccatctccat 3000ctgcccttaa atgctgtatg cttttttgcc accgtgcaac ttgcccaaca tcaatcttca 3060ccctcatccc taaaaaagta aaacagacaa ggttctgagt cctgtggtat gtcccctagc 3120aaatgtaact aggaacatgc actagatgac agattgcggg agggcctgag agaagcaggg 3180acaggaggga gcctggggat tgtggtttgg gaaggcagac acctggttct agaactagct 3240ctgcccttag ccccctgtat gaccctatgc aagtcctcct ccctcatctc aaagggtcct 3300caaagctctg acgatctaag atacaatgaa gccattttcc ccctgataag atgaggtaaa 3360gccaatgtaa ccaaaaggca aaaattacaa tcggttcaaa ggaactttga tgcagacaaa 3420atgctgctgc tgctgctcct gaaataccca cccctttcca ctacgggtgg gttcccaagg 3480acatgggaca ggcaaagtgt gagccaaagg atccttcctt attcctaagc agagcatctg 3540ctctgggccc tggcctcctt cccttcttgg gaaactgggc tgcatgaggt gggccctggt 3600agtttgtacc ccaggcccct atactcttcc ttcctatgtc cacagctgac cccaagcagc 3660cgttccccga ctcctcaccc ctgagcctca ccctgaactc cctcatcttg caaggccata 3720agtgttttcc aagcaaaatg cctctcccat cctctctcag gaagcttcta gagactttat 3780gccctccaga gctccaagat ataagccctc caagggatca gaagctccaa gttcctgtct 3840tctgttttat agaaattgat cttccctggg ggactttaac tcttgacctg tatgcagctg 3900ttggagtaat tccaggtctc ttgaaaaaaa agaggaagat aatggagaat gagaacatat 3960atatatatat attaagcccc aggctgaata ctcagggaca gcaattcaca gcctgcctct 4020ggttctataa acaagtcatt ctacctcttt gtgccctgct gtttattctg taaggggaag 4080gtggcaatgg gacccagctc catcagacac ttgtcaagct agcagaaact ccattttcaa 4140tgccaaagaa gaactgtaat gctgttttgg aatcatccca aggcatccca agacaccata 4200tcttcccatt tcaagcactg cctgggcaca ccccaacatc ccaggctgtg gtggctcctg 4260tgggaactac ctagatgaag agagtatcat ttataccttc taggagctcc tattgggaga 4320catgaaacat atgtaattga ctaccatgta atagaacaaa ccctgccaag tgctgctttg 4380gaaagtcatg gaggtaaaag aaagaccatt ctggtatgaa ggttttgggg gaggagatat 4440caatcaagaa ggcttcccag aagaggtgac tggaccagag ccttgtccac aggtaagacg 4500gaggaggcct tccacatgga gggagaacaa tagtaaatgt ccactcaaga tgtcctttat 4560tataccagct cctcccacaa aaacacatgt ccagtggact ctttttctgg gatcagaacc 4620aacaccaaaa agagcttttc tccttaaagt tagaattcta aacaggactt gaaatggcct 4680caaggtttgt gcacaaatac tgacttctgg ctggacccag cttattctgt ttatttctcc 4740aattgcaatt tcatccttat cctgagaaaa tgtctaaata ggccatggaa cccaggcttc 4800cccgtgacct acaagcactt attagctgtg ccagctcctg cactgctgct aaggtccaag 4860aaacccagat ctctcacaga gccatagaag cagagggctg gagtatctgt gaggacaaca 4920accttgtcta acttcgcgac ctcattcttg agcatttcta ctgatgagaa actcactacc 4980cccaattgca gctcattcaa ctttaaaatt gctgcttttt gaatcactga ttgtgaatat 5040taatttaaaa aaataagtaa gaaaatgttt taaatgtgct gcctctttaa aaggtctcct 5100ctttgtgcaa ccaaaaccca cctctctaga atacagtttg tataactgaa gctataattt 5160cataccatga gtgctgctgt tagcaataat aatcatgccc ggattttatt aacaacagaa 5220gctgttgctc gtatgaaaaa acaaacatta gttctaataa acatctgcat tgagtcaaag 5280ctccctgttt gtttgtatgt cttttatgca ctgatgatta tagtgagttg ctttcattta 5340ccaacatttt gttgtattcg tgtaggatca ctgtaccatg aagggagaga gactatgatg 5400ggaagattgt tgtagataca aaagcatgtc ttaggttttt gggtcagttc tgtttaaata 5460cctgtcctat tattcctgta aattatcaaa atatcccaga atgtcaatgt ttctgcatcc 5520acattacaat tattaaatgc cactcattta ttaaatttac tattatcagt ggcatttaat 5580aaatttgaat catatgttca gtgtttggtt tagaaaatat ggtgccatgt ctatgagtgg 5640cctgttctgg attggagtac atgccttctt tctgccttga gttaatctta ctcaatggag 5700aacaagaatc aaagaaacac caccaccaag aagcccttca agctagagtt gggcaagagt 5760cagggagggg aatgtagacc actcatatga cagaggtgga aaccaatctt ggtctagaat 5820aagtctcaaa atcaaaagac ttgaattcta gtgcagcgta ggttgactcc cttatttatt 5880taattttccc atctctacac cgctagaata acctctctcc tgaggctgtt gaatctgatg 5940aattagcaga taggaaagaa cttagaaaat tataagttca ctcaaatgta aaaggttata 6000tgggaaataa tcaccactaa catttttgag tacttactat ctgcttgtta tacacattct 6060ctaatttaat tttcacaaga aaattcatga aaggactata cttatccccc ttttacaggt 6120gagcaacctg gagtgcagtg aagtgtaaaa tgtgggccga cttaggagca caaatacccc 6180agcaacaatg agcactctta gtacacaggt cttggtttct aaaatatcat catccgataa 6240aaggaacaca ggctctttga agaaatgact gattccggga ttggggcaag aaacacacaa 6300gctgagactg gagcatcttg cagtcccaga aagtgaagaa acgctgagga tatgtcaaag 6360ggacacagga gccaaatgaa agagcttcca ctggccaaag ctggaatgtt gagcaacaaa 6420gcaacatagc attggataat aacccaaagt ataaaataaa tattcatgag tacata 64762699PRTHomo Sapiens 2Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu Ser Lys 1 5 10 15 Ala Ser Ala His Thr Val Glu Leu Asn Asn Met Phe Gly Gln Ile Gln 20 25 30 Ser Pro Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser Glu Val Thr Trp 35 40 45 Asn Ile Thr Val Pro Asp Gly Phe Arg Ile Lys Leu Tyr Phe Met His 50 55 60 Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val 65 70 75 80 Glu Thr Glu Asp Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95 Asp Thr Glu Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110 Phe Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120 125 Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys 130 135 140 Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His Asn Tyr 145 150 155 160 Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175 Asp Asn Arg Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190 Arg Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205 Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220 Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val 225 230 235 240 Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu 245 250 255 Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr Gln Ser 260 265 270 His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285 Gly Trp Arg Leu Ser Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu 290 295 300 Gln Pro Pro Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe 305 310 315 320 Phe Lys Asp Gln Val Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu 325 330 335 Lys Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu Lys Asp 340 345 350 Gly Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Ile Val Asp Cys Arg 355 360 365 Ala Pro Gly Glu Leu Glu His Gly Leu Ile Thr Phe Ser Thr Arg Asn 370 375 380 Asn Leu Thr Thr Tyr Lys Ser Glu Ile Lys Tyr Ser Cys Gln Glu Pro 385 390 395 400 Tyr Tyr Lys Met Leu Asn Asn Asn Thr Gly Ile Tyr Thr Cys Ser Ala 405 410 415 Gln Gly Val Trp Met Asn Lys Val Leu Gly Arg Ser Leu Pro Thr Cys 420 425 430 Leu Pro Val Cys Gly Leu Pro Lys Phe Ser Arg Lys Leu Met Ala Arg 435 440 445 Ile Phe Asn Gly Arg Pro Ala Gln Lys Gly Thr Thr Pro Trp Ile Ala 450 455 460 Met Leu Ser His Leu Asn Gly Gln Pro Phe Cys Gly Gly Ser Leu Leu 465 470 475 480 Gly Ser Ser Trp Ile Val Thr Ala Ala His Cys Leu His Gln Ser Leu 485 490 495 Asp Pro Glu Asp Pro Thr Leu Arg Asp Ser Asp Leu Leu Ser Pro Ser 500 505 510 Asp Phe Lys Ile Ile Leu Gly Lys His Trp Arg Leu Arg Ser Asp Glu 515 520 525 Asn Glu Gln His Leu Gly Val Lys His Thr Thr Leu His Pro Gln Tyr 530 535 540 Asp Pro Asn Thr Phe Glu Asn Asp Val Ala Leu Val Glu Leu Leu Glu 545 550 555 560 Ser Pro Val Leu Asn Ala Phe Val Met Pro Ile Cys Leu Pro Glu Gly 565 570 575 Pro Gln Gln Glu Gly Ala Met Val Ile Val Ser Gly Trp Gly Lys Gln 580 585 590 Phe Leu Gln Arg Phe Pro Glu Thr Leu Met Glu Ile Glu Ile Pro Ile 595 600 605 Val Asp His Ser Thr Cys Gln Lys Ala Tyr Ala Pro Leu Lys Lys Lys 610 615 620 Val Thr Arg Asp Met Ile Cys Ala Gly Glu Lys Glu Gly Gly Lys Asp 625 630 635 640 Ala Cys Ala Gly Asp Ser Gly Gly Pro Met Val Thr Leu Asn Arg Glu 645 650 655 Arg Gly Gln Trp Tyr Leu Val Gly Thr Val Ser Trp Gly Asp Asp Cys 660 665 670 Gly Lys Lys Asp Arg Tyr Gly Val Tyr Ser Tyr Ile His His Asn Lys 675 680 685 Asp Trp Ile Gln Arg Val Thr Gly Val Arg Asn 690 695 32471DNAHomo Sapiens 3agaccaggcc aggccagctg gacgggcaca ccatgaggct gctgaccctc ctgggccttc 60tgtgtggctc ggtggccacc cccttgggcc cgaagtggcc tgaacctgtg ttcgggcgcc 120tggcatcccc cggctttcca ggggagtatg ccaatgacca ggagcggcgc tggaccctga 180ctgcaccccc cggctaccgc ctgcgcctct acttcaccca cttcgacctg gagctctccc 240acctctgcga gtacgacttc gtcaagctga gctcgggggc caaggtgctg gccacgctgt 300gcgggcagga gagcacagac acggagcggg cccctggcaa ggacactttc tactcgctgg 360gctccagcct ggacattacc ttccgctccg actactccaa cgagaagccg ttcacggggt 420tcgaggcctt ctatgcagcc gaggacattg acgagtgcca ggtggccccg ggagaggcgc 480ccacctgcga ccaccactgc cacaaccacc tgggcggttt ctactgctcc tgccgcgcag 540gctacgtcct gcaccgtaac aagcgcacct gctcagccct gtgctccggc caggtcttca 600cccagaggtc tggggagctc agcagccctg aatacccacg gccgtatccc aaactctcca 660gttgcactta cagcatcagc ctggaggagg ggttcagtgt cattctggac tttgtggagt 720ccttcgatgt ggagacacac cctgaaaccc tgtgtcccta cgactttctc aagattcaaa 780cagacagaga agaacatggc ccattctgtg ggaagacatt gccccacagg attgaaacaa 840aaagcaacac ggtgaccatc acctttgtca cagatgaatc aggagaccac acaggctgga 900agatccacta cacgagcaca gcgcagcctt gcccttatcc gatggcgcca cctaatggcc 960acgtttcacc tgtgcaagcc aaatacatcc tgaaagacag cttctccatc ttttgcgaga 1020ctggctatga gcttctgcaa ggtcacttgc ccctgaaatc ctttactgca gtttgtcaga 1080aagatggatc ttgggaccgg ccaatgcccg cgtgcagcat tgttgactgt ggccctcctg 1140atgatctacc cagtggccga gtggagtaca tcacaggtcc tggagtgacc acctacaaag 1200ctgtgattca gtacagctgt gaagagacct tctacacaat gaaagtgaat gatggtaaat 1260atgtgtgtga ggctgatgga ttctggacga gctccaaagg agaaaaatca ctcccagtct 1320gtgagcctgt ttgtggacta tcagcccgca caacaggagg gcgtatatat ggagggcaaa 1380aggcaaaacc tggtgatttt ccttggcaag tcctgatatt aggtggaacc acagcagcag 1440gtgcactttt atatgacaac tgggtcctaa cagctgctca tgccgtctat gagcaaaaac 1500atgatgcatc cgccctggac attcgaatgg gcaccctgaa aagactatca cctcattata 1560cacaagcctg gtctgaagct gtttttatac atgaaggtta tactcatgat gctggctttg 1620acaatgacat agcactgatt aaattgaata acaaagttgt aatcaatagc aacatcacgc 1680ctatttgtct gccaagaaaa gaagctgaat cctttatgag gacagatgac attggaactg 1740catctggatg gggattaacc caaaggggtt ttcttgctag aaatctaatg tatgtcgaca 1800taccgattgt tgaccatcaa aaatgtactg ctgcatatga aaagccaccc tatccaaggg 1860gaagtgtaac tgctaacatg ctttgtgctg gcttagaaag tgggggcaag gacagctgca 1920gaggtgacag cggaggggca ctggtgtttc tagatagtga aacagagagg tggtttgtgg 1980gaggaatagt gtcctggggt tccatgaatt gtggggaagc aggtcagtat ggagtctaca 2040caaaagttat taactatatt ccctggatcg agaacataat tagtgatttt taacttgcgt 2100gtctgcagtc aaggattctt catttttaga aatgcctgtg aagaccttgg cagcgacgtg 2160gctcgagaag cattcatcat tactgtggac atggcagttg ttgctccacc caaaaaaaca 2220gactccaggt gaggctgctg tcatttctcc acttgccagt ttaattccag ccttacccat 2280tgactcaagg ggacataaac cacgagagtg acagtcatct ttgcccaccc agtgtaatgt 2340cactgctcaa attacatttc attaccttaa aaagccagtc tcttttcata ctggctgttg 2400gcatttctgt aaactgcctg tccatgctct ttgtttttaa acttgttctt attgaaaaaa 2460aaaaaaaaaa a 24714686PRTHomo Sapiens 4Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn 165 170 175 Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg 180 185 190 Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu 195 200 205 Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser

Val Ile 210 215 220 Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu 225 230 235 240 Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly 245 250 255 Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn 260 265 270 Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly 275 280 285 Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met 290 295 300 Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu 305 310 315 320 Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln 325 330 335 Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly 340 345 350 Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro 355 360 365 Pro Asp Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly 370 375 380 Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400 Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly 405 410 415 Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro 420 425 430 Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly Gly 435 440 445 Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu Gly 450 455 460 Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu Thr 465 470 475 480 Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp 485 490 495 Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala 500 505 510 Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly 515 520 525 Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val Ile 530 535 540 Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu Ser 545 550 555 560 Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu Thr 565 570 575 Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro Ile 580 585 590 Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro 595 600 605 Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly 610 615 620 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu 625 630 635 640 Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 645 650 655 Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val 660 665 670 Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe 675 680 685 59PRTArtificial SequenceSynthetic 5Xaa Cys Thr Xaa Xaa Xaa Cys Xaa Gln 1 5 636PRTArtificial SequenceSynthetic 6Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys 20 25 30 Leu Cys Tyr Gln 35 733PRTArtificial SequenceSynthetic 7Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg 1 5 10 15 Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr 20 25 30 Gln 820PRTArtificial SequenceSynthetic 8Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys 1 5 10 15 Leu Cys Tyr Gln 20 936PRTArtificial SequenceSynthetic 9Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu 20 25 30 Trp Cys Asn Gln 35 1033PRTArtificial SequenceSynthetic 10Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg 1 5 10 15 Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn 20 25 30 Gln 1120PRTArtificial SequenceSynthetic 11Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu 1 5 10 15 Trp Cys Asn Gln 20 12227PRTHomo Sapiens 12Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 1312PRTArtificial SequenceSynthetic 13Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Arg Ser 1 5 10 1410PRTArtificial SequenceSynthetic 14Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser 1 5 10 15888DNAArtificial SequenceSynthetic 15atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60ctcgaggtga catgtgaacc cggtacgacg tttaaggata agtgcaacac atgtaggtgc 120ggtagcgacg gcaaatcagc gttctgtacc cggaaattgt gctaccaggg aactggagga 180gggtcggggt cctcgtcaag atctgacaaa actcacacat gcccaccgtg cccagcacct 240gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 300atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 360gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 420gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 480tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 540gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 600ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 660tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 720accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 780gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca cgaggctctg 840cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga 88816275PRTArtificial SequenceSynthetic 16Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys 20 25 30 Leu Cys Tyr Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Arg Ser 35 40 45 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 50 55 60 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 65 70 75 80 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 85 90 95 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 100 105 110 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 115 120 125 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 130 135 140 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 145 150 155 160 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 165 170 175 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 180 185 190 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 195 200 205 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 210 215 220 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 225 230 235 240 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 245 250 255 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 260 265 270 Pro Gly Lys 275 17888DNAArtificial SequenceSynthetic 17atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60ttggaagtga cgtgtgagcc cggaacgaca ttcaaagaca agtgcaatac ttgtcggtgc 120ggttcagatg ggaaatcggc ggtctgcaca aagctctggt gtaaccaggg caccggtgga 180gggtcgggat ccagctcaag atctgacaaa actcacacat gcccaccgtg cccagcacct 240gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 300atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 360gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 420gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 480tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 540gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 600ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 660tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 720accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 780gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca cgaggctctg 840cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga 88818275PRTArtificial SequenceSynthetic 18Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu 20 25 30 Trp Cys Asn Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Arg Ser 35 40 45 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 50 55 60 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 65 70 75 80 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 85 90 95 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 100 105 110 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 115 120 125 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 130 135 140 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 145 150 155 160 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 165 170 175 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 180 185 190 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 195 200 205 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 210 215 220 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 225 230 235 240 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 245 250 255 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 260 265 270 Pro Gly Lys 275 1930DNAArtificial SequenceSynthetic 19ctactgcgcc aaactcgagg tgacatgtga 302030DNAArtificial SequenceSynthetic 20cgtggcccca tgcctggtag cacaatttcc 302130DNAArtificial SequenceSynthetic 21cgggggtggc agcttggaag tgacgtgtga 302230DNAArtificial SequenceSynthetic 22agccataata gtactggtta caccagagct 3023125PRTChicken 23Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Arg 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Asp Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ala Ala Gly Gly Ser Gly Tyr Cys Gly Ser Gly Ala Tyr Ile 100 105 110 Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 2430PRTChickenVARIANT(16)..(16)wherein Xaa at position 16 is Arg or Gly 24Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Xaa 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser 20 25 30 2514PRTChickenVARIANT(12)..(12)wherein Xaa at position 12 is Phe or Trp 25Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Xaa Val Ala 1 5 10 2632PRTChickenVARIANT(13)..(13)wherein Xaa at position 13 is Val or Leu 26Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Xaa Arg Leu Gln 1 5 10 15 Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Xaa Lys 20 25 30 275PRTChickenVARIANT(4)..(4)wherein Xaa at position 4 is Ala or Thr 27Tyr Tyr Cys Xaa Lys 1 5 2811PRTChicken 28Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 1 5 10 295PRTChicken 29Trp Gly His Gly Thr 1 5 30102PRTChicken 30Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Asp Ser Ser Tyr Tyr Gly Trp Tyr Gln Gln 20 25 30 Lys Ala Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Thr Asn 35 40 45 Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60 Thr Ala Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp Asn Ala Val 65 70 75 80 Tyr Tyr Cys Ala Ser Thr Asp Ser Ser Ser Thr Ala Phe Gly Ala Gly 85 90 95 Thr Thr Leu Thr Val Leu 100 3120PRTChickenVARIANT(6)..(6)wherein Xaa at position 6 is Ala or Ser 31Ala Leu Thr Gln Pro Xaa Ser Val Ser Ala Asn Xaa Gly Xaa Thr Val 1 5 10 15 Lys Ile Thr Cys 20 325PRTChicken 32Val Lys Ile Thr Cys 1 5 3316PRTChickenVARIANT(6)..(6)wherein Xaa at position 6 is Ala or Ser 33Trp Tyr Gln Gln Lys Xaa Pro Gly Ser Ala Pro Val Thr Xaa Ile Tyr 1 5 10 15 345PRTChicken

34Trp Tyr Gln Gln Lys 1 5 3532PRTChickenVARIANT(1)..(1)wherein Xaa at position 1 is Asn or Asp 35Xaa Ile Pro Ser Arg Phe Ser Gly Ser Xaa Ser Gly Ser Thr Xaa Thr 1 5 10 15 Leu Thr Ile Thr Gly Val Arg Ala Asp Asp Xaa Ala Val Tyr Xaa Cys 20 25 30 3610PRTChicken 36Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 1 5 10 376PRTArtificial SequenceSynthetic 37Ala Ala Gly Gly Ser Gly 1 5 3810PRTArtificial SequenceSynthetic 38Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala 1 5 10 394PRTArtificial SequenceSynthetic 39Tyr Ile Asp Ala 1 405PRTArtificial SequenceSynthetic 40Ala Tyr Ile Asp Ala 1 5 4114PRTArtificial SequenceSynthetic 41Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Tyr Ile Asp Ala 1 5 10 428PRTArtificial SequenceSynthetic 42Gly Ser Gly Ala Tyr Ile Asp Ala 1 5 4314PRTArtificial SequenceSynthetic 43Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala Tyr Ile Asp Ala 1 5 10 445PRTArtificial SequenceSynthetic 44Ser Gly Gly Gly Ser 1 5 454PRTArtificial SequenceSynthetic 45Tyr Tyr Tyr Gly 1 464PRTArtificial SequenceSynthetic 46Gly Ser Gly Ala 1 47330PRTHomo Sapiens 47Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 481488DNAArtificial SequenceSynthetic 48atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa actcgaggtg 360acatgtgaac ccggtacgac gtttaaggat aagtgcaaca catgtaggtg cggtagcgac 420ggcaaatcag cgttctgtac ccggaaattg tgctaccagg catggggcca cgggaccgaa 480gtcatcgtct cctccgctag caccaagggc ccatcggtct tccccctggc accctcctcc 540aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 600ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 660gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 720ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 780aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 840gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 900atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 960gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1020gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1140gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1200ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1380gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1440cacaaccact acacacagaa gagcctctcc ctgtctccgg gtaaatga 148849476PRTArtificial SequenceSynthetic 49Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys 100 105 110 Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr 115 120 125 Arg Lys Leu Cys Tyr Gln Ala Trp Gly His Gly Thr Glu Val Ile Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 501479DNAArtificial SequenceSynthetic 50atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa aacatgtgaa 360cccggtacga cgtttaagga taagtgcaac acatgtaggt gcggtagcga cggcaaatca 420gcgttctgta cccggaaatt gtgctaccag gcatggggcc acgggaccga agtcatcgtc 480tcctccgcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 540tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 600gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 660tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 720cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 780gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 840gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 900acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 960aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1020tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1080ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1140atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1200gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1260gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1320cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1380aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1440tacacacaga agagcctctc cctgtctccg ggtaaatga 147951473PRTArtificial SequenceSynthetic 51Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr 100 105 110 Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu 115 120 125 Cys Tyr Gln Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 145 150 155 160 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 210 215 220 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 225 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 521440DNAArtificial SequenceSynthetic 52atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa aacatgtagg 360tgcggtagcg acggcaaatc agcgttctgt acccggaaat tgtgctacca ggcatggggc 420cacgggaccg aagtcatcgt ctcctccgct agcaccaagg gcccatcggt cttccccctg 480gcaccctcct ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac 540tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 600accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 660ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac 720accaaggtgg acaagaaagt tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 780tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 840gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 900gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 960acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1020ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1080ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1140tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1200gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1260aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1320aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1380catgaggctc tgcacaacca ctacacacag aagagcctct ccctgtctcc gggtaaatga 144053460PRTArtificial SequenceSynthetic 53Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr 100 105 110 Arg Lys Leu Cys Tyr Gln Ala Trp Gly His Gly Thr Glu Val Ile

Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 130 135 140 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 145 150 155 160 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 165 170 175 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 180 185 190 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 195 200 205 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 210 215 220 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 225 230 235 240 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 325 330 335 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 355 360 365 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 541527DNAArtificial SequenceSynthetic 54atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa actcgaggtg 360acatgtgaac ccggtacgac gtttaaggat aagtgcaaca catgtaggtg cggtagcgac 420ggcaaatcag cgttctgtac ccggaaattg tgctaccagg gaactggagg agggtcgggg 480tcctcgtcat atatcgacgc atggggccac gggaccgaag tcatcgtctc ctccgctagc 540accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 600gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 660tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 720tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 780tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 840tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 900gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 960acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1020gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1080taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1140aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1200aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1260aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1320gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1380tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1440gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1500agcctctccc tgtctccggg taaatga 152755489PRTArtificial SequenceSynthetic 55Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys 100 105 110 Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr 115 120 125 Arg Lys Leu Cys Tyr Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser 130 135 140 Tyr Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala 145 150 155 160 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 165 170 175 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 180 185 190 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 195 200 205 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 210 215 220 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 225 230 235 240 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 245 250 255 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 260 265 270 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 275 280 285 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 290 295 300 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 305 310 315 320 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 325 330 335 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 340 345 350 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 355 360 365 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 370 375 380 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 385 390 395 400 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 405 410 415 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 420 425 430 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 435 440 445 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 450 455 460 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 465 470 475 480 Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 561557DNAArtificial SequenceSynthetic 56atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa aggcaccggt 360ggagggtcgg gatccagctc actcgaggtg acatgtgaac ccggtacgac gtttaaggat 420aagtgcaaca catgtaggtg cggtagcgac ggcaaatcag cgttctgtac ccggaaattg 480tgctaccagg gaactggagg agggtcgggg tcctcgtcat atatcgacgc atggggccac 540gggaccgaag tcatcgtctc ctccgctagc accaagggcc catcggtctt ccccctggca 600ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 660ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 720ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 780tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 840aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 900ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 960accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1020gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1080aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1140caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1200gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1260accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1320aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1380aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1440ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1500gaggctctgc acaaccacta cacacagaag agcctctccc tgtctccggg taaatga 155757499PRTArtificial SequenceSynthetic 57Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Leu Glu Val Thr 100 105 110 Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys 115 120 125 Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln 130 135 140 Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Tyr Ile Asp Ala Trp Gly 145 150 155 160 His Gly Thr Glu Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 165 170 175 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 180 185 190 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 195 200 205 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 210 215 220 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 225 230 235 240 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 245 250 255 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 260 265 270 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 275 280 285 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 290 295 300 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 305 310 315 320 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 335 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 370 375 380 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 405 410 415 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 420 425 430 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 450 455 460 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 465 470 475 480 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 485 490 495 Pro Gly Lys 581545DNAArtificial SequenceSynthetic 58atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa agccgctggt 360ggtagtggtc tcgaggtgac atgtgaaccc ggtacgacgt ttaaggataa gtgcaacaca 420tgtaggtgcg gtagcgacgg caaatcagcg ttctgtaccc ggaaattgtg ctaccaggga 480actggaggag ggtcggggtc ctcgtcatat atcgacgcat ggggccacgg gaccgaagtc 540atcgtctcct ccgctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 600agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 660gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 720ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 780ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 840aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 900ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 960tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1020aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1080gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1140ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1200aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1260tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1320cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1380acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1440aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1500aaccactaca cacagaagag cctctccctg tctccgggta aatga 154559495PRTArtificial SequenceSynthetic 59Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys Glu Pro Gly 100 105 110 Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly 115 120 125 Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Gly Thr Gly Gly 130 135 140 Gly Ser Gly Ser Ser Ser Tyr Ile Asp Ala Trp Gly His Gly Thr Glu 145 150 155 160 Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 165 170 175 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 180 185 190 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 195 200 205 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 210 215 220 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 225

230 235 240 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 245 250 255 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 260 265 270 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 275 280 285 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 290 295 300 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 305 310 315 320 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 325 330 335 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 340 345 350 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 355 360 365 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 370 375 380 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 385 390 395 400 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 405 410 415 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 420 425 430 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 435 440 445 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 450 455 460 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 465 470 475 480 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 495 601509DNAArtificial SequenceSynthetic 60atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa actcgaggtg 360acatgtgaac ccggtacgac gtttaaggat aagtgcaaca catgtaggtg cggtagcgac 420ggcaaatcag cgttctgtac ccggaaattg tgctaccagg gtagtggtgc ttatatcgac 480gcatggggcc acgggaccga agtcatcgtc tcctccgcta gcaccaaggg cccatcggtc 540ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct gggctgcctg 600gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 660ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg 720gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt gaatcacaag 780cccagcaaca ccaaggtgga caagaaagtt gagcccaaat cttgtgacaa aactcacaca 840tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 900aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1140aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1200ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 1260acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1320cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1380ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1440tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctccg 1500ggtaaatga 150961493PRTArtificial SequenceSynthetic 61Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Leu Glu Val Thr 100 105 110 Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys 115 120 125 Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln 130 135 140 Gly Ser Gly Ala Tyr Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile 145 150 155 160 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 165 170 175 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 180 185 190 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 195 200 205 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 210 215 220 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 225 230 235 240 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 245 250 255 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 260 265 270 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 275 280 285 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 290 295 300 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 305 310 315 320 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 325 330 335 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 340 345 350 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 355 360 365 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 370 375 380 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 385 390 395 400 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 405 410 415 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 420 425 430 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 435 440 445 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 450 455 460 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 465 470 475 480 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 621527DNAArtificial SequenceSynthetic 62atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa agccgctggt 360ggtagtggtc tcgaggtgac atgtgaaccc ggtacgacgt ttaaggataa gtgcaacaca 420tgtaggtgcg gtagcgacgg caaatcagcg ttctgtaccc ggaaattgtg ctaccagggt 480agtggtgctt atatcgacgc atggggccac gggaccgaag tcatcgtctc ctccgctagc 540accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 600gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 660tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 720tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 780tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 840tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 900gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 960acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1020gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1080taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1140aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1200aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1260aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1320gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1380tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1440gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1500agcctctccc tgtctccggg taaatga 152763489PRTArtificial SequenceSynthetic 63Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys Glu Pro Gly 100 105 110 Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly 115 120 125 Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Gly Ser Gly Ala 130 135 140 Tyr Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala 145 150 155 160 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 165 170 175 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 180 185 190 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 195 200 205 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 210 215 220 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 225 230 235 240 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 245 250 255 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 260 265 270 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 275 280 285 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 290 295 300 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 305 310 315 320 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 325 330 335 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 340 345 350 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 355 360 365 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 370 375 380 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 385 390 395 400 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 405 410 415 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 420 425 430 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 435 440 445 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 450 455 460 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 465 470 475 480 Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 641539DNAArtificial SequenceSynthetic 64atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa agccgctggt 360ggtagtggtg gtagtggtgc tctcgaggtg acatgtgaac ccggtacgac gtttaaggat 420aagtgcaaca catgtaggtg cggtagcgac ggcaaatcag cgttctgtac ccggaaattg 480tgctaccagg gtagtggtgc ttatatcgac gcatggggcc acgggaccga agtcatcgtc 540tcctccgcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 600tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 660gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 720tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 780cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 840gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 900gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 960acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1020aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1080tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1140ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1200atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1260gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1320gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1380cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1440aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1500tacacacaga agagcctctc cctgtctccg ggtaaatga 153965493PRTArtificial SequenceSynthetic 65Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala Leu Glu Val Thr 100 105 110 Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys 115 120 125 Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln 130 135 140 Gly Ser Gly Ala Tyr Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile 145 150 155 160 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 165 170 175 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 180 185 190 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 195 200 205 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 210 215 220 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 225 230 235 240 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 245 250 255 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 260 265 270 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 275 280 285 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 290 295 300 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

Pro Glu Val Lys 305 310 315 320 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 325 330 335 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 340 345 350 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 355 360 365 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 370 375 380 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 385 390 395 400 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 405 410 415 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 420 425 430 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 435 440 445 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 450 455 460 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 465 470 475 480 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 661527DNAArtificial SequenceSynthetic 66atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa actcgaggtg 360acatgtgaac ccggtacgac gtttaaggat aagtgcaaca catgtaggtg cggtagcgac 420ggcaaatcag cgttctgtac ccggaaattg tgctaccagg ccgctggtgg tagtggtggt 480agtggtgctt atatcgacgc atggggccac gggaccgaag tcatcgtctc ctccgctagc 540accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 600gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 660tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 720tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 780tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 840tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 900gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 960acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1020gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1080taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1140aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1200aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1260aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1320gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1380tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1440gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1500agcctctccc tgtctccggg taaatga 152767489PRTArtificial SequenceSynthetic 67Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys 100 105 110 Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr 115 120 125 Arg Lys Leu Cys Tyr Gln Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala 130 135 140 Tyr Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala 145 150 155 160 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 165 170 175 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 180 185 190 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 195 200 205 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 210 215 220 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 225 230 235 240 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 245 250 255 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 260 265 270 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 275 280 285 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 290 295 300 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 305 310 315 320 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 325 330 335 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 340 345 350 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 355 360 365 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 370 375 380 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 385 390 395 400 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 405 410 415 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 420 425 430 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 435 440 445 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 450 455 460 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 465 470 475 480 Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 681557DNAArtificial SequenceSynthetic 68atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa aggcaccggt 360ggagggtcgg gatccagctc actcgaggtg acatgtgaac ccggtacgac gtttaaggat 420aagtgcaaca catgtaggtg cggtagcgac ggcaaatcag cgttctgtac ccggaaattg 480tgctaccagg ccgctggtgg tagtggtggt agtggtgctt atatcgacgc atggggccac 540gggaccgaag tcatcgtctc ctccgctagc accaagggcc catcggtctt ccccctggca 600ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 660ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 720ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 780tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 840aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 900ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 960accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1020gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1080aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1140caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1200gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1260accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1320aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1380aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1440ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1500gaggctctgc acaaccacta cacacagaag agcctctccc tgtctccggg taaatga 155769499PRTArtificial SequenceSynthetic 69Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Leu Glu Val Thr 100 105 110 Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys 115 120 125 Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln 130 135 140 Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala Tyr Ile Asp Ala Trp Gly 145 150 155 160 His Gly Thr Glu Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 165 170 175 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 180 185 190 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 195 200 205 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 210 215 220 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 225 230 235 240 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 245 250 255 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 260 265 270 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 275 280 285 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 290 295 300 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 305 310 315 320 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 335 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 370 375 380 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 405 410 415 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 420 425 430 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 450 455 460 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 465 470 475 480 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 485 490 495 Pro Gly Lys 701545DNAArtificial SequenceSynthetic 70atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa agccgctggt 360ggtagtggtc tcgaggtgac atgtgaaccc ggtacgacgt ttaaggataa gtgcaacaca 420tgtaggtgcg gtagcgacgg caaatcagcg ttctgtaccc ggaaattgtg ctaccaggcc 480gctggtggta gtggtggtag tggtgcttat atcgacgcat ggggccacgg gaccgaagtc 540atcgtctcct ccgctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 600agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 660gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 720ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 780ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 840aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 900ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 960tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1020aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1080gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1140ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1200aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1260tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1320cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1380acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1440aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1500aaccactaca cacagaagag cctctccctg tctccgggta aatga 154571495PRTArtificial SequenceSynthetic 71Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys Glu Pro Gly 100 105 110 Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly 115 120 125 Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Ala Ala Gly Gly 130 135 140 Ser Gly Gly Ser Gly Ala Tyr Ile Asp Ala Trp Gly His Gly Thr Glu 145 150 155 160 Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 165 170 175 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 180 185 190 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 195 200 205 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 210 215 220 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 225 230 235 240 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 245 250 255 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 260 265 270 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 275 280 285 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 290 295 300 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 305 310 315 320 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 325 330 335 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 340 345 350 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 355 360 365 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 370 375 380 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

Val Tyr Thr Leu Pro 385 390 395 400 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 405 410 415 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 420 425 430 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 435 440 445 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 450 455 460 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 465 470 475 480 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 495 721557DNAArtificial SequenceSynthetic 72atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcacctact actgcgccaa agccgctggt 360ggtagtggtg gtagtggtgc tctcgaggtg acatgtgaac ccggtacgac gtttaaggat 420aagtgcaaca catgtaggtg cggtagcgac ggcaaatcag cgttctgtac ccggaaattg 480tgctaccagg ccgctggtgg tagtggtggt agtggtgctt atatcgacgc atggggccac 540gggaccgaag tcatcgtctc ctccgctagc accaagggcc catcggtctt ccccctggca 600ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 660ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 720ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 780tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 840aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 900ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 960accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1020gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1080aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1140caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1200gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1260accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1320aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1380aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1440ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1500gaggctctgc acaaccacta cacacagaag agcctctccc tgtctccggg taaatga 155773499PRTArtificial SequenceSynthetic 73Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala Leu Glu Val Thr 100 105 110 Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys 115 120 125 Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln 130 135 140 Ala Ala Gly Gly Ser Gly Gly Ser Gly Ala Tyr Ile Asp Ala Trp Gly 145 150 155 160 His Gly Thr Glu Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 165 170 175 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 180 185 190 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 195 200 205 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 210 215 220 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 225 230 235 240 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 245 250 255 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 260 265 270 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 275 280 285 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 290 295 300 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 305 310 315 320 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 335 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 370 375 380 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 405 410 415 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 420 425 430 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 450 455 460 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 465 470 475 480 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 485 490 495 Pro Gly Lys 74795DNAArtificial SequenceSynthetic 74atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gcttggaagt gacgtgtgag cccggaacga cattcaaaga caagtgcaat 180acttgtcggt gcggttcaga tgggaaatcg gcggtctgca caaagctctg gtgtaaccag 240tactattatg gctggtacca gcagaaggca cctggcagtg cccctgtcac tctgatctat 300tacaacaaca agagaccctc ggacatccct tcacgattct ccggttccct atccggctcc 360acaaacacat taaccatcac tggggtccga gccgatgacg aggctgtcta tttctgtggg 420agtgcagaca acagtggtgc tgcatttggg gccgggacaa ccctgacagt acttggtcag 480cccaaggctg ccccttcggt caccctgttc ccgccctcct ctgaggagct tcaagccaac 540aaggccacac tggtgtgtct cataagtgac ttctacccgg gagccgtgac agtggcctgg 600aaggcagata gcagccccgt caaggcggga gtggagacca ccacaccctc caaacaaagc 660aacaacaagt acgcggccag cagctatctg agcctgacgc ctgagcagtg gaagtcccac 720agaagctaca gctgccaggt cacgcatgaa gggagcaccg tggagaagac agtggcccct 780acagaatgtt catag 79575245PRTArtificial SequenceSynthetic 75Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Leu Glu Val Thr Cys Glu Pro 20 25 30 Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp 35 40 45 Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn Gln Tyr Tyr Tyr 50 55 60 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 65 70 75 80 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 85 90 95 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 100 105 110 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 115 120 125 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 130 135 140 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 145 150 155 160 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 165 170 175 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 180 185 190 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 195 200 205 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 210 215 220 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 225 230 235 240 Pro Thr Glu Cys Ser 245 76786DNAArtificial SequenceSynthetic 76atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gcacgtgtga gcccggaacg acattcaaag acaagtgcaa tacttgtcgg 180tgcggttcag atgggaaatc ggcggtctgc acaaagctct ggtgtaacca gtactattat 240ggctggtacc agcagaaggc acctggcagt gcccctgtca ctctgatcta ttacaacaac 300aagagaccct cggacatccc ttcacgattc tccggttccc tatccggctc cacaaacaca 360ttaaccatca ctggggtccg agccgatgac gaggctgtct atttctgtgg gagtgcagac 420aacagtggtg ctgcatttgg ggccgggaca accctgacag tacttggtca gcccaaggct 480gccccttcgg tcaccctgtt cccgccctcc tctgaggagc ttcaagccaa caaggccaca 540ctggtgtgtc tcataagtga cttctacccg ggagccgtga cagtggcctg gaaggcagat 600agcagccccg tcaaggcggg agtggagacc accacaccct ccaaacaaag caacaacaag 660tacgcggcca gcagctatct gagcctgacg cctgagcagt ggaagtccca cagaagctac 720agctgccagg tcacgcatga agggagcacc gtggagaaga cagtggcccc tacagaatgt 780tcatag 78677242PRTArtificial SequenceSynthetic 77Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Thr Cys Glu Pro Gly Thr Thr 20 25 30 Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser 35 40 45 Ala Val Cys Thr Lys Leu Trp Cys Asn Gln Tyr Tyr Tyr Gly Trp Tyr 50 55 60 Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr Tyr Asn 65 70 75 80 Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser 85 90 95 Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp Glu 100 105 110 Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala Ala Phe Gly 115 120 125 Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 130 135 140 Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala 145 150 155 160 Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val 165 170 175 Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr 180 185 190 Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200 205 Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln 210 215 220 Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu 225 230 235 240 Cys Ser 78747DNAArtificial SequenceSynthetic 78atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gcacttgtcg gtgcggttca gatgggaaat cggcggtctg cacaaagctc 180tggtgtaacc agtactatta tggctggtac cagcagaagg cacctggcag tgcccctgtc 240actctgatct attacaacaa caagagaccc tcggacatcc cttcacgatt ctccggttcc 300ctatccggct ccacaaacac attaaccatc actggggtcc gagccgatga cgaggctgtc 360tatttctgtg ggagtgcaga caacagtggt gctgcatttg gggccgggac aaccctgaca 420gtacttggtc agcccaaggc tgccccttcg gtcaccctgt tcccgccctc ctctgaggag 480cttcaagcca acaaggccac actggtgtgt ctcataagtg acttctaccc gggagccgtg 540acagtggcct ggaaggcaga tagcagcccc gtcaaggcgg gagtggagac caccacaccc 600tccaaacaaa gcaacaacaa gtacgcggcc agcagctatc tgagcctgac gcctgagcag 660tggaagtccc acagaagcta cagctgccag gtcacgcatg aagggagcac cgtggagaag 720acagtggccc ctacagaatg ttcatag 74779229PRTArtificial SequenceSynthetic 79Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Thr Cys Arg Cys Gly Ser Asp 20 25 30 Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn Gln Tyr Tyr Tyr 35 40 45 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 50 55 60 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 65 70 75 80 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 85 90 95 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 100 105 110 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 115 120 125 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 130 135 140 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 145 150 155 160 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 165 170 175 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 180 185 190 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 195 200 205 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 210 215 220 Pro Thr Glu Cys Ser 225 80795DNAArtificial SequenceSynthetic 80atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gcctcgaggt gacatgtgaa cccggtacga cgtttaagga taagtgcaac 180acatgtaggt gcggtagcga cggcaaatca gcgttctgta cccggaaatt gtgctaccag 240tactattatg gctggtacca gcagaaggca cctggcagtg cccctgtcac tctgatctat 300tacaacaaca agagaccctc ggacatccct tcacgattct ccggttccct atccggctcc 360acaaacacat taaccatcac tggggtccga gccgatgacg aggctgtcta tttctgtggg 420agtgcagaca acagtggtgc tgcatttggg gccgggacaa ccctgacagt acttggtcag 480cccaaggctg ccccttcggt caccctgttc ccgccctcct ctgaggagct tcaagccaac 540aaggccacac tggtgtgtct cataagtgac ttctacccgg gagccgtgac agtggcctgg 600aaggcagata gcagccccgt caaggcggga gtggagacca ccacaccctc caaacaaagc 660aacaacaagt acgcggccag cagctatctg agcctgacgc ctgagcagtg gaagtcccac 720agaagctaca gctgccaggt cacgcatgaa gggagcaccg tggagaagac agtggcccct 780acagaatgtt catag 79581245PRTArtificial SequenceSynthetic 81Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Leu Glu Val Thr Cys Glu Pro 20 25 30 Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp 35 40 45 Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Tyr Tyr Tyr 50 55 60 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 65 70 75 80 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 85 90 95 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 100 105 110 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 115 120 125 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 130 135 140 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 145 150 155 160 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 165 170 175 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

180 185 190 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 195 200 205 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 210 215 220 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 225 230 235 240 Pro Thr Glu Cys Ser 245 82786DNAArtificial SequenceSynthetic 82atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gcacatgtga acccggtacg acgtttaagg ataagtgcaa cacatgtagg 180tgcggtagcg acggcaaatc agcgttctgt acccggaaat tgtgctacca gtactattat 240ggctggtacc agcagaaggc acctggcagt gcccctgtca ctctgatcta ttacaacaac 300aagagaccct cggacatccc ttcacgattc tccggttccc tatccggctc cacaaacaca 360ttaaccatca ctggggtccg agccgatgac gaggctgtct atttctgtgg gagtgcagac 420aacagtggtg ctgcatttgg ggccgggaca accctgacag tacttggtca gcccaaggct 480gccccttcgg tcaccctgtt cccgccctcc tctgaggagc ttcaagccaa caaggccaca 540ctggtgtgtc tcataagtga cttctacccg ggagccgtga cagtggcctg gaaggcagat 600agcagccccg tcaaggcggg agtggagacc accacaccct ccaaacaaag caacaacaag 660tacgcggcca gcagctatct gagcctgacg cctgagcagt ggaagtccca cagaagctac 720agctgccagg tcacgcatga agggagcacc gtggagaaga cagtggcccc tacagaatgt 780tcatag 78683242PRTArtificial SequenceSynthetic 83Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Thr Cys Glu Pro Gly Thr Thr 20 25 30 Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser 35 40 45 Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Tyr Tyr Tyr Gly Trp Tyr 50 55 60 Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr Tyr Asn 65 70 75 80 Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser 85 90 95 Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp Glu 100 105 110 Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala Ala Phe Gly 115 120 125 Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 130 135 140 Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala 145 150 155 160 Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val 165 170 175 Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr 180 185 190 Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200 205 Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln 210 215 220 Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu 225 230 235 240 Cys Ser 84747DNAArtificial SequenceSynthetic 84atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gcacatgtag gtgcggtagc gacggcaaat cagcgttctg tacccggaaa 180ttgtgctacc agtactatta tggctggtac cagcagaagg cacctggcag tgcccctgtc 240actctgatct attacaacaa caagagaccc tcggacatcc cttcacgatt ctccggttcc 300ctatccggct ccacaaacac attaaccatc actggggtcc gagccgatga cgaggctgtc 360tatttctgtg ggagtgcaga caacagtggt gctgcatttg gggccgggac aaccctgaca 420gtacttggtc agcccaaggc tgccccttcg gtcaccctgt tcccgccctc ctctgaggag 480cttcaagcca acaaggccac actggtgtgt ctcataagtg acttctaccc gggagccgtg 540acagtggcct ggaaggcaga tagcagcccc gtcaaggcgg gagtggagac caccacaccc 600tccaaacaaa gcaacaacaa gtacgcggcc agcagctatc tgagcctgac gcctgagcag 660tggaagtccc acagaagcta cagctgccag gtcacgcatg aagggagcac cgtggagaag 720acagtggccc ctacagaatg ttcatag 74785229PRTArtificial SequenceSynthetic 85Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Thr Cys Arg Cys Gly Ser Asp 20 25 30 Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Tyr Tyr Tyr 35 40 45 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 50 55 60 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 65 70 75 80 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 85 90 95 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 100 105 110 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 115 120 125 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 130 135 140 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 145 150 155 160 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 165 170 175 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 180 185 190 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 195 200 205 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 210 215 220 Pro Thr Glu Cys Ser 225 86125PRTArtificial SequenceSynthetic 86Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys 85 90 95 Thr Lys Cys Ala Tyr Ser Ser Gly Cys Asp Tyr Glu Gly Gly Tyr Ile 100 105 110 Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 87107PRTArtificial SequenceSynthetic 87Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr 20 25 30 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 35 40 45 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 65 70 75 80 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 85 90 95 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 881563DNAArtificial SequenceSynthetic 88atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcctc 60gaggtgacat gtgaacccgg tacgacgttt aaggataagt gcaacacatg taggtgcggt 120agcgacggca aatcagcgtt ctgtacccgg aaattgtgct accagggaac tggaggaggg 180tcggggtcct cgtcagccgt gacgttggac gagtccgggg gcggcctcca gacgcccggg 240ggagcgctca gcctcgtctg caaggcctcc gggttcacct tcagcagtaa cgccatgggt 300tgggtgcgac aggcgcccgg caaggggctg gagtgggtcg ctggtattga tgatgatggt 360agtggcacaa gatacgcgcc ggcggtgaag ggccgtgcca ccatctcgag ggacaacggg 420cagagcacac tgaggctgca gctgaacaac ctcagggctg aggacaccgg catctactac 480tgcacgaaat gtgcttacag tagtggttgt gattatgaag gtggttatat cgacgcatgg 540ggccacggga ccgaagtcat cgtctcctcc gctagcacca agggcccatc ggtcttcccc 600ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag 660gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 720cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 780gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 840aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 900ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 960aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 1020cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1080aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1140gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1200ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1260gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1320ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1380gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1440agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1500atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tccgggtaaa 1560tga 156389501PRTArtificial SequenceSynthetic 89Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys 20 25 30 Leu Cys Tyr Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Ala Val 35 40 45 Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu 50 55 60 Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn Ala Met 65 70 75 80 Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 85 90 95 Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val Lys Gly 100 105 110 Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg Leu Gln 115 120 125 Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Lys 130 135 140 Cys Ala Tyr Ser Ser Gly Cys Asp Tyr Glu Gly Gly Tyr Ile Asp Ala 145 150 155 160 Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala Ser Thr Lys Gly 165 170 175 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 180 185 190 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 195 200 205 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 210 215 220 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 225 230 235 240 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 245 250 255 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 260 265 270 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 275 280 285 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 290 295 300 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 305 310 315 320 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 325 330 335 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 340 345 350 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 355 360 365 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 370 375 380 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 385 390 395 400 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 405 410 415 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 420 425 430 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 435 440 445 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 450 455 460 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 465 470 475 480 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 485 490 495 Leu Ser Pro Gly Lys 500 901563DNAArtificial SequenceSynthetic 90atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcttg 60gaagtgacgt gtgagcccgg aacgacattc aaagacaagt gcaatacttg tcggtgcggt 120tcagatggga aatcggcggt ctgcacaaag ctctggtgta accagggcac cggtggaggg 180tcgggatcca gctcagccgt gacgttggac gagtccgggg gcggcctcca gacgcccggg 240ggagcgctca gcctcgtctg caaggcctcc gggttcacct tcagcagtaa cgccatgggt 300tgggtgcgac aggcgcccgg caaggggctg gagtgggtcg ctggtattga tgatgatggt 360agtggcacaa gatacgcgcc ggcggtgaag ggccgtgcca ccatctcgag ggacaacggg 420cagagcacac tgaggctgca gctgaacaac ctcagggctg aggacaccgg catctactac 480tgcacgaaat gtgcttacag tagtggttgt gattatgaag gtggttatat cgacgcatgg 540ggccacggga ccgaagtcat cgtctcctcc gctagcacca agggcccatc ggtcttcccc 600ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag 660gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 720cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 780gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 840aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 900ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 960aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 1020cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1080aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1140gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1200ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1260gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1320ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1380gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1440agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1500atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tccgggtaaa 1560tga 156391501PRTArtificial SequenceSynthetic 91Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu 20 25 30 Trp Cys Asn Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Ala Val 35 40 45 Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu 50 55 60 Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn Ala Met 65 70 75 80 Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly 85 90 95 Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val Lys Gly 100 105 110 Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg Leu Gln 115 120 125 Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Lys 130 135 140 Cys Ala Tyr Ser Ser Gly Cys Asp Tyr Glu Gly Gly Tyr Ile Asp Ala 145 150 155 160 Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala Ser Thr Lys Gly 165 170 175 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 180 185 190 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 195 200 205 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 210 215 220 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 225 230

235 240 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 245 250 255 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 260 265 270 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 275 280 285 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 290 295 300 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 305 310 315 320 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 325 330 335 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 340 345 350 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 355 360 365 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 370 375 380 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 385 390 395 400 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 405 410 415 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 420 425 430 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 435 440 445 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 450 455 460 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 465 470 475 480 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 485 490 495 Leu Ser Pro Gly Lys 500 921563DNAArtificial SequenceSynthetic 92atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcatctact actgcacgaa atgtgcttac 360agtagtggtt gtgattatga aggtggttat atcgacgcat ggggccacgg gaccgaagtc 420atcgtctcct ccgctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 480agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 540gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 600ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 660ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 720aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 780ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1200cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1320aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380aaccactaca cacagaagag cctctccctg tctccgggta aagccgctgg tggtagtggt 1440ctcgaggtga catgtgaacc cggtacgacg tttaaggata agtgcaacac atgtaggtgc 1500ggtagcgacg gcaaatcagc gttctgtacc cggaaattgt gctaccaggg tagtggtgct 1560tga 156393501PRTArtificial SequenceSynthetic 93Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys 85 90 95 Thr Lys Cys Ala Tyr Ser Ser Gly Cys Asp Tyr Glu Gly Gly Tyr Ile 100 105 110 Asp Ala Trp Gly His Gly Thr Glu Val Ile 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 Lys Ala Ala Gly Gly Ser Gly Leu Glu Val 450 455 460 Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg 465 470 475 480 Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr 485 490 495 Gln Gly Ser Gly Ala 500 941563DNAArtificial SequenceSynthetic 94atggagttcg gcctgagctg gctgttcctg gtggccatcc ttaagggcgt gcagtgcgcc 60gtgacgttgg acgagtccgg gggcggcctc cagacgcccg ggggagcgct cagcctcgtc 120tgcaaggcct ccgggttcac cttcagcagt aacgccatgg gttgggtgcg acaggcgccc 180ggcaaggggc tggagtgggt cgctggtatt gatgatgatg gtagtggcac aagatacgcg 240ccggcggtga agggccgtgc caccatctcg agggacaacg ggcagagcac actgaggctg 300cagctgaaca acctcagggc tgaggacacc ggcatctact actgcacgaa atgtgcttac 360agtagtggtt gtgattatga aggtggttat atcgacgcat ggggccacgg gaccgaagtc 420atcgtctcct ccgctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 480agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 540gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 600ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 660ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 720aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 780ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1200cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1320aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380aaccactaca cacagaagag cctctccctg tctccgggta aagccgctgg tggtagtggt 1440ttggaagtga cgtgtgagcc cggaacgaca ttcaaagaca agtgcaatac ttgtcggtgc 1500ggttcagatg ggaaatcggc ggtctgcaca aagctctggt gtaaccaggg tagtggtgct 1560tga 156395501PRTArtificial SequenceSynthetic 95Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Asp Asp Asp Gly Ser Gly Thr Arg Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Leu Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys 85 90 95 Thr Lys Cys Ala Tyr Ser Ser Gly Cys Asp Tyr Glu Gly Gly Tyr Ile 100 105 110 Asp Ala Trp Gly His Gly Thr Glu Val Ile 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 Lys Ala Ala Gly Gly Ser Gly Leu Glu Val 450 455 460 Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg 465 470 475 480 Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn 485 490 495 Gln Gly Ser Gly Ala 500 96837DNAArtificial SequenceSynthetic 96atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccctc 60gaggtgacat gtgaacccgg tacgacgttt aaggataagt gcaacacatg taggtgcggt 120agcgacggca aatcagcgtt ctgtacccgg aaattgtgct accagggaac tggaggaggg 180tcggggtcct cgtcagcgct gactcagcct gcctcggtgt cagcaaaccc aggagaaacc 240gtcaagatca cctgctccgg gggtggcagc tatgctggaa gttactatta tggctggtac 300cagcagaagg cacctggcag tgcccctgtc actctgatct attacaacaa caagagaccc 360tcggacatcc cttcacgatt ctccggttcc ctatccggct ccacaaacac attaaccatc 420actggggtcc gagccgatga cgaggctgtc tatttctgtg ggagtgcaga caacagtggt 480gctgcatttg gggccgggac aaccctgaca gtacttggtc agcccaaggc tgccccttcg 540gtcaccctgt tcccgccctc ctctgaggag cttcaagcca acaaggccac actggtgtgt 600ctcataagtg acttctaccc gggagccgtg acagtggcct ggaaggcaga tagcagcccc 660gtcaaggcgg gagtggagac caccacaccc tccaaacaaa gcaacaacaa gtacgcggcc 720agcagctatc tgagcctgac gcctgagcag tggaagtccc acagaagcta cagctgccag 780gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttcatag 83797259PRTArtificial SequenceSynthetic 97Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys 20 25 30 Leu Cys Tyr Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Ala Leu 35 40 45 Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val Lys Ile 50 55 60 Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr Gly Trp 65 70 75 80 Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr Tyr 85 90 95 Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu 100 105 110 Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp 115 120 125 Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala Ala Phe 130 135 140 Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro 145 150 155 160 Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys 165 170 175 Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr 180 185 190 Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr 195 200 205 Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 210 215 220 Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys 225 230 235 240 Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr 245 250 255 Glu Cys Ser 98837DNAArtificial SequenceSynthetic 98atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccttg 60gaagtgacgt gtgagcccgg aacgacattc aaagacaagt gcaatacttg tcggtgcggt 120tcagatggga aatcggcggt ctgcacaaag ctctggtgta accagggcac cggtggaggg 180tcgggatcca gctcagcgct gactcagcct gcctcggtgt cagcaaaccc aggagaaacc 240gtcaagatca cctgctccgg gggtggcagc tatgctggaa gttactatta tggctggtac 300cagcagaagg cacctggcag tgcccctgtc actctgatct attacaacaa caagagaccc 360tcggacatcc cttcacgatt ctccggttcc ctatccggct ccacaaacac attaaccatc 420actggggtcc gagccgatga cgaggctgtc tatttctgtg ggagtgcaga caacagtggt 480gctgcatttg gggccgggac aaccctgaca gtacttggtc agcccaaggc tgccccttcg 540gtcaccctgt tcccgccctc ctctgaggag cttcaagcca acaaggccac actggtgtgt 600ctcataagtg acttctaccc gggagccgtg acagtggcct ggaaggcaga tagcagcccc 660gtcaaggcgg gagtggagac caccacaccc tccaaacaaa gcaacaacaa gtacgcggcc 720agcagctatc tgagcctgac gcctgagcag tggaagtccc acagaagcta cagctgccag 780gtcacgcatg

aagggagcac cgtggagaag acagtggccc ctacagaatg ttcatag 83799259PRTArtificial SequenceSynthetic 99Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu 20 25 30 Trp Cys Asn Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Ala Leu 35 40 45 Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val Lys Ile 50 55 60 Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr Gly Trp 65 70 75 80 Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr Tyr 85 90 95 Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu 100 105 110 Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp 115 120 125 Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala Ala Phe 130 135 140 Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro 145 150 155 160 Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys 165 170 175 Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr 180 185 190 Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr 195 200 205 Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 210 215 220 Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys 225 230 235 240 Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr 245 250 255 Glu Cys Ser 100837DNAArtificial SequenceSynthetic 100atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gctatgctgg aagttactat tatggctggt accagcagaa ggcacctggc 180agtgcccctg tcactctgat ctattacaac aacaagagac cctcggacat cccttcacga 240ttctccggtt ccctatccgg ctccacaaac acattaacca tcactggggt ccgagccgat 300gacgaggctg tctatttctg tgggagtgca gacaacagtg gtgctgcatt tggggccggg 360acaaccctga cagtacttgg tcagcccaag gctgcccctt cggtcaccct gttcccgccc 420tcctctgagg agcttcaagc caacaaggcc acactggtgt gtctcataag tgacttctac 480ccgggagccg tgacagtggc ctggaaggca gatagcagcc ccgtcaaggc gggagtggag 540accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta tctgagcctg 600acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca tgaagggagc 660accgtggaga agacagtggc ccctacagaa tgttcagccg ctggtggtag tggtctcgag 720gtgacatgtg aacccggtac gacgtttaag gataagtgca acacatgtag gtgcggtagc 780gacggcaaat cagcgttctg tacccggaaa ttgtgctacc agggtagtgg tgcttag 837101259PRTArtificial SequenceSynthetic 101Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr 20 25 30 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 35 40 45 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 65 70 75 80 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 85 90 95 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 100 105 110 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys 210 215 220 Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly 225 230 235 240 Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Gly 245 250 255 Ser Gly Ala 102837DNAArtificial SequenceSynthetic 102atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gctatgctgg aagttactat tatggctggt accagcagaa ggcacctggc 180agtgcccctg tcactctgat ctattacaac aacaagagac cctcggacat cccttcacga 240ttctccggtt ccctatccgg ctccacaaac acattaacca tcactggggt ccgagccgat 300gacgaggctg tctatttctg tgggagtgca gacaacagtg gtgctgcatt tggggccggg 360acaaccctga cagtacttgg tcagcccaag gctgcccctt cggtcaccct gttcccgccc 420tcctctgagg agcttcaagc caacaaggcc acactggtgt gtctcataag tgacttctac 480ccgggagccg tgacagtggc ctggaaggca gatagcagcc ccgtcaaggc gggagtggag 540accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta tctgagcctg 600acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca tgaagggagc 660accgtggaga agacagtggc ccctacagaa tgttcagccg ctggtggtag tggtttggaa 720gtgacgtgtg agcccggaac gacattcaaa gacaagtgca atacttgtcg gtgcggttca 780gatgggaaat cggcggtctg cacaaagctc tggtgtaacc agggtagtgg tgcttag 837103259PRTArtificial SequenceSynthetic 103Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr 20 25 30 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 35 40 45 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 65 70 75 80 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 85 90 95 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 100 105 110 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys 210 215 220 Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly 225 230 235 240 Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn Gln Gly 245 250 255 Ser Gly Ala 104259PRTArtificial SequenceSynthetic 104Leu Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15 Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu 20 25 30 Trp Cys Asn Gln Gly Thr Gly Gly Gly Ser Gly Ser Ser Ser Ala Leu 35 40 45 Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val Lys Ile 50 55 60 Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr Gly Trp 65 70 75 80 Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr Tyr 85 90 95 Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu 100 105 110 Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp 115 120 125 Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala Ala Phe 130 135 140 Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro 145 150 155 160 Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys 165 170 175 Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr 180 185 190 Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr 195 200 205 Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 210 215 220 Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys 225 230 235 240 Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr 245 250 255 Glu Cys Ser 105837DNAArtificial SequenceSynthetic 105atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gctatgctgg aagttactat tatggctggt accagcagaa ggcacctggc 180agtgcccctg tcactctgat ctattacaac aacaagagac cctcggacat cccttcacga 240ttctccggtt ccctatccgg ctccacaaac acattaacca tcactggggt ccgagccgat 300gacgaggctg tctatttctg tgggagtgca gacaacagtg gtgctgcatt tggggccggg 360acaaccctga cagtacttgg tcagcccaag gctgcccctt cggtcaccct gttcccgccc 420tcctctgagg agcttcaagc caacaaggcc acactggtgt gtctcataag tgacttctac 480ccgggagccg tgacagtggc ctggaaggca gatagcagcc ccgtcaaggc gggagtggag 540accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta tctgagcctg 600acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca tgaagggagc 660accgtggaga agacagtggc ccctacagaa tgttcagccg ctggtggtag tggtctcgag 720gtgacatgtg aacccggtac gacgtttaag gataagtgca acacatgtag gtgcggtagc 780gacggcaaat cagcgttctg tacccggaaa ttgtgctacc agggtagtgg tgcttag 837106259PRTArtificial SequenceSynthetic 106Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr 20 25 30 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 35 40 45 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 65 70 75 80 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 85 90 95 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 100 105 110 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys 210 215 220 Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly 225 230 235 240 Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg Lys Leu Cys Tyr Gln Gly 245 250 255 Ser Gly Ala 107837DNAArtificial SequenceSynthetic 107atggcctgga ttcctctact tctccccctc ctcactctct gcacaggatc cgaagccgcg 60ctgactcagc ctgcctcggt gtcagcaaac ccaggagaaa ccgtcaagat cacctgctcc 120gggggtggca gctatgctgg aagttactat tatggctggt accagcagaa ggcacctggc 180agtgcccctg tcactctgat ctattacaac aacaagagac cctcggacat cccttcacga 240ttctccggtt ccctatccgg ctccacaaac acattaacca tcactggggt ccgagccgat 300gacgaggctg tctatttctg tgggagtgca gacaacagtg gtgctgcatt tggggccggg 360acaaccctga cagtacttgg tcagcccaag gctgcccctt cggtcaccct gttcccgccc 420tcctctgagg agcttcaagc caacaaggcc acactggtgt gtctcataag tgacttctac 480ccgggagccg tgacagtggc ctggaaggca gatagcagcc ccgtcaaggc gggagtggag 540accaccacac cctccaaaca aagcaacaac aagtacgcgg ccagcagcta tctgagcctg 600acgcctgagc agtggaagtc ccacagaagc tacagctgcc aggtcacgca tgaagggagc 660accgtggaga agacagtggc ccctacagaa tgttcagccg ctggtggtag tggtttggaa 720gtgacgtgtg agcccggaac gacattcaaa gacaagtgca atacttgtcg gtgcggttca 780gatgggaaat cggcggtctg cacaaagctc tggtgtaacc agggtagtgg tgcttag 837108259PRTArtificial SequenceSynthetic 108Ala Leu Thr Gln Pro Ala Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Ala Gly Ser Tyr Tyr Tyr 20 25 30 Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile 35 40 45 Tyr Tyr Asn Asn Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val Arg Ala 65 70 75 80 Asp Asp Glu Ala Val Tyr Phe Cys Gly Ser Ala Asp Asn Ser Gly Ala 85 90 95 Ala Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Pro Lys Ala 100 105 110 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser Ala Ala Gly Gly Ser Gly Leu Glu Val Thr Cys 210 215 220 Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn Thr Cys Arg Cys Gly 225 230 235 240 Ser Asp Gly Lys Ser Ala Val Cys Thr Lys Leu Trp Cys Asn Gln Gly 245 250 255 Ser Gly Ala

Claims

1.-26. (canceled)

27. An isolated antibody, or antigen-binding fragment thereof, comprising one or more bioactive peptide amino acid sequence(s), wherein at least one bioactive peptide amino acid sequence is engrafted into at least one of: (i) a light chain variable region comprising one or more chicken framework regions and/or (ii) a heavy chain variable region comprising one or more chicken framework regions.

28. The isolated antibody of claim 27, wherein the bioactive peptide amino acid sequence is engrafted into at least one of CDR-H1, CDR-H2 or CDR-H3 of the heavy chain variable region.

29. The isolated antibody of claim 27, wherein the bioactive peptide amino acid sequence is engrafted into at least one of CDR-L1, CDR-L2 or CDR-L3 of the light chain variable region.

30. The isolated antibody of claim 28, wherein the heavy chain further comprises a human IgG1 constant region.

31. The isolated antibody of claim 29, wherein the light chain further comprises a human lambda light chain constant region.

32. The isolated antibody of claim 27, wherein the antibody further comprises at least one flexible amino acid linker sequence between the bioactive peptide amino acid sequence and a chicken framework region.

33. The isolated antibody of claim 27, wherein the antibody comprises a heavy chain variable region of general formula (I): N--X--B--Y--C (I) wherein: N is an amino terminal region of the heavy chain variable region, X is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; B is a bioactive peptide amino acid sequence and consists of an amino acid sequence of no more than 60, or no more than 50 amino acid residues to a minimum of at least 3 amino acid residues; Y is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; and C is a carboxy terminal region of the heavy chain variable region, and wherein at least one of the following applies: N comprises FR-1, set forth as SEQ ID NO:24, or a variant thereof, and C comprises FR-2, set forth as SEQ ID NO:25, or a variant thereof, or N comprises FR-2, set forth as SEQ ID NO:25, or a variant thereof, and C comprises FR-3, set forth as SEQ ID NO:26, or a variant thereof; or N comprises FR-3, set forth as SEQ ID NO:26, or a variant thereof, or flanking SEQ ID NO:27, and C comprises FR-4, set forth as SEQ ID NO:28, or a variant thereof, or flanking SEQ ID NO:29.

34. The isolated antibody of claim 33, wherein C further comprises the human IgG1 constant region, set forth as SEQ ID NO:47, or a variant thereof.

35. The isolated antibody of claim 33, wherein N comprises FR-3, set forth as SEQ ID NO:26, or a variant thereof, or flanking SEQ NO:27, and C comprises FR-4, set forth as SEQ ID NO:28, or a variant thereof, or flanking SEQ ID NO:29.

36. The isolated antibody of claim 33, wherein X and/or Y are linker sequences derived from the CDR of a generic parental chicken clone.

37. The isolated antibody of claim 33, wherein X and/or Y are linker sequences selected from TABLE 4.

38. The isolated antibody of claim 27, wherein the antibody comprises a light chain variable region of general formula (II): N--X--B--Y--C (II) wherein: N is an amino terminal region of the light chain variable region, X is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; B is a bioactive peptide amino acid sequence and consists of an amino acid sequence of no more than 60, or no more than 50 amino acid residues to a minimum of at least 3 amino acid residues; Y is a flexible amino acid linker region and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; and C is a carboxy terminal region of the light chain variable region, and wherein at least one of the following applies: N comprises FR-1, set forth as SEQ ID NO:31, or a variant thereof, or flanking SEQ ID NO:32, and C comprises FR-2, set forth as SEQ ID NO:33, or a variant thereof, or flanking SEQ ID NO:34; or N comprises FR-2, set forth as SEQ ID NO:33, or a variant thereof, and C comprises FR-3, set forth as SEQ ID NO:35, or a variant thereof; or N comprises FR-3, set forth as SEQ ID NO:35, or a variant thereof, and C comprises FR-4, set forth as SEQ ID NO:36, or a variant thereof.

39. The isolated antibody of claim 38, wherein C further comprises the human lambda light chain, set forth as SEQ ID NO:48, or a variant thereof.

40. The isolated antibody of claim 38, wherein N comprises FR-1, set forth as SEQ ID NO:31, or a variant thereof, or flanking SEQ NO:32, and C comprises FR-2, set forth as SEQ ID NO:33, or a variant thereof, or flanking SEQ NO:34.

41. The isolated antibody of claim 38, wherein X and/or Y is a linker sequence selected from TABLE 4.

42. The isolated antibody of claim 27, wherein the light chain variable region comprises the chicken framework regions VL-FR1, VL-FR2, VL-FR3 and VL-FR4 amino acid sequences set forth in SEQ ID NO:s 31, 33, 35 and 36, respectively, and variants thereof.

43. The isolated antibody claim 27, wherein the heavy chain variable region comprises the chicken framework regions VH-FR-1, VH-FR2, VH-FR3 and VH-FR4 amino acid sequences set forth in SEQ ID NO:S 24, 25, 26, and 28, respectively, and variants thereof.

44. The isolated antibody of claim 27, wherein the bioactive peptide is selected from the group consisting of (i) bioactive peptides that inhibit medically-important proteases, (ii) neuropeptides (iii) bioactive peptides that inhibit or activate neuropeptide activity, (iii) peptide hormones, (iv) bioactive peptides that inhibit or activate peptide hormone activity, (v) peptides that are ligands for Class A GPCRs, (vi) bioactive peptides the inhibit or activate Class A GPCRs, (vii) Class B GPCR ligands, and (viii) bioactive peptides that inhibit or activate Class B GPCR ligands.

45. The isolated antibody of claim 27, wherein the antibody comprises two bioactive peptide sequences, wherein one bioactive peptide sequence is engrafted into the light chain variable region and the second bioactive peptide sequence is engrafted into the heavy chain variable region.

46. The isolated antibody of claim 44, wherein the bioactive peptide is an inhibitor of the complement system.

47. The isolated antibody of claim 46, wherein the bioactive peptide comprises an SGMI core amino acid sequence according to TABLE-US-00019 (SEQ ID NO: 5) X₁CTX₂X₃X₄CX₅Q

where: X₁ is F oar V, X₂ is R or K, X₃ is K or L, X₄ is L or W, and X₅ is Y or N; and wherein the bioactive peptide inhibits the activity of at least one of MASP-1 or MASP-2.

48. The antibody of claim 47, wherein the bioactive peptide comprises at least one of the following sequences: SEQ ID NO:6 to SEQ ID NO:11.

49. The antibody of claim 47, wherein the polypeptide inhibits the activity of MASP-1.

50. The antibody of claim 49, wherein the bioactive peptide comprises at least one of SEQ ID NO: 6 to SEQ ID NO:8.

51. The antibody of claim 47, wherein the polypeptide inhibits the activity of MASP-2.

52. The antibody of claim 51, wherein the bioactive peptide comprises at least one of SEQ ID NO: 9 to SEQ ID NO:11.

53. The antibody of claim 45, wherein one bioactive peptide inhibits MASP-1 and the other bioactive peptide inhibits MASP-2.

54. The isolated antibody of claim 27, wherein the isolated antibody has substantially the same biological activity as the bioactive peptide

55. A nucleic acid molecule encoding the amino acid sequence of an isolated antibody of claim 27.

56. A cell comprising a nucleic acid molecule of claim 55.

57.-71. (canceled)

72. A pharmaceutical composition comprising the antibody according to claim 27 and a pharmaceutically acceptable excipient.

73. The pharmaceutical composition of claim 72, wherein the composition inhibits MASP-1 activity.

74. The pharmaceutical composition of claim wherein the composition inhibits MASP-2 activity.

75. The composition of claim 72, wherein the composition is formulated for systemic delivery.

76. The composition of claim 75, wherein the composition is formulated for intra-arterial, intravenous, intracranial, intramuscular, inhalational, nasal, or subcutaneous administration.

77. A method of inhibiting lectin pathway complement activation in a human subject comprising administering a composition of claim 72 in an amount sufficient to inhibit lectin pathway complement activation in said human subject.

78. The method of claim 77, wherein the composition is formulated to specifically inhibit MASP-1 or MASP-2 activity.

79.-85. (canceled)

Images