ANTIBODIES TO HUMAN GDF8

Abstract

The present invention provides isolated human or humanized antibodies or antigen-binding fragments thereof which specifically bind to Growth and Differentiation Factor-8 (GDF8) and block GDF8 activity. The antibodies and antibody fragments of the present invention may be used in therapeutic methods for treating conditions or disorders which are ameliorated or improved by inhibition of GDF8.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 14/974,147, filed Dec. 18, 2015, which is a continuation of U.S. patent application Ser. No. 14/462,085, filed Aug. 18, 2014, now U.S. Pat. No. 9,260,515, issued Feb. 16, 2016; which is a continuation of U.S. patent application Ser. No. 13/115,170, filed May 25, 2011, now U.S. Pat. No. 8,840,894, issued Sep. 23, 2014, which claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S. provisional application No. 61/348,559, filed on May 26, 2010; and 61/372,882, filed on Aug. 12, 2010, the disclosures of each of which are herein incorporated by reference in their entireties

FIELD OF THE INVENTION

[0002] The present invention relates to antibodies, and antigen-binding fragments thereof, which are specific for growth and differentiation factor-8 (GDF8).

BACKGROUND

[0003] Growth and Differentiation Factor-8 (GDF8), also known as myostatin, is a member of the TGF-.beta. superfamily of growth factors. GDF8 is a negative regulator of skeletal muscle mass, highly expressed in the developing and adult skeletal muscle.

[0004] GDF8 is highly conserved across species, and the amino acid sequences of murine and human GDF8 are identical (human GDF8 nucleic acid sequence and amino acid sequence shown in SEQ ID NO:338-339) (McPherron et al. 1977 Nature 387:83-90).

[0005] A number of human diseases are associated with loss or impairment of muscle tissue, for example, muscular dystrophy, muscle atrophy, muscle wasting syndrome, sarcopenia and cachexia, and inhibitors of GDF8 are applicable to treating these diseases or disorders.

[0006] Antibodies to GDF8 and therapeutic methods are disclosed in, e.g., U.S. Pat. No. 6,096,506, U.S. Pat. No. 7,320,789, U.S. Pat. No. 7,807,159, WO 2007/047112, WO 2005/094446, US 2007/0087000, U.S. Pat. No. 7,261,893, and WO 2010/070094.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides human or humanized antibodies and antigen-binding fragments of human or humanized antibodies that specifically bind human growth and differentiation factor 8 (GDF8). These antibodies are characterized by binding to GDF8 with high affinity and by the ability to neutralize GDF8 activity. The antibodies can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab').sub.2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933).

[0008] In one embodiment, the antibody of the invention comprises a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 360, and 376, or a substantially identical sequence thereof.

[0009] In one embodiment, the antibody of the invention comprises a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 322, 368, and 384 or a substantially identical sequence thereof.

[0010] In one embodiment, the antibody of the invention comprises a HCVR amino acid sequence and a LCVR amino acid sequence, wherein the HCVR/LCVR pair sequences are selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368, and 376/384.

[0011] The present invention also features a human or humanized antibody or antigen-binding fragment of an antibody comprising a heavy chain complementarity determining region 3 (HCDR3) amino acid sequence and a light chain CDR3 amino acid sequence (LCDR3), wherein the HCDR3 amino acid sequence is selected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 366, and 382, or a substantially identical sequence thereof, and the LCDR3 amino acid sequence is selected from the group consisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 328, 374, and 390, or a substantially identical sequence thereof. In another embodiment, the antibody or fragment thereof comprises an HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NO:8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160, 168/176, 184/192, 200/208, 216/224, 232/240, 248/256, 264/272, 280/288, 296/304, 312/320, 120/328, 366/374, and 382/390.

[0012] In a related embodiment, the antibody or fragment thereof further comprises heavy chain CDR1 (HCDR1) and CDR2 (HCDR2) amino acid sequences and light chain CDR1 (LCDR1) and CDR2 (LCDR2) amino acid sequences, wherein the HCDR1 amino acid sequence is selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 362, and 378, or a substantially identical sequence thereof; the HCDR2 amino acid sequence is selected from the group consisting of SEQ ID NO:6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 364, and 380, or a substantially identical sequence thereof; the LCDR1 amino acid sequence is selected from the group consisting of SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 324, 370, and 386 or a substantially identical sequence thereof; and the LCDR2 amino acid sequence is selected from the group consisting of SEQ ID NO:14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 326, 372, and 388 or a substantially identical sequence thereof. In another embodiment, the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO:36/38/40, 116/118/120, 228/230/232, 362/364/366, and 378/380/382; and LCDR1, LCDR2 and LCDR3 are selected from the group consisting of SEQ ID NO:44/46/48, 124/126/128, 236/238/240, 370/372/374, and 386/388/390. In yet another embodiment, the heavy and light chain CDRs are selected from the group consisting of SEQ ID NO: 36/38/40/44/46/48 (e.g. 21-E5), 116/118/120/124/126/128 (e.g. 8D12), 228/230/232/236/238/240 (e.g. 1A2), 362/364/366/370/372/374 (e.g. H4H1657N2), and 378/380/382/386/388/390 (e.g. H4H1669P).

[0013] In a related embodiment, the invention includes an antibody or antigen-binding fragment of an antibody which specifically binds GDF8, wherein the antibody or fragment comprises the heavy and light chain CDR domains contained within heavy and light chain variable domain sequences selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368, and 376/384. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

[0014] The present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments of the invention. Recombinant expression vectors carrying the antibody-encoding nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing the host cells of the invention.

[0015] In one embodiment, the antibody of the invention comprises a HCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 359, and 375, or a substantially similar sequence having at least 95% homology thereof.

[0016] In one embodiment, the antibody of the invention comprises a LCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 321, 367, and 383 or a substantially similar sequence having at least 95% homology thereof.

[0017] In one embodiment, the antibody of the invention comprises a HCVR amino acid sequence and a LCVR amino acid sequence, wherein the HCV/LCVR pair sequences are encoded by a nucleic acid molecule pair selected from the group consisting of SEQ ID NO: 1/9, 17/25, 33/41, 49/57, 65/73, 81/89, 97/105, 113/121, 129/137, 145/153, 161/169, 177/185, 193/201, 209/217, 225/233, 241/249, 257/265, 273/281, 289/297, 305/313, 113/321, 359/367, and 375/383.

[0018] The present invention also features a human or humanized antibody or antibody fragment comprising a HCDR3 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 295, 311, 365, and 381, or a substantially similar sequence having at least 95% homology thereof and a LCDR3 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 327, 373, and 389, or a substantially similar sequence having at least 95% homology thereof. In one embodiment, the HCDR3/LCDR3 set is encoded by a nucleotide sequence pair selected from the group consisting of SEQ ID NO:7/15, 23/31, 39/47, 55/63, 71/79, 87/95, 103/111, 119/127, 135/143, 151/159, 167/175, 183/191, 199/207, 215/223, 231/239, 247/255, 263/271, 279/287, 295/303, 311/319, 119/327, 365/373, and 381/389.

[0019] In a related embodiment, the antibody or antibody fragment further comprises a HCDR1 and HCDR2, and a LCDR1 and LCDR2, wherein the HCDR1 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 361, and 377, or a substantially similar sequence having at least 95% homology thereof, the HCDR2 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 363, and 379, or a substantially similar sequence having at least 95% homology thereof, the LCDR1 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 323, 369, and 385 or a substantially similar sequence having at least 95% homology thereof, and the LCDR2 encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 325, 371, and 387, or a substantially similar sequence having at least 95% homology thereof. In one embodiment, the antibody or antibody fragment comprises heavy and light chain CDRs encoded by a nucleic acid sequence set of SEQ ID NO:35/37/39/43/45/47, 115/117/119/123/125/127, 227/229/231/235/237/239, 361/363/365/369/371/373, or 377/379/381/385/387/389.

[0020] The present invention also features an isolated antibody or antibody fragment that specifically binds GDF8, comprising heavy and light chain CDRs selected from the group consisting of (a) a HCDR1 comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO:329), wherein X.sup.1 is Gly; X.sup.2 is Phe; X.sup.3 is Thr; X.sup.4 is Phe; X.sup.5 is Ser; X.sup.6 is Ala or Ser; X.sup.7 is Phe or Tyr; X.sup.8 is Gly or Ala; (b) a HCDR2 comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO:330), wherein X.sup.1 is Ile; X.sup.2 Gly or Ser; X.sup.3 is Tyr or Gly; X.sup.4 Ser or Asp; X.sup.5 is Gly; X.sup.6 is Gly; X.sup.7 is Ser or Asn; and X.sup.8 is Ala or Glu; (c) a HCDR3 comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10-X.sup.11-X.sup.12-X.sup.13-X.sup.14 (SEQ ID NO:331), wherein X.sup.1 is Ser or Ala; X.sup.2 is Thr or Lys; X.sup.3 is Asp or Ile; X.sup.4 is Gly or Ser; X.sup.5 is Ala or His; X.sup.6 is Trp or Tyr; X.sup.7 is Lys or Asp; X.sup.8 is Met or Ile; X.sup.9 is Ser or Leu; X.sup.10 is Gly or Ser; X.sup.11 is Leu or Gly; X.sup.12 is Asp or Met; X.sup.13 is Val or Asp; X.sup.14 is Val or absent; (d) a LCDR1 comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6 (SEQ ID NO:332), wherein X.sup.1 is Gln; X.sup.2 is Asp or Gly; X.sup.3 is Ile; X.sup.4 is Ser; X.sup.5 is Asp or Asn; and X.sup.6 is Tyr or Trp; (e) a LCDR2 comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3 (SEQ ID NO:333), wherein X.sup.1 is Thr or Ala; X.sup.2 is Thr or Ala; and X.sup.3 is Ser; and (f) a LCDR3 region comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9 (SEQ ID NO:334), wherein X.sup.1 is Gln; X.sup.2 is Lys or Gln; X.sup.3 is Ala or Tyr; X.sup.4 is Asp or Asn; X.sup.5 is Ser; X.sup.6 is Ala or Phe; X.sup.7 is Pro; X.sup.8 is Leu; and X.sup.9 is Thr.

[0021] The methodology for deriving the aforementioned consensus sequences (SEQ ID NOs: 329-334) is illustrated in FIGS. 4A and 4B.

[0022] The present invention also features a fully human or humanized antibody or antibody fragment which binds GDF8 with an affinity (expressed as a dissociation constant, "K.sub.D") of about 1 nM or less, as measured by surface plasmon resonance assay (for example, BIACORE.TM.). In certain embodiments, the antibody of the invention exhibits a K.sub.D of about 700 pM or less; about 500 pM or less; about 320 pM or less; about 160 pM or less; about 100 pM or less; about 50 pM or less; about 10 pM or less; or about 5 pM or less.

[0023] In one embodiment, the invention provides a fully human or humanized monoclonal antibody (mAb) which specifically binds and inhibits human GDF8 and exhibits an IC.sub.50 of less than or equal to about 10 nM; about 5 nM or less; about 3 nM or less; about 2 nM or less; about 1 nM or less; about 500 pM or less; or about 200 pM or less, as measured by GDF8 inducible luciferase assay. As shown in the experimental section below, some of the anti-GDF8 antibodies of the invention block the activity of closely related proteins, such as GDF11, with a much higher IC.sub.50 than GDF8 in a luciferase bioassay. In one embodiment, the invention provides an antibody or antigen-binding fragment of an antibody that exhibits at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, at least about 1000-fold, or at least about 1500-fold higher IC.sub.50 for blocking GDF11 activity relative to GDF8.

[0024] The invention encompasses anti-GDF8 antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of a galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[0025] The invention includes anti-GDF8 antibodies which bind specific epitopes of GDF8 and are capable of blocking the biological activity of GDF8. In a first embodiment, the antibody of the invention binds an epitope of the mature GDF8 protein (SEQ ID NO:340) within amino acids from about 1 to about 109; from about 1 to about 54; from about 1 to about 44; from about 1 to about 34; from about 1 to about 24; and from about 1 to about 14. In a second embodiment, the antibody of the invention binds one or more of an epitope of the mature GDF8 protein (SEQ ID NO:340) within amino acids from about 35 to about 109; from about 45 to about 109; from about 55 to about 109; from about 65 to about 109; from about 75 to about 109; from about 85 to about 109; from about 92 to about 109; or from about 95 to about 109. In a third embodiment, the antibody or antigen-binding fragment of the antibody binds within an epitope of the mature human GDF8 protein from about amino acid residue 48 to about 72; from about 48 to about 69; from about 48 to about 65; from about 52 to about 72; from about 52 to about 65; or from about 56 to about 65.

[0026] In a related embodiment, the invention provides an antibody or antigen-binding fragment thereof that competes for specific binding to GDF8 with another antibody comprising a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 amino acid sequence combination of SEQ ID NO:36/38/40/44/46/48, 116/118/120/124/126/128, 228/230/232/236/238/240, 362/364/366/370/372/374, or 378/380/382/386/388/390. In one embodiment, the antibody or antigen-binding fragment of the invention competes for specific binding to GDF8 with another antibody comprising a HCVR/LCVR amino acid sequence pair of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368, or 376/384. In yet another related embodiment, the invention provides an antibody or antigen-binding fragment thereof that recognizes the epitope on GDF8 that is recognized by another antibody comprising a HCDRs/LCDRs amino acid sequence combination of SEQ ID NO: 36/38/40/44/46/48, 116/118/120/124/126/128, 228/230/232/236/238/240, 362/364/366/370/372/374, or 378/380/382/386/388/390. In one embodiment, the antibody or antigen-binding fragment of the invention recognizes the epitope on GDF8 that is recognized by another antibody comprising a HCVR/LCVR amino acid sequence pair of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368, or 376/384.

[0027] The present invention also features a composition comprising a recombinant human or humanized anti-human GDF8 antibody and an acceptable carrier. Further included in the invention are vectors and host cells comprising vectors which contain nucleic acid molecules encoding the human anti-GDF8 antibody of the invention, as well as methods of producing these novel antibodies, comprising growing a host cell comprising nucleic acid encoding the anti-GDF8 antibody of the invention or an antibody fragment, under conditions permitting production of the protein and recovering the protein so produced.

[0028] The present invention also features methods for inhibiting GDF8 activity using an antibody, or antigen-binding portion thereof, of the invention. In one embodiment, the method comprises administering an antibody or antibody fragment of the invention, to a human subject suffering from a disorder which is ameliorated by inhibition of GDF8 activity. In preferred embodiments, the human subject treated with the antibody or antibody fragment of the invention is in need of improving glucose homeostasis, decreasing fat mass, increasing insulin sensitivity, improving kidney function and/or decreasing fat accumulation. The antibody or antibody fragment of the invention is useful for treating, preventing or inhibiting a disease or condition characterized by bone loss, including osteoporosis, osteopenia, osteoarthritis and bone fractures, treating metabolic syndrome, counteracting muscle wasting from sustained administration of a glucocorticoid or a steroid hormone or muscle loss related to muscle dystrophy, muscle atrophy, muscle wasting syndrome, sarcopenia and cachexia.

[0029] Other objects and advantages will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1. Immunoblot of Limited Proteolysis of Human GDF8 with Proteinase K. Gels were nonreducing 18% SDS-PAGE with 0.2 .mu.g GDF8 loaded in each lane, and 2 .mu.g/ml of antibodies either control I (A), 1A2 (B) or 21-E9 (C). Lane 1: digest time 10 min, 1 .mu.g GDF8, 0 .mu.g Proteinase K; Lane 2: digest time 10 min, GDF8 1 .mu.g, 1 .mu.g Proteinase K; Lane 3: digest time 10 min, 1 .mu.g GDF8, 6 .mu.g Proteinase K; Lane 4: digest time 45 min, 1 .mu.g GDF8, 0 .mu.g Proteinase K; Lane 5: digest time 45 min, GDF8 1 .mu.g, 1 .mu.g Proteinase K; Lane 6: digest time 45 min, 1 .mu.g GDF8, 6 .mu.g Proteinase K.

[0031] FIG. 2. Immunoblot of Limited Proteolysis of Human GDF8 with High Doses of Proteinase K. Gels were nonreducing 18% SDS-PAGE with 0 or 4 .mu.g GDF8 loaded in each lane, and 2 .mu.g/ml of either control I (A) or 1A2 (B). Lane 1: digest time 16 hr, 0 .mu.g GDF8, 96 .mu.g Proteinase K; Lane 2: digest time 16 hr, GDF8 4 .mu.g, 0 .mu.g Proteinase K; Lane 3: digest time 16 hr, 4 .mu.g GDF8, 24 .mu.g Proteinase K; Lane 4: digest time 16 hr, 4 .mu.g GDF8, 96 .mu.g Proteinase K; Lane 5: digest time 1 hr, GDF8 4 .mu.g, 24 .mu.g Proteinase K; Lane 6: digest time 1 hr, 4 .mu.g GDF8, 96 .mu.g Proteinase K; Lane 7: digest time 10 min, GDF8 4 .mu.g, 0 .mu.g Proteinase K; Lane 8: digest time 10 min, 4 .mu.g GDF8, 24 .mu.g Proteinase K.

[0032] FIGS. 3A and 3B. Graphs illustrating the percent starting glucose levels over time in mice subjected to an insulin tolerance test before (FIG. 3A) and after (FIG. 3B) antibody treatment.

[0033] FIGS. 4A and 4B. Alignment of the amino acid sequences of the heavy chain CDRs (FIG. 4A) and light chain CDRs (FIG. 4B) from exemplary anti-GDF8 antibodies H4H1657N2 and H4H1669P, illustrating the consensus sequences shared between these sequences.

DETAILED DESCRIPTION

[0034] Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0035] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0037] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties.

Definitions

[0038] "Human Growth Differentiation Factor-8", "GDF8" and "myostatin" are used interchangeably to refer to the protein encoded by the nucleic acid sequence of SEQ ID NO:338 and the protein having the amino acid sequence of SEQ ID NO:339 (propeptide) and 340 (mature protein).

[0039] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V.sub.H) and a heavy chain constant region. The heavy chain constant region comprises three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V.sub.L) and a light chain constant region. The light chain constant region comprises one domain (C.sub.L1). The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-GDF8 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0040] The term "antibody," as used herein, also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0041] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab').sub.2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0042] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V.sub.H domain associated with a V.sub.L domain, the V.sub.H and V.sub.L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V.sub.H-V.sub.H, V.sub.H-V.sub.L or V.sub.L-V.sub.L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V.sub.H or V.sub.L domain.

[0043] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V.sub.H-C.sub.H1; (ii) V.sub.H-C.sub.H2; (iii) V.sub.H-C.sub.H3; (iv) V.sub.H-C.sub.H1-C.sub.H2; (v) V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3; V.sub.H-C.sub.H2-C.sub.H3; V.sub.H-C.sub.L; V.sub.L-C.sub.H1; (ix) V.sub.L-C.sub.H2; (x) V.sub.L-C.sub.H3; (xi) V.sub.L-C.sub.H1-C.sub.H2; (xii) V.sub.L-C.sub.H1-C.sub.H2-C.sub.H3; (xiii) V.sub.L-C.sub.H2-C.sub.H3; and (xiv) V.sub.L-C.sub.L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V.sub.H or V.sub.L domain (e.g., by disulfide bond(s)).

[0044] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

[0045] The antibodies of the present invention may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998)).

[0046] The term "specifically binds," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a dissociation constant of 1.times.10.sup.-6 M or less. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" human GDF8, as used in the context of the present invention, includes antibodies that bind human GDF8 or portion thereof (e.g., a peptide comprising at least 6 contiguous amino acids of SEQ ID NO:340) with a K.sub.D of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less than about 0.5 nM, as measured in a surface plasmon resonance assay. (See, e.g., Example 3, herein). An isolated antibody that specifically binds human GDF8 may, however, have cross-reactivity to other antigens, such as GDF8 molecules from other species.

[0047] The term "high affinity" antibody refers to those antibodies capable of binding to GDF8 with a dissociation constant (K.sub.D) of about 10.sup.-8 M or less, about 10.sup.-9 M or less, about 10.sup.-10 M or less, about 10.sup.-11M or less, or about 10.sup.-12 M or less, as measured by surface plasmon resonance, e.g., BIACORE.TM. or solution-affinity ELISA.

[0048] By the term "slow off rate" or "Koff" is meant an antibody that dissociates from GDF8 with a rate constant of 1.times.10.sup.-3 s.sup.-1 or less, preferably 1.times.10.sup.-4s.sup.-1 or less, as determined by surface plasmon resonance, e.g., BIACORE.TM..

[0049] A "neutralizing" or "blocking" antibody, is intended to refer to an antibody whose binding to GDF8 results in inhibition of the biological activity of GDF8. This inhibition of the biological activity of GDF8 can be assessed by measuring one or more indicators of GDF8 biological activity. These indicators of GDF8 biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see examples below).

[0050] The fully-human anti-GDF8 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are back-mutated to the corresponding germline residue(s) or to a conservative amino acid substitution (natural or non-natural) of the corresponding germline residue(s) (such sequence changes are referred to herein as "germline back-mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline back-mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V.sub.H and/or V.sub.L domains are mutated back to the germline sequence. In other embodiments, only certain residues are mutated back to the germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. Furthermore, the antibodies of the present invention may contain any combination of two or more germline back-mutations within the framework and/or CDR regions, i.e., wherein certain individual residues are mutated back to the germline sequence while certain other residues that differ from the germline sequence are maintained. Once obtained, antibodies and antigen-binding fragments that contain one or more germline back-mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0051] The present invention also includes anti-GDF8 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-GDF8 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. In one embodiment, the antibody comprises an HCVR having an amino acid sequence selected from SEQ ID NO:360 and 376 with 8 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having an amino acid sequence selected from SEQ ID NO:360 and 376 with 6 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having an amino acid sequence selected from SEQ ID NO:360 and 376 with 4 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having an amino acid sequence selected from SEQ ID NO:360 and 376 with 2 or fewer conservative amino acid substitutions. In one embodiment, the antibody comprises an LCVR having an amino acid sequence selected from SEQ ID NO:368 and 384 with 8 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having an amino acid sequence selected from SEQ ID NO:368 and 384 with 6 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having an amino acid sequence selected from SEQ ID NO:368 and 384 with 4 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having an amino acid sequence selected from SEQ ID NO:368 and 384 with 2 or fewer conservative amino acid substitutions.

[0052] In certain embodiments, antibody or antibody fragment of the invention may be conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, and immunosuppressant or a radioisotope.

[0053] An "isolated antibody," as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, tissue or cell in which the antibody naturally exists or is naturally produced is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell, as well as an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0054] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE.TM. system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0055] The term "K.sub.D", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0056] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. For example, an antibody is said to specifically bind an antigen when the K.sub.D is less than or equal to 10.sup.-8 M, less than or equal to 10.sup.-9 M, or less than or equal to 10.sup.-10 M.

[0057] A protein or polypeptide is "substantially pure," "substantially homogeneous" or "substantially purified" when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60, 70%, 80% or 90% w/w of a protein sample, usually about 95%, and preferably over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0058] The term "polypeptide analog or variant" as used herein refers to a polypeptide that is comprised of a segment of at least 25 amino acids that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to GDF8 under suitable binding conditions, or (2) ability to block the biological activity of GDF8. Typically, polypeptide analogs or variants comprise a conservative amino acid substitution (or insertion or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

[0059] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutations of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton 1984 W. H. Freeman and Company, New York; Introduction to Protein Structure (Branden & Tooze, eds., 1991, Garland Publishing, NY); and Thornton et at. 1991 Nature 354:105, which are each incorporated herein by reference.

[0060] Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (see, for example, Fauchere (1986) J. Adv. Drug Res. 15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which are incorporated herein by reference. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo et al. (1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0061] The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides or more, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (1990) Methods Enzymol. 183:63-98 and (2000) Methods Mol. Biol. 132:185-219, each herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0062] A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Generally, the art uses the terms "percent sequence identity", "percent sequence similarity" and "percent sequence homology" interchangeably. In this application, these terms shall have the same meaning with respect to nucleic acid sequences.

[0063] The term "substantial similarity", or "substantial sequence similarity," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0064] As applied to polypeptides, the term "substantial identity" or "substantially identical" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 80% sequence identity, at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0065] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000), supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389 402, each of which is herein incorporated by reference.

[0066] The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, at least about 20 residues, at least about 24 residues, at least about 28 residues, or at least about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

[0067] The term "effective amount" is a concentration or amount of an antibody or antigen-binding fragment of an antibody which results in achieving a particular stated purpose. An "effective amount" of an anti-GDF8 antibody or antigen-binding fragment of an antibody thereof may be determined empirically. Furthermore, a "therapeutically effective amount" is a concentration or amount of an anti-GDF8 antibody or antigen-binding fragment thereof which is effective for achieving a stated therapeutic effect. This amount may also be determined empirically.

Preparation of Human Antibodies

[0068] Methods for generating monoclonal antibodies, including fully human monoclonal antibodies are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to GDF8.

[0069] Using VELOCIMMUNE.TM. technology or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to GDF8 are initially isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

[0070] In general, the antibodies of the instant invention possess very high affinities, typically possessing K.sub.D of from about 10.sup.-12 through about 10.sup.-9 M, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention, for example wild-type IgG1 (SEQ ID NO:335) or IgG4 (SEQ ID NO:336), or modified IgG1 or IgG4 (for example, SEQ ID NO:337). While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

Bioequivalents

[0071] The anti-GDF8 antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind human GDF8. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the anti-GDF8 antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an anti-GDF8 antibody or antibody fragment that is essentially bioequivalent to an anti-GDF8 antibody or antibody fragment of the invention. Examples of such variant amino acid and DNA sequences are discussed above.

[0072] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0073] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0074] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0075] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0076] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0077] Bioequivalent variants of anti-GDF8 antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include anti-GDF8 antibody variants comprising amino acid changes which modify the glycosylation characteristics of the antibodies, e.g., mutations which eliminate or remove glycosylation.

Epitope Mapping and Related Technologies

[0078] To screen for antibodies which bind to a particular epitope (e.g., those which block binding of IgE to its high affinity receptor), a routine cross-blocking assay such as that described in Harlow and Lane (1990) supra can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63) (herein specifically incorporated by reference in its entirety), or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9:487-496) (herein specifically incorporated by reference in its entirety).

[0079] The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0080] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the anti-GDF8 antibodies of the invention into groups of antibodies binding different epitopes.

[0081] The invention includes anti-GDF8 antibodies and antigen-binding fragments of antibodies which bind specific epitopes of human GDF8 (SEQ ID NO:340) and are capable of blocking the biological activity of GDF8. In one embodiment, the antibody or antigen-binding fragment thereof binds within an epitope comprising amino acids residues 1 to 109; 1 to 54; 1 to 44; 1 to 34; 1 to 24; and 1 to 14. In another embodiment, the antibody or antigen-binding fragment thereof binds within an epitope comprising of amino acid residues 65 to 72; 35 to 109; 45 to 109; 55 to 109; 65 to 109; 75 to 109; 85 to 109; 92 to 109; or 95 to 109. In another embodiment, the antibody or antigen-binding fragment thereof binds within an epitope comprising amino acid residue 48 to 72; 48 to 69; 48 to 65; 52 to 72; 52 to 65; or 56 to 65. In specific embodiments, the antibody or antigen-binding fragment thereof may bind within 2 or more epitopes.

[0082] The present invention also includes antibodies and antigen-binding fragments thereof that bind wild-type mature GDF8 (SEQ ID NO:340) but do not bind isolated peptides having less than the full amino acid sequence of SEQ ID NO:340. For example, the invention includes anti-GDF8 antibodies that bind wild-type mature GDF8 (SEQ ID NO:340) but do not bind isolated peptides consisting of 10 to 40 contiguous amino acids of SEQ ID NO:340. The invention also includes anti-GDF8 antibodies that do not bind any linear epitopes within wild-type mature GDF8. In certain embodiments of the present invention, the anti-GDF8 antibodies bind wild-type mature human GDF8 comprising SEQ ID NO:340 but do not bind one or more isolated GDF8 peptides having an amino acid sequence selected from the group consisting of amino acids 1-14, 1-18, 17-42, 48-65, 48-69, 48-72, 52-65, 52-72, 56-65, 56-72, 65-72, 73-90, 75-105 and 91-105, of SEQ ID NO:340. In certain embodiments, the anti-GDF8 antibodies do not bind any of the aforementioned GDF8 peptides. Methods for determining whether a given antibody is able to bind a particular GDF8 peptide are known to persons of ordinary skill in the art. One exemplary method is illustrated by Example 7 herein, in which GDF8 peptides are attached to microspheres, antibodies are added to the peptide-conjugated microspheres, and, following washing steps, antibody-bound microspheres are detected. The absence of bound antibodies indicates that the antibodies do not bind the particular peptides being tested.

[0083] The present invention also includes isolated human antibodies, or antigen-binding fragments thereof, that specifically bind to wild-type mature human GDF8 (e.g., a protein or polypeptide comprising SEQ ID NO:340), but do not bind to a chimeric GDF8 construct in which certain amino acids of GDF8 are replaced with the corresponding amino acid sequence(s) from a non-identical but related protein such as TGF.beta.-1. In one example, the chimeric construct is a GDF8/TGF.beta.-1 chimera in which amino acids 48-72 of mature GDF8 are replaced with the corresponding amino acid sequence of TGF.beta.-1 (e.g., amino acids 49-76 of TGF.beta.-1). An example of one such chimera is represented by SEQ ID NO:352 (see Examples 4 and 6 herein). Thus, in certain embodiments, the antibodies of the invention specifically bind to wild-type mature human GDF8 (SEQ ID NO:340) but do not bind to the chimeric GDF8/TGF.beta.-1 construct of SEQ ID NO:352, indicating that the epitope to which such antibodies bind includes or encompasses amino acids located within residues 48 to 72 of SEQ ID NO:340. Blocking bioassays, such as the assay set forth in Example 4 herein, can also be used to indirectly ascertain if an antibody binds wild-type mature human GDF8 (SEQ ID NO:340) and does not bind a chimeric GDF8/TGF.beta.-1 construct, e.g., the construct of SEQ ID NO:352. For example, an antibody which blocks the bioactivity of wild-type mature human GDF8 but does not block the bioactivity of a chimeric GDF8/TGF.beta.-1 is deemed to bind to the portion of GDF8 that is replaced by the corresponding TGF.beta.-1 sequence in the chimeric construct.

[0084] Similarly, the present invention also includes isolated human antibodies, or antigen-binding fragments thereof, that block wild-type mature GDF8-mediated activity in a bioassay but do not block activity of a chimeric GDF8 construct (e.g., a GDF8/TGF.beta.-1 chimera in which amino acids 48-72 of mature GDF8 are replaced with the corresponding amino acid sequence of TGF.beta.-1 (e.g., SEQ ID NO:352)). An exemplary GDF8 bioassay that can be used in the context of this aspect of the invention is the GDF8-inducible luciferase assay set forth in Example 4 herein, although other similar bioassays capable of measuring the cellular activity of GDF8 are contemplated herein as well.

[0085] The present invention includes anti-GDF8 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein. Likewise, the present invention also includes anti-GDF8 antibodies that cross-compete for binding to GDF8 or a GDF8 fragment with any of the specific exemplary antibodies described herein.

[0086] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-GDF8 antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-GDF8 antibody of the invention, the reference antibody is allowed to bind to a GDF8 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the GDF8 molecule is assessed. If the test antibody is able to bind to GDF8 following saturation binding with the reference anti-GDF8 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-GDF8 antibody. On the other hand, if the test antibody is not able to bind to the GDF8 molecule following saturation binding with the reference anti-GDF8 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-GDF8 antibody of the invention. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0087] To determine if an antibody competes for binding with a reference anti-GDF8 antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a GDF8 molecule under saturating conditions followed by assessment of binding of the test antibody to the GDF8 molecule. In a second orientation, the test antibody is allowed to bind to a GDF8 molecule under saturating conditions followed by assessment of binding of the reference antibody to the GDF8 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the GDF8 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to GDF8. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Species Selectivity and Species Cross-Reactivity

[0088] According to certain embodiments of the invention, the anti-GDF8 antibodies bind to human GDF8 but not to GDF8 from other species. Alternatively, the anti-GDF8 antibodies of the invention, in certain embodiments, bind to human GDF8 and to GDF8 from one or more non-human species. For example, the anti-GDF8 antibodies of the invention may bind to human GDF8 and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee GDF8.

Immunoconjugates

[0089] The invention encompasses a human or humanized anti-GDF8 monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxin agents include any agent that is detrimental to cells. Examples of suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081, which is herein specifically incorporated by reference).

Multispecific Antibodies

[0090] The antibodies of the present invention may be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-GDF8 antibodies of the present invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity. For example, the present invention includes bi-specific antibodies wherein one arm of an immunoglobulin is specific for human GDF8 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.

[0091] An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) C.sub.H3 domain and a second Ig C.sub.H3 domain, wherein the first and second Ig C.sub.H3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C.sub.H3 domain binds Protein A and the second Ig C.sub.H3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C.sub.H3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C.sub.H3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

Therapeutic Administration and Formulations

[0092] The invention provides therapeutic compositions comprising the antibodies or antigen-binding fragments thereof of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN.TM.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0093] The dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the antibody of the present invention is used for treating various conditions and diseases associated with GDF8, for example, muscular dystrophy, muscle atrophy, muscle wasting syndrome, sarcopenia and cachexia, in an adult patient, it is advantageous to intravenously administer the antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, or about 0.1 to about 5 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In other parenteral administration and oral administration, the antibody can be administered in a dose corresponding to the dose given above. When the condition is especially severe, the dose may be increased according to the condition up to the amount that causes significant side effects, if any.

[0094] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0095] The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533).

[0096] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0097] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN.TM. (Owen Mumford, Inc., Woodstock, UK), DISETRONIC.TM. pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25.TM. pen, HUMALOG.TM. pen, HUMALIN 70/30.TM. pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN.TM. I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR.TM. (Novo Nordisk, Copenhagen, Denmark), BD.TM. pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN.TM., OPTIPEN PROT.TM., OPTIPEN STARLET.TM., and OPTICLIK.TM. (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR.TM. pen (sanofi-aventis), the FLEXPEN.TM. (Novo Nordisk), and the KWIKPEN.TM. (Eli Lilly), the SURECLICK.TM. Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET.TM. (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA.TM. Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

[0098] In certain situations, the pharmaceutical composition can be delivered in a controlled release system, for example, with the use of a pump or polymeric materials. In another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.

[0099] Examples of the composition for oral administration include solid or liquid dosage forms, specifically, tablets (including dragees and film-coated tablets), pills, granules, powdery preparations, capsules (including soft capsules), syrup, emulsions, suspensions, etc. Such a composition is manufactured by publicly known methods and contains a vehicle, a diluent or an excipient conventionally used in the field of pharmaceutical preparations. Examples of the vehicle or excipient for tablets are lactose, starch, sucrose, magnesium stearate, and the like.

[0100] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0101] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to 100 mg and in about 10 to 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

[0102] The antibodies of the present invention are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with GDF8 activity. More specifically, the antibodies of the present invention are useful for the treatment of any condition or affliction which can be improved by increasing muscle strength/power and/or muscle mass and/or muscle function in an individual, or by favorably altering metabolism (carbohydrate, lipid and protein processing) by blocking GDF8 activity. Exemplary diseases, disorders and conditions that can be treated with the anti-GDF8 antibodies of the present invention include, but are not limited to, sarcopenia, cachexia (either idiopathic or secondary to other conditions, e.g., cancer, chronic renal failure, or chronic obstructive pulmonary disease), muscle injury, muscle wasting and muscle atrophy, e.g., muscle atrophy or wasting caused by or associated with disuse, immobilization, bed rest, injury, medical treatment or surgical intervention (e.g., hip fracture, hip replacement, knee replacement, etc.) or by necessity of mechanical ventilation. The anti-GDF8 antibodies of the invention may also be used to treat, prevent or ameliorate diseases such as cancer, obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndromes (including, but not limited to diabetes, obesity, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease, and anorexia).

[0103] The present invention includes therapeutic administration regimens which comprise administering an anti-GDF8 antibody of the present invention in combination with at least one additional therapeutically active component. Non-limiting examples of such additional therapeutically active components include other GDF8 antagonists (e.g., small molecule inhibitors of GDF8 or other GDF8 antibodies or binding molecules), growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and cytotoxic/cytostatic agents. The additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the anti-GDF8 antibody of the present invention.

Diagnostic Uses of the Antibodies

[0104] The anti-GDF8 antibodies of the present invention may also be used to detect and/or measure GDF8 in a sample, e.g., for diagnostic purposes. For example, an anti-GDF8 antibody, or fragment thereof, may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of expression, etc.) of GDF8. Exemplary diagnostic assays for GDF8 may comprise, e.g., contacting a sample, obtained from a patient, with an anti-GDF8 antibody of the invention, wherein the anti-GDF8 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-GDF8 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as .sup.3H, .sup.14C, .sup.32P, .sup.35S, or .sup.125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, .beta.-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure GDF8 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0105] Samples that can be used in GDF8 diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient which contains detectable quantities of GDF8 protein, or fragments thereof, under normal or pathological conditions. Generally, levels of GDF8 in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease or condition associated with abnormal GDF8 levels or activity) will be measured to initially establish a baseline, or standard, level of GDF8. This baseline level of GDF8 can then be compared against the levels of GDF8 measured in samples obtained from individuals suspected of having a GDF8 related disease or condition.

EXAMPLES

Example 1. Generation of Human Antibodies to Human GDF8

[0106] Mice may be immunized by any method known in the art (see, for example, Harlow and Lane supra). In one embodiment, GDF8 antigen is administered directly, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE.RTM. mouse comprising DNA encoding human Ig heavy and kappa light chain variable regions. Suitable adjuvants include complete and incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptides) (see O'Hagan 2000 Vaccine Adjuvant, by Human Press, Totawa, N.J.). The antibody immune response is monitored by standard antigen-specific immunoassay. When a desired immune response is achieved, in one embodiment, antibody-expressing B cells are harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines are screened and selected to identify cell lines that produce antigen-specific antibodies.

[0107] Alternatively, antigen-specific hybridoma cells may be isolated by flow cytometry. Briefly, after fusion to myeloma cells, pooled hybridoma cells are grown for 10 days in HAT medium. The cells are then harvested and stained with biotin-labeled GDF8 at 2 .mu.g/ml for one hour, followed by addition of phycoerythrin-streptavidin. The fluorescence-labeled cells are sorted by flow cytometry (single cell per well into 96 well plates containing hybridoma growth medium), cultured for 8-10 days, and conditioned media screened for the presence of functionally desirable monoclonal antibodies.

[0108] In another embodiment, anti-GDF8 antibodies generated via direct isolation of splenocytes. Antigen-specific antibodies are isolated directly from antigen-immunized B cells without fusion to myeloma cells, as described in U.S. 2007/0280945A1, herein specifically incorporated by reference in its entirety. Stable recombinant antibody-expressing CHO cell lines are established from the isolated proper recombinants.

Example 2. Gene Utilization Analysis

[0109] To analyze the structure of antibodies produced, the nucleic acids encoding antibody variable regions were cloned and sequenced. From the nucleic acid sequence and predicted amino acid sequence of the antibodies, gene usage was identified for each antibody chain. Table 1 sets forth the gene usage for selected antibodies in accordance with the invention. Antibody identifier (HCVR/LCVR): 21-E5 (SEQ ID NO:34/42); 21-B9 (SEQ ID NO:18/26); 21-E9 (SEQ ID NO:98/106); 21-A2 (SEQ ID NO:2/10); 22-D3 (SEQ ID NO:50/58); 22-E6 (SEQ ID NO:66/74); 22-G10 (SEQ ID NO:82/90); 1A2 (SEQ ID NO:226/234); 20B12 (SEQ ID NO:274/282); 58C8 (SEQ ID NO:242/250); 19F2 (SEQ ID NO:258/266); 8D12-1 (SEQ ID NO:114/122); 4E3-7 (SEQ ID NO:194/202); 9B11-12 (SEQ ID NO:162/170); 4B9 (SEQ ID NO:226/234); 1H4-5 (SEQ ID NO:210/218); 9B4-3 (SEQ ID NO:178/186); 3E2-1 (SEQ ID NO:290/298); 4G3-25 (SEQ ID NO:306/314); 4B6-6 (SEQ ID NO:130/138); H4H1657N2 (SEQ ID NO:360/368); H4H1669P (SEQ ID NO:376/384).

TABLE-US-00001 TABLE 1 Heavy Chain Light Chain Variable Region Variable Region Antibody VH D JH VK JK 21-E5 4-39 3-22 5 1-17 1 21-B9 4-39 3-22 5 1-17 1 21-E9 4-39 3-22 5 1-17 1 21-A2 3-23 1-7 4 3-15 4 22-D3 3-21 5-5 4 1-17 2 22-E6 4-39 3-22 5 1-17 1 22-G10 2-5 1-7 4 1-16 4 1A2 3-23 1-7 3 3-15 4 20B12 3-23 6-13 6 3-15 4 58C8 3-23 1-7 3 3-15 2 19F2 3-30 1-26 4 2-28 3 8D12-1 3-30 1-7 4 2-137* 5* 4E3-7 3-23 1-7 4 3-15 4 9B11-12 3-23 1-7 3 3-15 4 4B9 3-23 1-7 3 3-15 4 1H4-5 3-30 6-13 6 3-15 4 9B4-3 3-23 1-7 4 3-15 4 3E2-1 4-34 4-4 4 1-9 4 3A4-3 3-21 5-5 4 1-17 1 4G3-25 3-30 3-3 4 2-28 5 4B6-6 3-30 1-7 4 2-137* 5* H4H1657N2 3-23 2-21 6 1-27 4 H4H1669P 3-33 3-9 6 1-12 4

Control Constructs Used in the Following Examples

[0110] Various control constructs (anti-GDF8 antibodies and other GDF8 antagonists) were included in the following experiments for comparative purposes. The control constructs are designated as follows: Control I: a human anti-GDF8 antibody with heavy and light chain variable regions having the amino acid sequences of the corresponding domains of "Myo29" SEQ ID NOs: 16 and 18) as set forth in U.S. Pat. No. 7,261,893; Control II: a human anti-GDF8 antibody with heavy and light chain variable regions having the amino acid sequences of the corresponding domains of "2_112_K" SEQ ID NOs: 118 and 120) as set forth in US 2006/0263354; Control III: ActRIIB-Fc fusion construct having the amino acid sequence of SEQ ID NO:391; and Control IV: variant ActRIIB-Fc fusion construct, identical to Control III except that alanine at position 64 of SEQ ID NO:391 [A64] is replaced with an arginine [R64]. (Not all control constructs were used in every Example).

Example 3. Antigen Binding Affinity Determination

[0111] Equilibrium dissociation constants (K.sub.D values) for antigen binding to selected antibodies were determined by surface kinetics using a real-time biosensor surface plasmon resonance assay (BIACORE.TM. 2000). Each selected antibody was captured on either a goat anti-mouse IgG polyclonal antibody surface or a goat anti-hFc polyclonal antibody (Jackson Immuno Research Lab) surface created through direct chemical coupling to a BIACORE.TM. chip to form a captured antibody surface. Human GDF8 homodimer, hGDF11 homodimer, or hGDF5 homodimer at 25 nM was injected over the captured antibody surfaces, and antigen-antibody binding and dissociation were monitored in real time at room temperature. Kinetic analysis was performed to calculate K.sub.D, dissociation rate constants (kd), association rate constants (ka) and half-life of antigen/antibody complex dissociation (Table 2).

TABLE-US-00002 TABLE 2 GDF8 GDF11 Antibody K.sub.D (nM) T.sub.1/2 (min) K.sub.D (nM) T.sub.1/2 (min) 21-E5 0.26 138 0.12 116 21-B9 0.12 126 0.064 133 21-E9 0.14 155 0.07 221 21-A2 0.40 78 0.90 21 22-D3 1.23 34 1.09 21 22-E6 0.26 148 0.12 87 22-G10 0.250 71 0.73 50 1A2 0.32 60 0.30 28 20B12 0.86 39 2.08 2 58C8 0.62 56 0.44 30 19F2 0.50 38 -- -- 8D12-1 0.66 95 1.87 23 4E3-7 1.89 27 1.33 29 9B11-12 1.45 39 1.41 29 4B9 0.55 55 1.09 34 1H4-5 0.95 54 1.48 24 9B4-3 1.18 50 1.08 32 3E2-1 2.55 45 0.70 79 3A4-3 1.07 137 0.51 71 4G3-25 3.90 25 1.41 39 4B6-6 0.95 121 0.55 82 Control I 0.05 191 0.08 136 Control II 0.3 41 -- --

[0112] The foregoing experiment was also carried out with GDF8 applied over a captured antibody surface of candidate antibodies H4H1669P or H4H1657N2. Preliminary data showed a very slow off rate for both antibodies, suggesting a K.sub.D of 1-2 pM or less.

[0113] K.sub.D values for antigen binding to selected antibodies were also determined as described above with a modified running buffer that does not contain BSA.

TABLE-US-00003 TABLE 3 GDF8 GDF11 Antibody K.sub.D (nM) T.sub.1/2 (min) K.sub.D (nM) T.sub.1/2 (min) 1A2-mIgG 0.018 152 0.926 1 1A2-hIgG 0.006 340 0.640 6 8D12 0.016 840 NB Control I 0.002 1301 0.001 105 Control II 0.071 62 1.580 7

[0114] Additional antigen binding experiments were conducted in which GDF8 and GDF11 were applied over a surface of selected anti-GDF8 antibodies and control antibodies at 25.degree. C. and 37.degree. C. Equilibrium dissociation constants (K.sub.D values) for antigen binding to selected antibodies were determined by surface kinetics using a real-time biosensor surface plasmon resonance assay (BIACORE.TM. T100). Each selected antibody or control was captured on a goat anti-hFc polyclonal antibody (Jackson Immuno Research Lab Cat#109-005-098) surface created through direct chemical coupling to a BIACORE.TM. CM5 sensor chip to form a captured antibody surface. Various concentrations (2.5-0.625 nM, 2-fold dilutions) of hGDF8 homodimer or hGDF11 homodimer (or in some experiments, Activin A) were injected over the captured antibody surfaces, and antigen-antibody binding and dissociation were monitored in real time. Kinetic analysis was performed to calculate K.sub.D, dissociation rate constants (kd), association rate constants (ka) and half-life of antigen/antibody complex dissociation. Results are summarized in Table 4. (NB=no binding observed).

TABLE-US-00004 TABLE 4 25.degree. C. 37.degree. C. Antigen K.sub.D T1/2 K.sub.D T1/2 Inhibitor Tested (M) (min) (M) (min) H4H1669P GDF8 3.93E-11 131 5.84E-11 40 GDF11 NB NB NB NB H4H1657N2 GDF8 2.83E-11 202 3.79E-11 91 GDF11 NB NB NB NB Activin A NB NB not determined 1A2-hIgG1 GDF8 6.23E-11 96 4.62E-11 39 GDF11 NB NB NB NB Control I GDF8 1.03E-11 273 2.44E-11 70 (Myo29) GDF11 1.46E-11 221 2.74E-11 56 Activin A NB NB not determined Control III GDF8 3.07E-11 111 6.40E-12 104 (ActRIIB-hFc GDF11 2.38E-11 132 9.92E-12 53 [A64]) Activin A 8.50E-12 196 not determined Control IV GDF8 2.13E-11 238 not determined (ActRIIB-hFc GDF11 3.00E-12 231 [R64]) Activin A 3.00E-12 439 Isotype GDF8 NB NB NB NB negative control GDF11 NB NB NB NB antibody Activin A NB NB not determined

[0115] As shown above, antibodies H4H1669P, H4H1657N2, and 1A2-hIgG1 of the present invention all exhibited strong binding to GDF8 but no binding to GDF11. By contrast, the control molecules showed binding to both GDF8 and GDF11.

Example 4. Antibody Blocking of Smad2/Luciferase Response

[0116] GDF8-Inducible Luciferase Assay.

[0117] A bioassay was developed to determine the ability of selected anti-GDF8 antibodies to neutralize GDF8-mediated or GDF11-mediated cellular function in vitro using an engineered A204 cell line (human rhabdomyosarcoma cells, ATCC) that contains a GDF8 or GDF11-responsive promoter driving luciferase expression. Inhibition of GDF8 or GDF11-inducible luciferase activity was determined as follows: Cells were seeded onto 96-well plate at 2.times.10.sup.4 cells/well in media and incubated overnight at 37.degree. C., 5% CO.sub.2. Antibody protein (in serial dilutions starting from 25 nM in cell media) was added to the wells of A204/Smad2 cells in triplicate on two plates; GDF8 or GDF11 (0.8 nM) was added to each well. The plates were incubated at 37.degree. C., 5% CO.sub.2 for 6 hours. Luciferase activity was determined by adding BRIGHT-GLO.RTM. Substrate (Promega) and IC.sub.50 values determined (Table 5).

TABLE-US-00005 TABLE 5 IC50 (nM) Antibody GDF8 GDF11 21-E5 8.50 35 21-B9 0.62 1.2 21-E9 0.99 1.2 21-A2 9.70 >10 22-D3 >20 >25 22-E6 2.20 22.4 22-G10 10.50 >25 1A2 0.80 1400 20B12 >20 >25 58C8 1.80 >25 19F2 >20 >25 8D12-1 2.40 >25 4E3-7 10.40 >25 9B11-12 5.50 >1000 4B9 0.47 >25 3A4-3 3.10 >20 4G3-25 >25 >25 Control I 0.62 0.94

[0118] The ability of selected anti-GDF8 antibodies to neutralize GDF8-mediated or GDF11-mediated cellular function was further analyzed as described above with varied concentrations of GDF8 or GDF11. (Table 6). (nd=not determined; NB=no binding).

TABLE-US-00006 TABLE 6 IC.sub.50 (nM) GDF8 GDF11 Antibody 0.5 nM 1 nM 0.4 nM 0.8 nM 1A2 0.196 0.363 ~600 ~800 8D12 3.11 5.55 >1000 >1000 21-E5 2.34 2.98 6.9 10.5 H4H1657N2 0.78 nd NB nd H4H1669P 0.90 nd NB nd Control I 0.172 0.398 0.255 0.459 Control II 1.6 4.15 >1000 >1000

[0119] The bioassay described above was repeated using a GDF8/TGF.beta.-1 chimeric construct as the activating peptide. In particular, a chimera consisting of mature GDF8 with amino acids 48-72 replaced with the corresponding amino acids of TGF.beta.-1 was used in this experiment (SEQ ID NO:352, also referred to herein as "GDF8/TGF.beta.[48-72]"). This was produced from an expression construct encoding the entire human GDF8 precursor with the human TGF.beta.-1 sequence replacing the corresponding GDF8 sequence. Bioactivity was assayed in conditioned medium produced by transient transfection of the GDF8/TGF.beta.[48-72] construct in CHO cells. Expression and processing were assessed by Western blot. The conditioned medium was concentrated 20-fold, heated to 80.degree. C. for 5 minutes to inactivate the bound GDF8 propeptide, and assessed for activity in serial dilution in the bioassay. While the precise concentration of chimeric protein was not determined in these experiments, the typical concentration was in the range of 1-10 ug/ml prior to concentration. As described above, cells containing a GDF8-responsive promoter driving luciferase expression were seeded onto a 96-well plate. Selected antibodies (in serial dilutions starting at 100 nM in cell media) were added to an amount of the GDF8/TGF.beta.[48-72] chimeric protein conditioned medium determined to give maximal response. This mixture was preincubated for 45 minutes and added to the reporter cells. Luciferase activity was measured and approximate IC.sub.50 values were calculated, as shown in Table 7 below. (NB=no blocking).

TABLE-US-00007 TABLE 7 Antibody Blocking of GDF8/TGF.beta.[48-72] Bioactivity Antibody IC.sub.50 (nM) 1A2-hFc 0.357 8D12-mFc NB H2M1657N2 NB H1H1669P NB Control I 0.402

[0120] The GDF8/TGF.beta.[48-72] chimeric construct was able to activate luciferase expression in this assay, and 1A2-hFc and Control I were able to block bioactivity of this construct. However, antibodies H2M1657N2 and H1H1669P failed to block the bioactivity of the GDF8/TGF.beta.[48-72] chimeric construct. Since these two antibodies were shown to block the bioactivity of wild-type GDF8 in this assay system (see Table 6), it can be concluded that H2M1657N2 and H1H1669P most likely exert their biological effects by interacting with an epitope within amino acids 48-72 of GDF8.

Example 5. Immunoblotting hGDF8 Fragments Generated by Proteinase K Digestion

[0121] Western blot analysis was used to determine the immunoreactivity of test and control mAbs human GDF8 proteolytically digested with Proteinase K. Enzyme reactions containing 1 .mu.g human GDF8 and 0, 1 or 6 .mu.g Proteinase K were incubated for either 10 or 45-min in digestion buffer. Equal aliquots containing 20% of the amount of GDF8 present in the reaction mixture (200 ng) was loaded into 3 separate 18% SDS-PAGE non-reducing gels and electroblotted to PVDF membranes. Each membrane was incubated with primary antibody at 2 .mu.g/ml followed by the appropriate secondary antibody conjugated to HRP. Shown in FIG. 1A-C, is the resulting immunoreactivity detected for the control mAb I, 1A2 mAb and 21-E9 mAb, respectively. Results show a loss of GDF8 reactivity for the control mAb (A) in lanes 3 and 6. In marked contrast, antibody 1A2 retains immunoreactivity to a smaller GDF8 fragment of approximately 17-19 kD in molecular weight (FIG. 1B).

[0122] The experiment was repeated with digestion times of up 10 min, 1 hr or 16 hr, in the presence of 0 or 4 .mu.g of GDF8, and 0, 24 or 96 .mu.g Proteinase K. The results (FIG. 2A-B) show that in the absence of Proteinase K, both control and 1A2 mAbs were immunoreactive with full length mature (undigested) GDF8 (see FIG. 2A-B, Lanes 2 and 7). In the presence of Proteinase K, immunoreactivity is lost for the control I mAb at all time points. In marked contrast, 1A2 mAb remained immunoreactive with digested human GDF8 fragment (FIG. 2B, Lanes 3-6 and 8). These results indicate that 1A2 mAb is immunoreactive with a smaller fragment of GDF8 that remains intact in the presence of Proteinase K, whereas control mAb loses immunoreactivity to the smaller fragment (17-19 kD).

[0123] A modified Western blot analysis was used to further determine the hGDF8 epitope for selected anti-hGDF8 antibodies. The modification being that before the hGDF8 specific primary antibody was incubated with the membrane, each anti-hGDF8 antibody was pre-incubated with 1000 fold or 50 fold molar excess of hGDF8 peptide fragments of 1-14 amino acids, 17-42 amino acids, 48-72 amino acids, or 75-105 amino acids. The results show that pre-incubation of peptide fragment 48-72 amino acids at 50 fold molar excess was able to block the binding of antibody 8D12 to hGDF8.

Example 6. Antibody Binding to GDF8 Chimeras

[0124] Twelve chimeric GDF8 pro-proteins were made. Table 8 shows the mature chimeric GDF8 protein structures. The chimeric GDF8 proteins comprised two sets: one set having various substitutions of GDF8 sequences with BMP2 sequences, the other set having various substitutions of GDF8 sequences with TGF.beta.1 sequences. These chimeric proteins were used to test and localize antibody binding.

TABLE-US-00008 TABLE 8 Substituted Chi- Mature SEQ mera GDF8 ID Name Fragment Mature Chimeric GDF8 Structure NO: B1 1-15 BMP2.1-13.GDF8.16-109 347 T1 1-15 TGFb1.1-15.GDF8.16-109 348 B17 17-42 GDF8.1-16.BMP2.15-42.GDF8.43-109 349 T17 17-42 GDF8.1-16, TGF.beta.1.17-43.GDF8.43-109 350 B48 48-72 GDF8.1-47.BMP2.48-77.GDF8.73-109 351 T48 48-72 GDF8.1-47.TGF.beta.1.49-76.GDF8.73-109 352 B65 65-72 GDF8.1-64.BMP2.68-77GDF8.73-109 353 T65 65-72 GDF8.1-64.TGF.beta.1.69-76.GDF8.73-109 354 B75 75-105 GDF8.1-74.BMP2.80-110DGF8.106-109 355 T75 75-105 GDF8.1-74.TGF.beta.1.79-108.GDF8.106-109 356 B91 91-105 GDF8.1-90.BMP2.96-110.GDF8.106-109 357 T91 91-105 GDF8.1-90.TGF.beta.1.95-10GDF8.106-109 358

[0125] The various chimeric GDF8 pro-proteins were transiently transfected in an engineered stable CHO.hFurin cell line. A similar Western Blot analysis as described above was used to detect the binding of various anti-hGDF8 antibodies to each of the chimeric GDF8. Briefly, 10 .mu.g of CHO supernatant were loaded onto each lane of an SDS-PAGE (non-reducing or reducing) gel and electroblotted onto a PVDF membrane. The membrane was then incubated with an anti-GDF8 antibody at 2 .mu.g/ml followed by exposure to the appropriate secondary antibody conjugated to HRP. As shown in Table 9, antibody 8D12 was not able to bind either B48 or T48. The result indicated that amino acids 48 to 72 of mature GDF8 participate in the binding of antibody 8D12 to GDF8.

TABLE-US-00009 TABLE 9 Non Reducing Reducing GDF8 Protein Control I 8D12 Control II 1A2 4A7 8D2 Wild Type GDF8 + + + + + + B48 + - + + + - B65 + + + + + + B91 + + + + + + T48 + - + + + - T65 + + + + + +

Example 7. Antibody Binding to hGDF8 Peptides

[0126] Fourteen peptides (Table 10) were generated from mature hGDF8 (SEQ ID NO:340). Unmodified peptides, N-terminal biotinylated peptides (N-term), or C-terminal biotinylated peptides (C-term) were used to test and localize antibody binding. Full-length hGDF8, hGDF11 and unmodified peptides were each individually amine-coupled to xMAP.RTM. Multi-Analyte COOH Microspheres (or beads). Each of the biotinylated peptides was bound to xMAP.RTM. Multi-Analyte LumAvidin Microspheres. Peptide-bound beads suspension were then mixed with an equal volume of blocking buffer (PBS, 1% BSA, 0.05% Tween20, 0.05% Sodium azide) and then distributed into a 96 well filter plate (Millipore, MULTISCREEN.RTM. BV). Control and test anti-hGDF8 antibodies, at 2.5 .mu.g/ml were then added to the peptide-bound beads suspension and were allowed to bind to the beads at RT, overnight. The antibody-incubated beads were then washed twice with PBST (PBS+0.05% Tween20) and incubated with either Phycoerythrin (PE) conjugated anti-hFC or PE-conjugated anti-mFC antibodies at RT for 45 min. The beads were washed again and the antibody binding signal to various peptides were detected with either LUMINEX.RTM. 100.TM. or 200 .TM. instruments. As shown in Table 10, anti-hGDF8 antibody 8D12 is able to bind to peptides 4, 5, 6, 7, 8, and 9. By contrast, anti-hGDF8 antibody H4H1657N2 did not bind any of the peptides (data not shown).

TABLE-US-00010 TABLE 10 Con- Con- No. Peptide Modification trol I trol II 21-E5 1A2 8D12 1 1-14 Unmodified - - - - - N-term - - - - - C-term - - - - - 2 1-18 Unmodified - - - - - N-term - - - - - C-term - ++ - +++ +/- 3 17-42 Unmodified - - - - - N-term - - - - - C-term - - - - - 4 48-65 Unmodified - - - - +++ C-term - - - - +++++ 5 48-69 Unmodified - - - - +++ C-term - - - - +++++ 6 48-72 Unmodified - + + + +++ N-term - - - - + C-term - - - - +++++ 7 52-65 Unmodified - - - - +++ C-term - - - - +++++ 8 52-72 Unmodified - - - - ++ C-term - - - - +++++ 9 56-65 C-term - - - - ++ 10 56-72 Unmodified - - - - - C-term - - - - + 11 65-72 Unmodified - - - - - N-term - - - - - C-term - - - - - 12 73-90 Unmodified - - - - +/- N-term - +/- - + +/- C-term + - - - + 13 75-105 Unmodified - - - - - N-term - - - - - C-term - - - - - 14 91-105 Unmodified - - - - - N-term - - - - - C-term - - - - -

Example 8. Effect of Human Anti-GDF8 Antibodies on the Binding GDF8 to Activin RIIB

[0127] Mature hGDF8 was first amine-coupled to Luminex.RTM. beads. The hGDF8-coated Luminex.RTM. beads were then incubated with various anti-hGDF8 antibodies, at 1.25 .mu.g/ml, for 2 hr at room temperature. Human Activin RIIB-mFc was then added to the bead-antibody mixture and incubated for an additional 2 hr at room temperature. The beads were then washed and stained with R-phycoerythrin (R-PE)-conjugated anti-mFc polyclonal antibody and mean fluorescence intensity (MFI) was measured. As shown in Table 11, although Control mAb I and antibody 21-E5 were both able to block the binding between hGDF8 and hActivin RIIB, Control mAb II was only able to partially block binding. Antibody 1A2 was not able to block the binding of hGDF8 to its receptor, Activin RIIB (Table 11, n=3).

TABLE-US-00011 TABLE 11 Antibody MFI Negative Control 4,560 Control mAb I 58 Control mAb II 1,653 1A2-hIgG 4,037 21-E5-hIgG 275

Example 9. Antibody 8D12 Variants

[0128] Antibody 8D12 variants with modified LCVR were generated by modifying one or more of the following amino acids of the LCVR of 8D12: A7S or T, A8P, P9L, S18P, V19A, M21I, K27Q, F41Y, V42L, R44K, R55L or T, M56G or L, N58Y, L59R, A75D, R79K, A105G, L109V, and L111I.

[0129] The binding affinity (K.sub.D) of the antibody variants with respect to hGDF8 was determined using a real-time biosensor surface plasma resonance assay (BIAcore.TM.3000) described above in modified running buffer that does not contain BSA (Table 12).

TABLE-US-00012 TABLE 12 Antibody K.sub.D (nM) T.sub.1/2 (min) 8D12 0.071 139 8D12-v2 0.520 25 8D12-v3 0.380 46

[0130] The binding between antibody variants and hGDF8 peptides was also tested as described above in Example 7. Antibody variants 8D12-v2 and 8D12-v3 showed strong binding to C-terminal biotinylated peptides 4, 5, 7, and 10.

Example 10. Effect of Anti-GDF8 Antibodies on Skeletal Muscle Mass

[0131] The efficacy of selected anti-GDF8 mAbs for inducing skeletal muscle hypertrophy was determined in vivo. Briefly, 20 male CB17 SCID mice, approximately 9 weeks old, were divided evenly according to body weight into 4 groups. A selected mAb (Control I, 1A2, 21-E5, 8D12, 1A2-hlgG, or Control II) was injected at three increasing doses of 2.5 mg/kg/dose, 5 mg/kg/dose, and 10 mg/kg/dose. The Fc fragment of human IgG was used as negative control. Antibodies were administered intraperitoneally twice for the first week and once a week for the following three weeks. On day 28, mice were euthanized and weighed, and the tibialis anterior (TA) muscles, the gastrocnemius (GA) muscles, and quadriceps (Quad) muscles, heart, spleen, and kidney, were dissected and weighed. Tissues were normalized to starting body weight, and percent change in weight over the negative control was calculated. Six separate experiments were repeated with antibodies: Control I, 1A2, 21-E5, 8D12, 1A2-hlgG, and Control II (Table 13-18). In addition, the experiment was also repeated with higher increasing doses of control I antibody at 10 mg/kg/dose, 30 mg/kg/dose, and 50 mg/kg/dose (Table 19). Results are expressed as percent increase over negative control.+-.standard deviation.

TABLE-US-00013 TABLE 13 Negative Control I Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg Body Weight 7.66 .+-. 1.37 9.92 .+-. 1.82 13.99 .+-. 1.21 0.00 .+-. 1.41 TA Muscle 13.84 .+-. 2.23 16.99 .+-. 2.16 13.68 .+-. 0.96 0.00 .+-. 2.71 GA Muscle 10.58 .+-. 1.67 11.31 .+-. 2.44 12.90 .+-. 3.0 0.00 .+-. 3.36 Quad Muscle 14.79 .+-. 1.55 15.99 .+-. 2.72 20.84 .+-. 2.09 0.00 .+-. 1.84 Heart 2.05 .+-. 3.04 2.96 .+-. 2.96 7.55 .+-. 1.61 0.00 .+-. 2.45 Kidney 3.53 .+-. 1.39 3.56 .+-. 3.51 5.66 .+-. 4.59 0.00 .+-. 2.82 Spleen 39.82 .+-. 6.78 45.26 .+-. 19.10 9.02 .+-. 3.08 0.00 .+-. 5.47

TABLE-US-00014 TABLE 14 Negative 1A2 Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg Body Weight 6.08 .+-. 1.08 9.96 .+-. 1.32 9.92 .+-. 1.09 0.00 .+-. 1.76 TA Muscle 15.56 .+-. 1.54 18.24 .+-. 4.49 20.69 .+-. 3.13 0.00 .+-. 4.47 GA Muscle 20.49 .+-. 1.84 21.36 .+-. 2.79 24.36 .+-. 3.46 0.00 .+-. 3.59 Quad Muscle 26.92 .+-. 3.07 30.15 .+-. 3.56 33.09 .+-. 4.46 0.00 .+-. 4.05 Heart 3.70 .+-. 1.31 6.37 .+-. 2.27 12.42 .+-. 2.70 0.00 .+-. 4.10 Kidney 1.28 .+-. 2.89 2.89 .+-. 3.30 5.31 .+-. 3.29 0.00 .+-. 4.39 Spleen -8.07 .+-. 5.75 -10.00 .+-. 4.68 9.68 .+-. 9.19 0.00 .+-. 6.84

TABLE-US-00015 TABLE 15 Negative 21-E5 Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg Body Weight 4.16 .+-. 0.87 4.14 .+-. 2.82 5.21 .+-. 1.58 0.00 .+-. 1.15 TA Muscle 13.86 .+-. 1.66 14.01 .+-. 2.41 10.32 .+-. 2.54 0.00 .+-. 1.65 GA Muscle 7.70 .+-. 1.86 12.94 .+-. 1.10 8.13 .+-. 2.36 0.00 .+-. 1.41 Quad Muscle 8.64 .+-. 1.31 13.57 .+-. 1.79 8.46 .+-. 3.22 0.00 .+-. 1.94 Heart -7.11 .+-. 1.00 -7.14 .+-. 3.17 -5.51 .+-. 1.58 0.00 .+-. 2.23 Kidney -6.8 .+-. 2.83 -3.2 .+-. 3.57 -0.32 .+-. 2.07 0.00 .+-. 3.93 Spleen 29.81 .+-. 9.83 49.76 .+-. 7.86 10.85 .+-. 6.63 0.00 .+-. 5.76

TABLE-US-00016 TABLE 16 Negative 8D12 Antibody Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg Body Weight 10.06 .+-. 0.86 12.76 .+-. 1.01 11.41 .+-. 1.35 0.00 .+-. 0.66 TA Muscle 17.99 .+-. 1.53 21.30 .+-. 2.06 22.11 .+-. 3.20 0.00 .+-. 1.85 GA Muscle 21.14 .+-. 1.19 23.10 .+-. 0.99 23.40 .+-. 3.72 0.00 .+-. 1.44 Quad Muscle 26.74 .+-. 1.53 31.00 .+-. 1.61 28.80 .+-. 2.72 0.00 .+-. 0.94 Heart -1.61 .+-. 2.06 3.63 .+-. 1.93 3.42 .+-. 2.52 0.00 .+-. 1.08 Kidney -1.06 .+-. 2.02 -4.26 .+-. 2.25 -5.52 .+-. 3.83 0.00 .+-. 3.66 Spleen 4.33 .+-. 6.34 -9.04 .+-. 2.64 -1.85 .+-. 6.26 0.00 .+-. 4.25

TABLE-US-00017 TABLE 17 Negative 1A2-hIgG Antibody Control Dose 2.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg Body 6.73 .+-. 1.51 3.84 .+-. 2.32 7.45 .+-. 2.91 0.00 .+-. 0.76 Weight TA 15.62 .+-. 2.4 12.09 .+-. 1.81 10.91 .+-. 3.33 0.00 .+-. 2.80 Muscle GA 16.29 .+-. 1.02 15.20 .+-. 2.54 15.67 .+-. 3.60 0.00 .+-. 2.13 Muscle Quad 19.39 .+-. 2.92 20.03 .+-. 2.54 19.73 .+-. 3.83 0.00 .+-. 1.98 Muscle Heart 5.91 .+-. 3.81 5.39 .+-. 2.77 3.52 .+-. 3.16 0.00 .+-. 3.14 Kidney 1.70 .+-. 4.01 1.20 .+-. 1.73 1.39 .+-. 3.70 0.00 .+-. 3.27 Spleen -15.44 .+-. 5.6 -30.08 .+-. 6.63 -23.90 .+-. 2.36 0.00 .+-. 11.32

TABLE-US-00018 TABLE 18 Control II Negative Control Dose 2.5 mg/kg 5 mg/kg 5 mg/kg Body Weight 10.19 .+-. 1.20 10.83 .+-. 1.58 0.00 .+-. 1.17 TA Muscle 14.70 .+-. 3.28 13.44 .+-. 4.13 0.00 .+-. 3.87 GA Muscle 19.44 .+-. 2.08 14.35 .+-. 3.36 0.00 .+-. 1.80 Quad Muscle 18.92 .+-. 4.86 13.00 .+-. 2.18 0.00 .+-. 2.03 Heart 2.78 .+-. 5.09 2.71 .+-. 6.72 0.00 .+-. 0.69 Kidney 1.84 .+-. 2.65 0.41 .+-. 3.04 0.00 .+-. 2.12 Spleen 12.32 .+-. 5.60 8.41 .+-. 3.38 0.00 .+-. 4.31

TABLE-US-00019 TABLE 19 Negative Control I Control Dose 10 mg/kg 30 mg/kg 50 mg/kg 10 mg/kg Body Weight 9.20 .+-. 0.73 13.62 .+-. 1.38 12.55 .+-. 1.71 0.00 .+-. 1.18 TA Muscle 18.16 .+-. 1.51 25.58 .+-. 3.14 23.03 .+-. 2.00 0.00 .+-. 3.81 GA Muscle 16.91 .+-. 2.08 23.72 .+-. 3.14 24.68 .+-. 2.73 0.00 .+-. 3.58 Quad Muscle 20.77 .+-. 1.01 26.54 .+-. 3.68 25.67 .+-. 3.32 0.00 .+-. 4.63 Heart 1.55 .+-. 3.07 3.11 .+-. 2.58 2.11 .+-. 2.23 0.00 .+-. 1.96 Kidney 3.09 .+-. 1.90 10.38 .+-. 5.08 8.89 .+-. 3.59 0.00 .+-. 1.35 Spleen 8.22 .+-. 5.80 6.90 .+-. 5.87 -0.74 .+-. 1.85 0.00 .+-. 7.38

[0132] A similar experiment was carried out using antibodies H4H1657N2 and H4H1669P and controls administered to SCID mice. In particular, male SCID mice at 10 weeks of age were administered antibody subcutaneously at 10 mg/kg according to the following dosing schedule: 2.times. on week 1 and 1.times./week on weeks 2 and 3. The total treatment time was 28 days. For this experiment, 5 mice were administered an isotype negative control antibody; 5 mice were administered Control I (Myo29); 6 mice were administered H4H1657N2; and 6 mice were administered H4H1669P. Results are summarized in Table 20 and are expressed as percent increase over negative control.+-.standard deviation.

TABLE-US-00020 TABLE 20 Isotype Control Control I H4H1657N2 H4H1669P Dose 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg Body Weight 0.00 .+-. 1.97 14.52 .+-. 2.68 10.28 .+-. 0.95 10.70 .+-. 1.26 TA Muscle 0.00 .+-. 4.10 17.47 .+-. 3.09 25.53 .+-. 3.96 13.77 .+-. 2.01 GA Muscle 0.00 .+-. 1.97 19.46 .+-. 2.92 21.69 .+-. 1.67 13.39 .+-. 1.30 Quad Muscle 0.00 .+-. 3.59 14.05 .+-. 3.03 22.15 .+-. 3.47 9.87 .+-. 2.55 Heart 0.00 .+-. 2.49 9.42 .+-. 1.63 5.40 .+-. 2.28 12.70 .+-. 2.67 White 0.00 .+-. 9.72 25.14 .+-. 19 -8.05 .+-. 7.53 4.22 .+-. 6.19 Adipose Tissue

[0133] A similar experiment was also carried out using antibodies H4H1657N2 and H4H1669P and controls administered to C57 mice. In particular, male C57 mice at 10 weeks of age were administered antibody subcutaneously at 10 mg/kg according to the following dosing schedule: 2.times. per week for two weeks. For this experiment, 5 mice were administered an isotype negative control antibody; 5 mice were administered Control I (Myo29); 5 mice were administered H4H1657N2; and 6 mice were administered H4H1669P. Results are summarized in Table 21 and are expressed as percent increase over negative control.+-.standard deviation.

TABLE-US-00021 TABLE 21 Isotype Control Control I H4H1657N2 H4H1669P Dose 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg Body 0.00 .+-. 0.92 4.64 .+-. 0.99 5.11 .+-. 0.73 2.75 .+-. 1.07 Weight TA 0.00 .+-. 4.26 8.92 .+-. 1.56 14.92 .+-. 3.09 7.62 .+-. 2.90 Muscle GA 0.00 .+-. 2.39 6.28 .+-. 2.10 14.20 .+-. 1.77 6.07 .+-. 3.80 Muscle Quad 0.00 .+-. 2.46 3.53 .+-. 2.11 12.85 .+-. 2.69 1.24 .+-. 1.74 Muscle Heart 0.00 .+-. 2.28 -3.18 .+-. 2.70 1.39 .+-. 2.83 2.45 .+-. 3.95 White 0.00 .+-. 8.20 19.05 .+-. 7.72 -5.23 .+-. 8.34 -4.53 .+-. 10.28 Adipose Tissue

[0134] Next, dose response experiments were carried out using antibodies H4H1657N2 and H4H1669P in SCID mice. In particular, male SCID mice at 10 weeks of age were administered control antibodies subcutaneously at 30 mg/kg, and H4H1657N2 or H4H1669P at 2.5, 10 or 30 mg/kg, according to the following dosing schedule: 2.times. per week on week 1 and 1.times. per week thereafter. Results are summarized in Tables 22 and 23 and are expressed as percent increase over negative control.+-.standard deviation.

TABLE-US-00022 TABLE 22 Isotype Control Control I H4H1657N2 Dose 30 mg/kg 30 mg/kg 2.5 mg/kg 10 mg/kg 30 mg/kg Number of Mice (n) 5 5 5 5 5 Body Weight 0.00 .+-. 1.38 7.46 .+-. 2.44 10.16 .+-. 0.92 6.51 .+-. 0.91 10.12 .+-. 1.68 TA Muscle 0.00 .+-. 2.55 16.13 .+-. 5.62 22.22 .+-. 2.75 19.79 .+-. 1.19 24.10 .+-. 3.62 GA Muscle 0.00 .+-. 2.66 13.50 .+-. 5.20 21.94 .+-. 3.18 21.84 .+-. 2.94 25.01 .+-. 4.15 Quad Muscle 0.00 .+-. 2.06 16.89 .+-. 4.11 26.30 .+-. 3.72 26.28 .+-. 2.39 30.81 .+-. 2.98 Heart 0.00 .+-. 2.67 3.23 .+-. 3.29 14.46 .+-. 1.55 2.70 .+-. 1.51 4.06 .+-. 2.53 White Adipose Tissue 0.00 .+-. 13.91 5.08 .+-. 9.02 -23.70 .+-. 10.29 -16.77 .+-. 7.54 -15.44 .+-. 11.43

TABLE-US-00023 TABLE 23 Isotype Control Control I H4H1669P Dose 30 mg/kg 30 mg/kg 2.5 mg/kg 10 mg/kg 30 mg/kg Number of Mice (n) 5 5 5 5 5 Body Weight 0.00 .+-. 1.47 13.77 .+-. 1.99 2.87 .+-. 1.66 5.72 .+-. 0.56 4.88 .+-. 1.11 TA Muscle 0.00 .+-. 2.38 25.79 .+-. 2.37 7.82 .+-. 2.25 12.38 .+-. 2.56 11.86 .+-. 2.59 GA Muscle 0.00 .+-. 2.12 22.82 .+-. 1.25 2.68 .+-. 2.06 12.46 .+-. 1.30 10.55 .+-. 3.33 Quad Muscle 0.00 .+-. 3.08 25.69 .+-. 3.65 4.10 .+-. 2.32 12.27 .+-. 1.63 9.43 .+-. 2.67 Heart 0.00 .+-. 0.91 7.78 .+-. 1.62 -0.01 .+-. 3.31 5.37 .+-. 1.71 2.84 .+-. 4.73 White Adipose Tissue 0.00 .+-. 9.41 12.60 .+-. 8.08 9.00 .+-. 4.12 -8.30 .+-. 3.71 -1.23 .+-. 7.03

[0135] In a separate experiment, 5 groups of 6 male SCID mice at 10 weeks of age were administered an isotype negative control antibody subcutaneously at 10 mg/kg, and H4H1657N2 at 0.1, 0.75, 2.5, or 10 mg/kg, according to the following dosing schedule: 2.times. per week on week 1 and 1.times. per week thereafter for a total 28 days of treatment. Results are summarized in Table 24, expressed as percent increase over negative control.+-.standard deviation.

TABLE-US-00024 TABLE 24 Isotype Control H4H1657N2 Dose 10 mg/kg 0.1 mg/kg 0.75 mg/kg 2.5 mg/kg 10 mg/kg Body Weight 0.00 .+-. 0.55 4.24 .+-. 0.42 2.84 .+-. 1.03 6.87 .+-. 1.21 7.23 .+-. 1.66 TA Muscle 0.00 .+-. 3.89 3.10 .+-. 2.06 4.20 .+-. 3.78 18.63 .+-. 2.52 11.54 .+-. 2.15 GA Muscle 0.00 .+-. 1.65 2.75 .+-. 0.82 0.71 .+-. 3.15 15.86 .+-. 2.11 18.28 .+-. 2.84 Quad Muscle 0.00 .+-. 3.00 0.84 .+-. 1.69 -0.62 .+-. 1.75 15.01 .+-. 1.37 16.87 .+-. 2.85 Heart 0.00 .+-. 2.24 6.51 .+-. 1.53 1.35 .+-. 2.85 3.25 .+-. 1.21 3.08 .+-. 2.66 White Adipose 0.00 .+-. 13.65 -7.75 .+-. 8.36 -7.81 .+-. 10.76 -13.53 .+-. 8.00 -28.33 .+-. 4.51 Tissue

[0136] As shown in this Example, antibodies H4H1657N2 and H4H1669P produced significant increases in muscle mass when administered to mice over a range of doses.

Example 11. Effect of Anti-GDF8 Antibodies on Glucose Homeostasis

[0137] Anti-GDF8 antibodies of the invention were tested for their effects on glucose homeostasis and insulin sensitivity in a diet-induced-obesity (DIO) mouse model. In this experiment, DIO mice were obtained by feeding C57BL6 mice a high-fat diet (45% kcal fat) for 7 weeks starting at 9 weeks of age. Starting at week 8, antibodies were administered at 30 mg/kg twice a week for two more weeks, and the study was terminated a week later (21 days post-treatment). The antibodies used in this experiment were H4H1669P and H4H1657N2, as well as an isotype negative control antibody and Control I (anti-GDF8 antibody corresponding to Myo29). Insulin-tolerance tests were performed before and after antibody treatment. Insulin (2 IU/kg) was administered by intraperitoneal injection following a 4 hour fast and glucose levels were measured. The results are illustrated in FIGS. 3A (prior to antibody treatment) and 3B (after antibody treatment).

[0138] As demonstrated in this experiment, myostatin inhibition by administration of an anti-GDF8 antibody improved glucose homeostasis in DIO mice. A significant difference in glucose lowering in response to an insulin bolus was noted between the anti-GDF8 antibody groups (H4H1669P, H4H1657N2 or Control I), and the isotype control antibody group.

Example 12. In Vivo Blockade of Muscle Atrophy by Administration of H4H1657N2

[0139] C57 mice were used to determine the in vivo properties of H4H1657N2 in preventing muscle atrophy induced by casting/immobilization and dexamethasone administration.

[0140] In the casting example, three groups of 8 C57 mice were anesthetized, and the ankle joint was immobilized at a 90.degree. angle with casting material for 14 days. A fourth group was left unperturbed and used as a non-immobilized group control. During the 14 days of immobilization, mice were administered with an isotype negative control antibody; Control I (Myo29); or H4H1657N2. The three groups were injected subcutaneously with antibody at 30 mg/kg, 2.times. per week for two weeks, starting at the time of the immobilization. Results are summarized in Table 25, expressed as percent change over negative control.+-.standard deviation. The results showed that after 14 days of immobilization, the group that received treatment with H4H1657N2 antibody showed a significant reduction in skeletal muscle loss versus the isotype control group.

TABLE-US-00025 TABLE 25 (Muscle Atrophy Induced by Immobilization) Non- Immobilized Isotype Immobilized Control Isotype Control Control I H4H1657N2 TA 0.00 .+-. 1.74 -32.97 .+-. 4.98 -23.36 .+-. 5.80 -13.77 .+-. 4.23 Muscle GA 0.00 .+-. 1.85 -24.34 .+-. 4.95 -7.64 .+-. 0.97 3.91 .+-. 6.02 Muscle

[0141] In the dexamethasone example, two groups of 5 C57 mice were anesthetized and implanted with osmotic pumps subcutaneously that delivered 1 .mu.g/g/day of dexamethasone. Two additional groups were implanted with osmotic pumps that delivered saline and were used as controls. During the 14 days of treatment, two groups of mice (one saline and one dexamethasone) were administered an isotype negative control antibody; and two groups of mice (one saline and one dexamethasone) were administered the H4H1657N2 antibody. Antibodies were injected subcutaneously at 30 mg/kg, 2.times. per week for two weeks. Antibody treatment started at the time of the pump implantation. Results are summarized in Table 26, expressed as percent change over negative control.+-.standard deviation. Comparison of the dexamethasone group that received the H4H1657N2 antibody versus the group that received the isotype control indicates that treatment with the H4H1657N2 antibody prevents the loss of muscle weights induced by dexamethasone treatment.

TABLE-US-00026 TABLE 26 (Muscle Atrophy Induced by Dexamethasone) Saline Treated Isotype Dexamethasone Treated Control H4H1657N2 Isotype Control H4H1657N2 TA Muscle 0.00 .+-. 2.73 14.63 .+-. 1.35 -17.78 .+-. 2.11 3.25 .+-. 2.09 GA Muscle 0.00 .+-. 3.02 20.73 .+-. 1.38 -18.97 .+-. 2.11 -5.08 .+-. 2.07 Quad Muscle 0.00 .+-. 3.91 20.46 .+-. 2.40 -23.94 .+-. 2.68 -4.26 .+-. 2.34

Example 13. Specificity of H4H1657N2 In Vivo

[0142] To examine the specificity of H4H1657N2 in vivo, C57BL6 mice were injected with drug, and serum from treated mice was subjected to a mass spectrometry based ligand "fishing" experiment. Briefly, drug (H4H1657N2, Control IV or isotype negative control) was injected multiple times (Day 0, D3, D7, D10) over a 14-day period into C57BL6 mice. Animals were sacrificed on D14 and serum was incubated with anti human Fc beads. The beads were washed and bound material was eluted with SDS-PAGE loading buffer. Eluted material was subjected to SDS-PAGE and gel slices corresponding to a molecular weight range of 5-20 kDa were excised. Samples were processed for mass spectrometry using standard reduction, alkylation and trypsinization conditions. Digests were separated on a nano C18 column and automatically spotted onto Bruker Anchor Targets. MALDI-MS (MS/MS) analysis (Bruker Ultraflextreme) was performed in an automated fashion, using LC-WARP (Bruker Daltonics). The mass spectra were searched using MASCOT (Matrix Science) and results were evaluated relative to the isotype control. The eluted proteins are listed in Table 27.

TABLE-US-00027 TABLE 27 Antibody Administered: H4H1657N2 Control IV Protein(s) Eluted: GDF8 GDF8 GDF11 Inhibin beta A chain Inhibin beta B chain Inhibin beta C chain

[0143] As shown in Table 27, only mouse GDF8 was identified as a binding partner for H4H1657N2 by this experiment. (The sequence of mouse and human GDF8 are identical.) By contrast, Control IV bound several other members of the TGF beta ligand family besides GDF8, including, inter alia, GDF11. This experiment confirms the specificity of H4H1657N2 for GDF8 in an in vivo context.

[0144] The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Sequence CWU 1

1

3911366DNAArtificial SequenceSynthetic 1caggtgcagc tggtgcagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttaac acctatgcca taagctgggt ccgccaggct 120ccagggaagg ggctggaatg ggtctcaact attactggta gtggttataa cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacactgtat 240ctacagatga gcagcctgag agccgaggac acggccgtat tttactgtgc gaaagactct 300cggtataact ggaattacgg aatttttgac tactggggcc agggaaccac ggtcaccgtc 360tcctca 3662122PRTArtificial SequenceSynthetic 2Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Thr Gly Ser Gly Tyr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Lys Asp Ser Arg Tyr Asn Trp Asn Tyr Gly Ile Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 324DNAArtificial SequenceSynthetic 3ggattcacct ttaacaccta tgcc 2448PRTArtificial SequenceSynthetic 4Gly Phe Thr Phe Asn Thr Tyr Ala1 5 524DNAArtificial SequenceSynthetic 5attactggta gtggttataa caca 2468PRTArtificial SequenceSynthetic 6Ile Thr Gly Ser Gly Tyr Asn Thr1 5 745DNAArtificial SequenceSynthetic 7gcgaaagact ctcggtataa ctggaattac ggaatttttg actac 45815PRTArtificial SequenceSynthetic 8Ala Lys Asp Ser Arg Tyr Asn Trp Asn Tyr Gly Ile Phe Asp Tyr1 5 10 15 9324DNAArtificial SequenceSynthetic 9gacatccaga tgacccagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca tcagaaacct 120ggccaggctc ccaggctcct catctatggt gtatccacca gggccactgg tatcccagcc 180aggttcagtg gcaatgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag cataataact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa acga 32410108PRTArtificial SequenceSynthetic 10Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr His Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Val Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Asn Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 1118DNAArtificial SequenceSynthetic 11cagagtgtta gcagcaac 18126PRTArtificial SequenceSynthetic 12Gln Ser Val Ser Ser Asn1 5 139DNAArtificial SequenceSynthetic 13ggtgtatcc 9143PRTArtificial SequenceSynthetic 14Gly Val Ser1 1527DNAArtificial SequenceSynthetic 15cagcagcata ataactggcc gctcact 27169PRTArtificial SequenceSynthetic 16Gln Gln His Asn Asn Trp Pro Leu Thr1 5 17375DNAArtificial SequenceSynthetic 17caggtgcagc tggtgcagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc aatagtaatt actactgggg ctggatccgc 120cagcccccag ggaaggggct ggagtggatt gggactacct attatagtgg gaccacctac 180tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagagat 300tattatgata gtagtggtta ttattacaac tggttcgatc cctggggcca gggaaccacg 360gtcaccgtct cctca 37518125PRTArtificial SequenceSynthetic 18Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Ser 20 25 30 Asn Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Thr Thr Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Asn Trp Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 1930DNAArtificial SequenceSynthetic 19ggtggctcca tcagcaatag taattactac 302010PRTArtificial SequenceSynthetic 20Gly Gly Ser Ile Ser Asn Ser Asn Tyr Tyr1 5 10 2121DNAArtificial SequenceSynthetic 21acctattata gtgggaccac c 21227PRTArtificial SequenceSynthetic 22Thr Tyr Tyr Ser Gly Thr Thr1 5 2351DNAArtificial SequenceSynthetic 23gcgagagatt attatgatag tagtggttat tattacaact ggttcgatcc c 512417PRTArtificial SequenceSynthetic 24Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Asn Trp Phe Asp1 5 10 15 Pro25324DNAArtificial SequenceSynthetic 25gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcagtctca cactcagcag cctgcagcct 240gaagattttg caacttattt ctgtctacag catcatattt acccgtggac gttcggccaa 300gggaccaagc tggagatcaa acga 32426108PRTArtificial SequenceSynthetic 26Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Leu Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His His Ile Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 2718DNAArtificial SequenceSynthetic 27caggacatta gaaatgat 18286PRTArtificial SequenceSynthetic 28Gln Asp Ile Arg Asn Asp1 5 299DNAArtificial SequenceSynthetic 29gctgcatcc 9303PRTArtificial SequenceSynthetic 30Ala Ala Ser1 3127DNAArtificial SequenceSynthetic 31ctacagcatc atatttaccc gtggacg 27329PRTArtificial SequenceSynthetic 32Leu Gln His His Ile Tyr Pro Trp Thr1 5 33375DNAArtificial SequenceSynthetic 33caagttcagc tggtggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctatggtgg ctccatcagc agtggtaatt actactgggg ctggatccgc 120cagcccccag ggaagggact ggagtggatt gggactatct attatagtgg aagcgcctac 180tacaacccgt ccctcaagag tcgagtcacc atgtccgtag acacgtccaa gaaccagttc 240tccctgaaac tgagctctgt gaccgccgca gacacggctg tttattactg tgtgagagat 300tactatgata gtagtggtca ttattacaac tggttcgacc cctggggcca gggaaccacg 360gtcaccgtct cctca 37534125PRTArtificial SequenceSynthetic 34Gln Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Tyr Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asn Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Thr Ile Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Asp Tyr Tyr Asp Ser Ser Gly His Tyr Tyr Asn Trp Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 3530DNAArtificial SequenceSynthetic 35ggtggctcca tcagcagtgg taattactac 303610PRTArtificial SequenceSynthetic 36Gly Gly Ser Ile Ser Ser Gly Asn Tyr Tyr1 5 10 3721DNAArtificial SequenceSynthetic 37atctattata gtggaagcgc c 21387PRTArtificial SequenceSynthetic 38Ile Tyr Tyr Ser Gly Ser Ala1 5 3951DNAArtificial SequenceSynthetic 39gtgagagatt actatgatag tagtggtcat tattacaact ggttcgaccc c 514017PRTArtificial SequenceSynthetic 40Val Arg Asp Tyr Tyr Asp Ser Ser Gly His Tyr Tyr Asn Trp Phe Asp1 5 10 15 Pro41324DNAArtificial SequenceSynthetic 41gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga catgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag cataatactt acccgtggac gttcggccaa 300gggaccaagg tggagatcaa acga 32442108PRTArtificial SequenceSynthetic 42Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg His Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 4318DNAArtificial SequenceSynthetic 43caggacatta gacatgat 18446PRTArtificial SequenceSynthetic 44Gln Asp Ile Arg His Asp1 5 459DNAArtificial SequenceSynthetic 45gctgcatcc 9463PRTArtificial SequenceSynthetic 46Ala Ala Ser1 4727DNAArtificial SequenceSynthetic 47ctacagcata atacttaccc gtggacg 27489PRTArtificial SequenceSynthetic 48Leu Gln His Asn Thr Tyr Pro Trp Thr1 5 49357DNAArtificial SequenceSynthetic 49caggtgcagc tggtgcagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac 180gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatcgt 300ggatacacct ttggggttga ctactggggc cagggaacca cggtcaccgt ctcctca 35750119PRTArtificial SequenceSynthetic 50Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Tyr Thr Phe Gly Val Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 5124DNAArtificial SequenceSynthetic 51ggattcacct tcagtagcta tagc 24528PRTArtificial SequenceSynthetic 52Gly Phe Thr Phe Ser Ser Tyr Ser1 5 5324DNAArtificial SequenceSynthetic 53attagtagta gtagtagtta cata 24548PRTArtificial SequenceSynthetic 54Ile Ser Ser Ser Ser Ser Tyr Ile1 5 5536DNAArtificial SequenceSynthetic 55gcgagagatc gtggatacac ctttggggtt gactac 365612PRTArtificial SequenceSynthetic 56Ala Arg Asp Arg Gly Tyr Thr Phe Gly Val Asp Tyr1 5 10 57324DNAArtificial SequenceSynthetic 57gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag cataatagtt acccgtacac ttttggccag 300gggaccaagg tggagatcaa acga 32458108PRTArtificial SequenceSynthetic 58Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 5918DNAArtificial SequenceSynthetic 59cagggcatta gaaatgat 18606PRTArtificial SequenceSynthetic 60Gln Gly Ile Arg Asn Asp1 5 619DNAArtificial SequenceSynthetic 61gctgcatcc 9623PRTArtificial SequenceSynthetic 62Ala Ala Ser1 6327DNAArtificial SequenceSynthetic 63ctacagcata atagttaccc gtacact 27649PRTArtificial SequenceSynthetic 64Leu Gln His Asn Ser Tyr Pro Tyr Thr1 5 65375DNAArtificial SequenceSynthetic 65caggtgcagc tggtgcagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcatc acttatagtt actactgggg ctggatccgc 120cagcccccag ggaaggggct ggagtggatt gggactatcc atcatagtgg gagcacctac 180tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240tccctgacac tgagttctgt gaccgccgca gacacggctg tgtattactg tgcgagagac 300tactatgata gtagtggtta ttattataac tggttcgacc cctggggcca gggaaccatg 360gtcaccgtct cctca 37566125PRTArtificial SequenceSynthetic 66Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ile Thr Tyr 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Thr Ile His His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80 Ser Leu Thr Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85

90 95 Cys Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Asn Trp Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 6730DNAArtificial SequenceSynthetic 67ggtggctcca tcatcactta tagttactac 306810PRTArtificial SequenceSynthetic 68Gly Gly Ser Ile Ile Thr Tyr Ser Tyr Tyr1 5 10 6921DNAArtificial SequenceSynthetic 69atccatcata gtgggagcac c 21707PRTArtificial SequenceSynthetic 70Ile His His Ser Gly Ser Thr1 5 7151DNAArtificial SequenceSynthetic 71gcgagagact actatgatag tagtggttat tattataact ggttcgaccc c 517217PRTArtificial SequenceSynthetic 72Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Asn Trp Phe Asp1 5 10 15 Pro73324DNAArtificial SequenceSynthetic 73gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag cataatagtt ccccgtggac gttcggccaa 300gggaccaagg tggagatcaa acga 32474108PRTArtificial SequenceSynthetic 74Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 7518DNAArtificial SequenceSynthetic 75cagggcatta gaaatgat 18766PRTArtificial SequenceSynthetic 76Gln Gly Ile Arg Asn Asp1 5 779DNAArtificial SequenceSynthetic 77gctgcatcc 9783PRTArtificial SequenceSynthetic 78Ala Ala Ser1 7927DNAArtificial SequenceSynthetic 79ctacagcata atagttcccc gtggacg 27809PRTArtificial SequenceSynthetic 80Leu Gln His Asn Ser Ser Pro Trp Thr1 5 81369DNAArtificial SequenceSynthetic 81caggtgcact tgaaggagtc tggtcctacg ctggtgaaac ccacacagac cctcacgctg 60acctgcacct tctctgggtt ctcactcagc actagtggag tgggtgtggg ctggatccgt 120cagcccccag gaaaggccct ggagtggctt gcactcattt attggaatga tgataagcgc 180tacagcccat ctctgaagag caggctcacc atcaccaagg acacctccaa aaaccaggtg 240gtccttacaa tgaccaacat ggaccctgtg gacacagcca catattactg tacacacacc 300tcccgttata actggcacta cggcttcctt gactactggg gccagggaac cacggtcacc 360gtctcctca 36982123PRTArtificial SequenceSynthetic 82Gln Val His Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Thr His Thr Ser Arg Tyr Asn Trp His Tyr Gly Phe Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 8330DNAArtificial SequenceSynthetic 83gggttctcac tcagcactag tggagtgggt 308410PRTArtificial SequenceSynthetic 84Gly Phe Ser Leu Ser Thr Ser Gly Val Gly1 5 10 8521DNAArtificial SequenceSynthetic 85atttattgga atgatgataa g 21867PRTArtificial SequenceSynthetic 86Ile Tyr Trp Asn Asp Asp Lys1 5 8745DNAArtificial SequenceSynthetic 87acacacacct cccgttataa ctggcactac ggcttccttg actac 458815PRTArtificial SequenceSynthetic 88Thr His Thr Ser Arg Tyr Asn Trp His Tyr Gly Phe Leu Asp Tyr1 5 10 15 89324DNAArtificial SequenceSynthetic 89gccatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagc aattatttag cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacaa tataatagtt acccgctcac tttcggcgga 300gggaccaagg tggaaatcaa acga 32490108PRTArtificial SequenceSynthetic 90Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 9118DNAArtificial SequenceSynthetic 91cagggcatta gcaattat 18926PRTArtificial SequenceSynthetic 92Gln Gly Ile Ser Asn Tyr1 5 939DNAArtificial SequenceSynthetic 93gctgcatcc 9943PRTArtificial SequenceSynthetic 94Ala Ala Ser1 9527DNAArtificial SequenceSynthetic 95caacaatata atagttaccc gctcact 27969PRTArtificial SequenceSynthetic 96Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1 5 97375DNAArtificial SequenceSynthetic 97gaggtgcagc tggtgcagtt gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc aatagtaatt actactgggg ctggatccgc 120cagcccccag ggaaggggct ggagtggatt gggactacct attatagtgg gaccacctac 180tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagagat 300tattatgata gtagtggtta ttattacaac tggttcgatc cctggggcca gggaaccacg 360gtcaccgtct cctca 37598125PRTArtificial SequenceSynthetic 98Glu Val Gln Leu Val Gln Leu Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Ser 20 25 30 Asn Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Thr Thr Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Asn Trp Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 9930DNAArtificial SequenceSynthetic 99ggtggctcca tcagcaatag taattactac 3010010PRTArtificial SequenceSynthetic 100Gly Gly Ser Ile Ser Asn Ser Asn Tyr Tyr1 5 10 10121DNAArtificial SequenceSynthetic 101acctattata gtgggaccac c 211027PRTArtificial SequenceSynthetic 102Thr Tyr Tyr Ser Gly Thr Thr1 5 10351DNAArtificial SequenceSynthetic 103gcgagagatt attatgatag tagtggttat tattacaact ggttcgatcc c 5110417PRTArtificial SequenceSynthetic 104Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Asn Trp Phe Asp1 5 10 15 Pro105324DNAArtificial SequenceSynthetic 105gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcagtctca cactcagcag cctgcagcct 240gaagattttg caacttattt ctgtctacag catcatattt acccgtggac gttcggccaa 300gggaccaagg tggagatcaa acga 324106108PRTArtificial SequenceSynthetic 106Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Leu Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His His Ile Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 10718DNAArtificial SequenceSynthetic 107caggacatta gaaatgat 181086PRTArtificial SequenceSynthetic 108Gln Asp Ile Arg Asn Asp1 5 1099DNAArtificial SequenceSynthetic 109gctgcatcc 91103PRTArtificial SequenceSynthetic 110Ala Ala Ser1 11127DNAArtificial SequenceSynthetic 111ctacagcatc atatttaccc gtggacg 271129PRTArtificial SequenceSynthetic 112Leu Gln His His Ile Tyr Pro Trp Thr1 5 113355DNAArtificial SequenceSynthetic 113caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt cgctatggca ttcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggctgtt atatcttatg atggaagtga tgaatactat 180gtagactccg tgaagggccg attcagcatc tcccgagaca attccaagaa cacgctttat 240ctacaaatga acagtctgag gcctgcggac tcggctgttt attactgtgt gaaaggagat 300ctggaacttg gttttgacta ctggggccag ggaaccctgg tcaccgtctc ctcaa 355114118PRTArtificial SequenceSynthetic 114Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asp Glu Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Ala Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Lys Gly Asp Leu Glu Leu Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 11524DNAArtificial SequenceSynthetic 115ggattcacct tcagtcgcta tggc 241168PRTArtificial SequenceSynthetic 116Gly Phe Thr Phe Ser Arg Tyr Gly1 5 11724DNAArtificial SequenceSynthetic 117atatcttatg atggaagtga tgaa 241188PRTArtificial SequenceSynthetic 118Ile Ser Tyr Asp Gly Ser Asp Glu1 5 11933DNAArtificial SequenceSynthetic 119gtgaaaggag atctggaact tggttttgac tac 3312011PRTArtificial SequenceSynthetic 120Val Lys Gly Asp Leu Glu Leu Gly Phe Asp Tyr1 5 10 121337DNAArtificial SequenceSynthetic 121gatattgtga tgactcaggc tgcaccctct atacctgtca ttccaggaga gtcagtatcc 60atgtcctgca ggtctagtaa gagtctcctg tacagtaatg gacatactta cgtgtattgg 120tttgtgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 180tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc 240agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaaaatct agaatttccg 300ctcacgttcg gtgctgggac caagctggag ctgaaac 337122112PRTArtificial SequenceSynthetic 122Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro Val Ile Pro Gly1 5 10 15 Glu Ser Val Ser Met Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser 20 25 30 Asn Gly His Thr Tyr Val Tyr Trp Phe Val Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Asn 85 90 95 Leu Glu Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 12333DNAArtificial SequenceSynthetic 123aagagtctcc tgtacagtaa tggacatact tac 3312411PRTArtificial SequenceSynthetic 124Lys Ser Leu Leu Tyr Ser Asn Gly His Thr Tyr1 5 10 1259DNAArtificial SequenceSynthetic 125cggatgtcc 91263PRTArtificial SequenceSynthetic 126Arg Met Ser1 12727DNAArtificial SequenceSynthetic 127atgcaaaatc tagaatttcc gctcacg 271289PRTArtificial SequenceSynthetic 128Met Gln Asn Leu Glu Phe Pro Leu Thr1 5 129355DNAArtificial SequenceSynthetic 129caggtgcagc tggtggaggc ggggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt cgctatggca ttcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggctgtt atatcttatg atggaactga tgaatactat 180gcagactccg tgaagggccg attcaccatc tcccgagaca attccaagaa cacgctttat 240ctacaaatga acagtctgag acctgcggac tcggctgtat attactgtgc gaaaggagat 300ctggaacttg gttttgacta ctggggccag ggaaccctgg tcaccgtctc ctcaa 355130118PRTArtificial SequenceSynthetic 130Gln Val Gln Leu Val Glu Ala Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Thr Asp Glu Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Ala Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Leu Glu Leu Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 13124DNAArtificial SequenceSynthetic 131ggattcacct tcagtcgcta tggc 241328PRTArtificial SequenceSynthetic 132Gly Phe Thr Phe Ser Arg Tyr Gly1 5 13324DNAArtificial SequenceSynthetic 133atatcttatg atggaactga tgaa 241348PRTArtificial SequenceSynthetic 134Ile Ser Tyr Asp Gly Thr Asp Glu1 5 13533DNAArtificial SequenceSynthetic 135gcgaaaggag atctggaact tggttttgac tac 3313611PRTArtificial SequenceSynthetic 136Ala Lys Gly Asp Leu Glu Leu Gly Phe Asp Tyr1 5 10 137322DNAArtificial SequenceSynthetic 137gccatccggt tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60atcacttgtc gggcgagtca ggatattagt atttggttag cctggtatca gcagagtcca 120gggaaagccc ctaaactcct gatcaatgtt gcatcccgtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacgg tctgcagcct 240gaagattttg taacttacta ttgtcaacag

gctaacagtt tcccgatcac cttcggccaa 300gggacacgac tggcgaccaa ac 322138107PRTArtificial SequenceSynthetic 138Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Ser Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Asn Val Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Gly Leu Gln Pro65 70 75 80 Glu Asp Phe Val Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Ala Thr Lys 100 105 13918DNAArtificial SequenceSynthetic 139caggatatta gtatttgg 181406PRTArtificial SequenceSynthetic 140Gln Asp Ile Ser Ile Trp1 5 1419DNAArtificial SequenceSynthetic 141gttgcatcc 91423PRTArtificial SequenceSynthetic 142Val Ala Ser1 14327DNAArtificial SequenceSynthetic 143caacaggcta acagtttccc gatcacc 271449PRTArtificial SequenceSynthetic 144Gln Gln Ala Asn Ser Phe Pro Ile Thr1 5 145358DNAArtificial SequenceSynthetic 145caggtgcagc tggtgcagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt catcttcaat acctatacca tgaattgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcatcc atcactagtc gtggtactta tatattctac 180tcagactcac ttaagggccg attcaccatt tccagagaca acgccaataa ctcactgttt 240ctgcaaatga acagcctgag agtcgaagac acggctgttt attactgttc gagagatcgt 300ggatacacct ttggtcctga ctactggggc cagggaaccc tggtcaccgt ctcttcag 358146119PRTArtificial SequenceSynthetic 146Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asn Thr Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Ser Arg Gly Thr Tyr Ile Phe Tyr Ser Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser Leu Phe65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Asp Arg Gly Tyr Thr Phe Gly Pro Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 14724DNAArtificial SequenceSynthetic 147ggattcatct tcaataccta tacc 241488PRTArtificial SequenceSynthetic 148Gly Phe Ile Phe Asn Thr Tyr Thr1 5 14924DNAArtificial SequenceSynthetic 149atcactagtc gtggtactta tata 241508PRTArtificial SequenceSynthetic 150Ile Thr Ser Arg Gly Thr Tyr Ile1 5 15136DNAArtificial SequenceSynthetic 151tcgagagatc gtggatacac ctttggtcct gactac 3615212PRTArtificial SequenceSynthetic 152Ser Arg Asp Arg Gly Tyr Thr Phe Gly Pro Asp Tyr1 5 10 153322DNAArtificial SequenceSynthetic 153gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagggcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatca ctgtctacat tatgattttc atcctcggac gttcggccaa 300gggaccaagg tggaaatcaa gc 322154107PRTArtificial SequenceSynthetic 154Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Gly Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr His Cys Leu His Tyr Asp Phe His Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 15518DNAArtificial SequenceSynthetic 155caggacatta gaaatgat 181566PRTArtificial SequenceSynthetic 156Gln Asp Ile Arg Asn Asp1 5 1579DNAArtificial SequenceSynthetic 157gctgcatcc 91583PRTArtificial SequenceSynthetic 158Ala Ala Ser1 15927DNAArtificial SequenceSynthetic 159ctacattatg attttcatcc tcggacg 271609PRTArtificial SequenceSynthetic 160Leu His Tyr Asp Phe His Pro Arg Thr1 5 161391DNAArtificial SequenceSynthetic 161caggtgcaac tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatgcca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaactaa ttattactat 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatccc 300ctatattacg atattttgac tggttattcc cccgactact actacggtat ggacgtctgg 360ggccaaggga ccacggtcac cgtctcctca g 391162130PRTArtificial SequenceSynthetic 162Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Thr Asn Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Leu Tyr Tyr Asp Ile Leu Thr Gly Tyr Ser Pro Asp 100 105 110 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser 130 16324DNAArtificial SequenceSynthetic 163ggattcacct tcagtagcta tgcc 241648PRTArtificial SequenceSynthetic 164Gly Phe Thr Phe Ser Ser Tyr Ala1 5 16524DNAArtificial SequenceSynthetic 165atatggtatg atggaactaa ttat 241668PRTArtificial SequenceSynthetic 166Ile Trp Tyr Asp Gly Thr Asn Tyr1 5 16769DNAArtificial SequenceSynthetic 167gcgagagatc ccctatatta cgatattttg actggttatt cccccgacta ctactacggt 60atggacgtc 6916823PRTArtificial SequenceSynthetic 168Ala Arg Asp Pro Leu Tyr Tyr Asp Ile Leu Thr Gly Tyr Ser Pro Asp1 5 10 15 Tyr Tyr Tyr Gly Met Asp Val 20 169322DNAArtificial SequenceSynthetic 169gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gacttttagc agcaacttag cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataagt ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac 322170107PRTArtificial SequenceSynthetic 170Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Phe Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Lys Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 17118DNAArtificial SequenceSynthetic 171cagactttta gcagcaac 181726PRTArtificial SequenceSynthetic 172Gln Thr Phe Ser Ser Asn1 5 1739DNAArtificial SequenceSynthetic 173ggtgcatcc 91743PRTArtificial SequenceSynthetic 174Gly Ala Ser1 17527DNAArtificial SequenceSynthetic 175cagcagtata ataagtggcc gctcact 271769PRTArtificial SequenceSynthetic 176Gln Gln Tyr Asn Lys Trp Pro Leu Thr1 5 177367DNAArtificial SequenceSynthetic 177gaggtgcagc tgttggagtc tgggggaggc ttggtccagc ctggggagtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agctatgcca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcaact atcagtggta gtggtggtta tatatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat atttctgtgc gaaagattcc 300aggtataact ggaactacgg caattttgac tactggggcc agggaaccct ggtcaccgtc 360tcctcag 367178122PRTArtificial SequenceSynthetic 178Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Gly Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Ser Arg Tyr Asn Trp Asn Tyr Gly Asn Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 17924DNAArtificial SequenceSynthetic 179ggattcacct ttagcagcta tgcc 241808PRTArtificial SequenceSynthetic 180Gly Phe Thr Phe Ser Ser Tyr Ala1 5 18124DNAArtificial SequenceSynthetic 181atcagtggta gtggtggtta tata 241828PRTArtificial SequenceSynthetic 182Ile Ser Gly Ser Gly Gly Tyr Ile1 5 18345DNAArtificial SequenceSynthetic 183gcgaaagatt ccaggtataa ctggaactac ggcaattttg actac 4518415PRTArtificial SequenceSynthetic 184Ala Lys Asp Ser Arg Tyr Asn Trp Asn Tyr Gly Asn Phe Asp Tyr1 5 10 15 185322DNAArtificial SequenceSynthetic 185gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gaatgttagc agcaacttag cctggaacaa gcagaaacct 120ggccaggctc ccagactcct catctatgct acatccacca gggccactgg tgtcccagcc 180aggttcagtg ccagtgggtc tgggacagac ttcgctctca ccatcaacag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact ggcctctcac tttcggcgga 300gggaccaagg tggagatcaa ac 322186107PRTArtificial SequenceSynthetic 186Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Ser Ser Asn 20 25 30 Leu Ala Trp Asn Lys Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Thr Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Ala 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Asn Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 18718DNAArtificial SequenceSynthetic 187cagaatgtta gcagcaac 181886PRTArtificial SequenceSynthetic 188Gln Asn Val Ser Ser Asn1 5 1899DNAArtificial SequenceSynthetic 189gctacatcc 91903PRTArtificial SequenceSynthetic 190Ala Thr Ser1 19127DNAArtificial SequenceSynthetic 191cagcagtata ataactggcc tctcact 271929PRTArtificial SequenceSynthetic 192Gln Gln Tyr Asn Asn Trp Pro Leu Thr1 5 193367DNAArtificial SequenceSynthetic 193gaggtgcaac tgttggaatc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgtag cctctcgatt caccttcagc agcaatgcca tgagttgggt ccgccaggct 120ccagggacgg ggctggagtg ggtctcagct attactggta gtggtagtag gacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacggtgtat 240ctgcaaatga acagcctgag aggcgaggac acggccgtat attactgtgc gaaagatcaa 300gggggtacct ggaactacgg agattttgac tactggggcc agggaaccct ggtcaccgtc 360tcctcag 367194122PRTArtificial SequenceSynthetic 194Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Thr Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Gly Ser Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gln Gly Gly Thr Trp Asn Tyr Gly Asp Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 19524DNAArtificial SequenceSynthetic 195cgattcacct tcagcagcaa tgcc 241968PRTArtificial SequenceSynthetic 196Arg Phe Thr Phe Ser Ser Asn Ala1 5 19724DNAArtificial SequenceSynthetic 197attactggta gtggtagtag gaca 241988PRTArtificial SequenceSynthetic 198Ile Thr Gly Ser Gly Ser Arg Thr1 5 19945DNAArtificial SequenceSynthetic 199gcgaaagatc aagggggtac ctggaactac ggagattttg actac 4520015PRTArtificial SequenceSynthetic 200Ala Lys Asp Gln Gly Gly Thr Trp Asn Tyr Gly Asp Phe Asp Tyr1 5 10 15 201322DNAArtificial SequenceSynthetic 201gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tctcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact ggcctctcac tttcggcgga 300gggaccaagg tggagatcaa gc 322202107PRTArtificial SequenceSynthetic 202Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Leu Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 20318DNAArtificial SequenceSynthetic 203cagagtgtta gcagcaac

182046PRTArtificial SequenceSynthetic 204Gln Ser Val Ser Ser Asn1 5 2059DNAArtificial SequenceSynthetic 205ggtgcatcc 92063PRTArtificial SequenceSynthetic 206Gly Ala Ser1 20727DNAArtificial SequenceSynthetic 207cagcagtata ataactggcc tctcact 272089PRTArtificial SequenceSynthetic 208Gln Gln Tyr Asn Asn Trp Pro Leu Thr1 5 209373DNAArtificial SequenceSynthetic 209caggtgcagc tggtggagtc tgggggagac gtggtccagc ctgggaggtc cctgagactc 60tcctgtacag cctctggatt caccttcagt agttatggca tgcactgggt ccgccaggct 120ccaggcaggg ggctggagtg ggtggcagtt atatcatttg atggaaaaaa taaatactat 180gcagactccg tgaagggccg attcaccgtc tccagagaca attccaagaa cacgctgttt 240ctgcaaatga acagcctgag agctgaggac acggctctat attactgtgc gaaaaggata 300gcagcaactg gttactacta cttctacggt ttggacgtct ggggccaagg gaccacggtc 360accgtctcct cag 373210124PRTArtificial SequenceSynthetic 210Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Phe Asp Gly Lys Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Ala Ala Thr Gly Tyr Tyr Tyr Phe Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 21124DNAArtificial SequenceSynthetic 211ggattcacct tcagtagtta tggc 242128PRTArtificial SequenceSynthetic 212Gly Phe Thr Phe Ser Ser Tyr Gly1 5 21324DNAArtificial SequenceSynthetic 213atatcatttg atggaaaaaa taaa 242148PRTArtificial SequenceSynthetic 214Ile Ser Phe Asp Gly Lys Asn Lys1 5 21551DNAArtificial SequenceSynthetic 215gcgaaaagga tagcagcaac tggttactac tacttctacg gtttggacgt c 5121617PRTArtificial SequenceSynthetic 216Ala Lys Arg Ile Ala Ala Thr Gly Tyr Tyr Tyr Phe Tyr Gly Leu Asp1 5 10 15 Val217322DNAArtificial SequenceSynthetic 217gaaataatga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagaggcacc 60ctctcctgca gggccagtca gagtgttagt agcaacttag cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccagtgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccgtcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac 322218107PRTArtificial SequenceSynthetic 218Glu Ile Met Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Gly Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Val Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 21918DNAArtificial SequenceSynthetic 219cagagtgtta gtagcaac 182206PRTArtificial SequenceSynthetic 220Gln Ser Val Ser Ser Asn1 5 2219DNAArtificial SequenceSynthetic 221ggtgcatcc 92223PRTArtificial SequenceSynthetic 222Gly Ala Ser1 22327DNAArtificial SequenceSynthetic 223cagcagtata ataactggcc gctcact 272249PRTArtificial SequenceSynthetic 224Gln Gln Tyr Asn Asn Trp Pro Leu Thr1 5 225367DNAArtificial SequenceSynthetic 225gaggttcagc tgttggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctctatc acctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtgtta gtggtactaa tacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca agtccaagaa catgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatctc 300ctacataact ggaaatacgg gacttttgat atctggggcc aagggacaat ggtcaccgtc 360tcttcag 367226122PRTArtificial SequenceSynthetic 226Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ile Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Val Ser Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Met Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu Leu His Asn Trp Lys Tyr Gly Thr Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 22724DNAArtificial SequenceSynthetic 227ggattcacct ctatcaccta tgcc 242288PRTArtificial SequenceSynthetic 228Gly Phe Thr Ser Ile Thr Tyr Ala1 5 22924DNAArtificial SequenceSynthetic 229attagtgtta gtggtactaa taca 242308PRTArtificial SequenceSynthetic 230Ile Ser Val Ser Gly Thr Asn Thr1 5 23145DNAArtificial SequenceSynthetic 231gcgaaagatc tcctacataa ctggaaatac gggacttttg atatc 4523215PRTArtificial SequenceSynthetic 232Ala Lys Asp Leu Leu His Asn Trp Lys Tyr Gly Thr Phe Asp Ile1 5 10 15 233322DNAArtificial SequenceSynthetic 233gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttgac agcaacttag tctggtacca acaaaaacct 120ggccaggttc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataagt ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac 322234107PRTArtificial SequenceSynthetic 234Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asp Ser Asn 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Lys Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 23518DNAArtificial SequenceSynthetic 235cagagtgttg acagcaac 182366PRTArtificial SequenceSynthetic 236Gln Ser Val Asp Ser Asn1 5 2379DNAArtificial SequenceSynthetic 237ggtgcatcc 92383PRTArtificial SequenceSynthetic 238Gly Ala Ser1 23927DNAArtificial SequenceSynthetic 239cagcagtata ataagtggcc gctcact 272409PRTArtificial SequenceSynthetic 240Gln Gln Tyr Asn Lys Trp Pro Leu Thr1 5 241367DNAArtificial SequenceSynthetic 241gaggttcagc tgttggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctctatc acctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtgtta gtggtactaa tacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca agtccaagaa catgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatctc 300ctacataact ggaaatacgg gacttttgat atctggggcc aagggacaat ggtcaccgtc 360tcttcag 367242122PRTArtificial SequenceSynthetic 242Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ile Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Val Ser Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Met Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu Leu His Asn Trp Lys Tyr Gly Thr Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 24324DNAArtificial SequenceSynthetic 243ggattcacct ctatcaccta tgcc 242448PRTArtificial SequenceSynthetic 244Gly Phe Thr Ser Ile Thr Tyr Ala1 5 24524DNAArtificial SequenceSynthetic 245attagtgtta gtggtactaa taca 242468PRTArtificial SequenceSynthetic 246Ile Ser Val Ser Gly Thr Asn Thr1 5 24745DNAArtificial SequenceSynthetic 247gcgaaagatc tcctacataa ctggaaatac gggacttttg atatc 4524815PRTArtificial SequenceSynthetic 248Ala Lys Asp Leu Leu His Asn Trp Lys Tyr Gly Thr Phe Asp Ile1 5 10 15 249325DNAArtificial SequenceSynthetic 249gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggaacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccacggg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataacaact ggcccatgta cacttttggc 300caggggacca agctggagat caaac 325250108PRTArtificial SequenceSynthetic 250Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Asn Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 25118DNAArtificial SequenceSynthetic 251cagagtgtta gcagcaac 182526PRTArtificial SequenceSynthetic 252Gln Ser Val Ser Ser Asn1 5 2539DNAArtificial SequenceSynthetic 253ggtgcatcc 92543PRTArtificial SequenceSynthetic 254Gly Ala Ser1 25530DNAArtificial SequenceSynthetic 255cagcagtata acaactggcc catgtacact 3025610PRTArtificial SequenceSynthetic 256Gln Gln Tyr Asn Asn Trp Pro Met Tyr Thr1 5 10 257361DNAArtificial SequenceSynthetic 257caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgaag cctctggatt caccttcagt agttctggca tgcactgggt ccgccaggct 120ccaggcaagg ggctgcagtg ggtggctgtt atatcatatg atggaaataa taaattttat 180gaagactccg tgaagggccg attgaccatt tccagagaca attccaacaa cactctgtgg 240ctgcaaatga acagcctgag agttgaagac acggctgttt attactgtgc gaaatcagga 300ggtagagtgg gagccgcctt tgcctactgg ggccagggaa ccctggtcac cgtctcctca 360g 361258120PRTArtificial SequenceSynthetic 258Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Phe Tyr Glu Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Asn Asn Thr Leu Trp65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Gly Gly Arg Val Gly Ala Ala Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 25924DNAArtificial SequenceSynthetic 259ggattcacct tcagtagttc tggc 242608PRTArtificial SequenceSynthetic 260Gly Phe Thr Phe Ser Ser Ser Gly1 5 26124DNAArtificial SequenceSynthetic 261atatcatatg atggaaataa taaa 242628PRTArtificial SequenceSynthetic 262Ile Ser Tyr Asp Gly Asn Asn Lys1 5 26339DNAArtificial SequenceSynthetic 263gcgaaatcag gaggtagagt gggagccgcc tttgcctac 3926413PRTArtificial SequenceSynthetic 264Ala Lys Ser Gly Gly Arg Val Gly Ala Ala Phe Ala Tyr1 5 10 265337DNAArtificial SequenceSynthetic 265gatattgtga acactcagtc tccactctct ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg tatggtaatg gatacaacta tttggattgg 120tacctgcaga agccagggca ctctccacag ctcctgatct atttgggttc taatcggggc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaagatc 240agcagagtgg aggctgaaga tgttggcatt tattactgca tgcaaactct acaaactcca 300ttcactttcg gccctgggac caaaatgtat atcaaac 337266112PRTArtificial SequenceSynthetic 266Asp Ile Val Asn Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Gly 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly His Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Gly Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Thr 85 90 95 Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Met Tyr Ile Lys 100 105 110 26733DNAArtificial SequenceSynthetic 267cagagcctcc tgtatggtaa tggatacaac tat 3326811PRTArtificial SequenceSynthetic 268Gln Ser Leu Leu Tyr Gly Asn Gly Tyr Asn Tyr1 5 10 2699DNAArtificial SequenceSynthetic 269ttgggttct 92703PRTArtificial SequenceSynthetic 270Leu Gly Ser1 27127DNAArtificial SequenceSynthetic 271atgcaaactc tacaaactcc attcact 272729PRTArtificial SequenceSynthetic 272Met Gln Thr Leu Gln Thr Pro Phe Thr1 5 273376DNAArtificial SequenceSynthetic 273gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag gctctggaat cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta atggtggtac cacaaactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca actccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagaaaga 300atccttacca gcagctggac gaggtacggt attatggacg tctggggcca agggaccacg 360gtcaccgtct cctcag

376274125PRTArtificial SequenceSynthetic 274Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Ile Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Asn Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Arg Ile Leu Thr Ser Ser Trp Thr Arg Tyr Gly Ile Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 27524DNAArtificial SequenceSynthetic 275ggaatcacct ttagcagcta tgcc 242768PRTArtificial SequenceSynthetic 276Gly Ile Thr Phe Ser Ser Tyr Ala1 5 27724DNAArtificial SequenceSynthetic 277attagtggta atggtggtac caca 242788PRTArtificial SequenceSynthetic 278Ile Ser Gly Asn Gly Gly Thr Thr1 5 27954DNAArtificial SequenceSynthetic 279gcgaaagaaa gaatccttac cagcagctgg acgaggtacg gtattatgga cgtc 5428018PRTArtificial SequenceSynthetic 280Ala Lys Glu Arg Ile Leu Thr Ser Ser Trp Thr Arg Tyr Gly Ile Met1 5 10 15 Asp Val281322DNAArtificial SequenceSynthetic 281gaaatagtga tgacgcagtc tccagccacc ctgtctatgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact ggcctctcac tttcggcgga 300gggaccaagt tagagatcaa ac 322282107PRTArtificial SequenceSynthetic 282Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Met Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 28318DNAArtificial SequenceSynthetic 283cagagtgtta gcagcaac 182846PRTArtificial SequenceSynthetic 284Gln Ser Val Ser Ser Asn1 5 2859DNAArtificial SequenceSynthetic 285ggtgcatcc 92863PRTArtificial SequenceSynthetic 286Gly Ala Ser1 28727DNAArtificial SequenceSynthetic 287cagcagtata ataactggcc tctcact 272889PRTArtificial SequenceSynthetic 288Gln Gln Tyr Asn Asn Trp Pro Leu Thr1 5 289358DNAArtificial SequenceSynthetic 289caggtgcaac tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaaacac caactacaac 180ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aaactgaact ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagagaggct 300acagtaactc catactttga ctactggggc cagggaaccc tggtcaccgt ctcctcag 358290119PRTArtificial SequenceSynthetic 290Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80 Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Glu Ala Thr Val Thr Pro Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 29124DNAArtificial SequenceSynthetic 291ggtgggtcct tcagtggtta ctac 242928PRTArtificial SequenceSynthetic 292Gly Gly Ser Gly Ser Gly Tyr Tyr1 5 29321DNAArtificial SequenceSynthetic 293atcaatcata gtggaaacac c 212947PRTArtificial SequenceSynthetic 294Ile Asn His Ser Gly Asn Thr1 5 29539DNAArtificial SequenceSynthetic 295gcgagaagag aggctacagt aactccatac tttgactac 3929613PRTArtificial SequenceSynthetic 296Ala Arg Arg Glu Ala Thr Val Thr Pro Tyr Phe Asp Tyr1 5 10 297322DNAArtificial SequenceSynthetic 297gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgct gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag cttaatagtt atccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac 322298107PRTArtificial SequenceSynthetic 298Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 29918DNAArtificial SequenceSynthetic 299cagggcatta gcagttat 183006PRTArtificial SequenceSynthetic 300Gln Gly Ile Ser Ser Tyr1 5 3019DNAArtificial SequenceSynthetic 301gctgcatcc 93023PRTArtificial SequenceSynthetic 302Ala Ala Ser1 30327DNAArtificial SequenceSynthetic 303caacagctta atagttatcc gctcact 273049PRTArtificial SequenceSynthetic 304Gln Gln Leu Asn Ser Tyr Pro Leu Thr1 5 305355DNAArtificial SequenceSynthetic 305caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgtag tctctggatt caacttcagt aggaatggca tacactgggt ccgccaggct 120ccaggcaagg ggctggaatg ggtggcagtt atatcatatg atggaagaaa taaattttat 180gtagagtccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagtctgag agttgaggac acggctgtat attactgtgc gaaatcctca 300attggagggt tttttgaata ctggggccag ggaaccctgg tcaccgtctc ctcag 355306118PRTArtificial SequenceSynthetic 306Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Asn Phe Ser Arg Asn 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Arg Asn Lys Phe Tyr Val Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ser Ile Gly Gly Phe Phe Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 30724DNAArtificial SequenceSynthetic 307ggattcaact tcagtaggaa tggc 243088PRTArtificial SequenceSynthetic 308Gly Phe Asn Phe Ser Arg Asn Gly1 5 30924DNAArtificial SequenceSynthetic 309atatcatatg atggaagaaa taaa 243108PRTArtificial SequenceSynthetic 310Ile Ser Tyr Asp Gly Arg Asn Lys1 5 31133DNAArtificial SequenceSynthetic 311gcgaaatcct caattggagg gttttttgaa tac 3331211PRTArtificial SequenceSynthetic 312Ala Lys Ser Ser Ile Gly Gly Phe Phe Glu Tyr1 5 10 313337DNAArtificial SequenceSynthetic 313gatattgtga tgactcagtc tccactctcc ctgcccgtca ctcctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120tacctgcaga agccaggaca gtctccacaa ctcatgatct atttgggttc tcatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggagtc tattactgca ttcaagttca acaaactccg 300atcaccttcg gccaagggac acggctggag attaaac 337314112PRTArtificial SequenceSynthetic 314Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Met Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ile Gln Val 85 90 95 Gln Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 31533DNAArtificial SequenceSynthetic 315cagagcctcc tgcatagtaa tggatacaac tat 3331611PRTArtificial SequenceSynthetic 316Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr1 5 10 3179DNAArtificial SequenceSynthetic 317ttgggttct 93183PRTArtificial SequenceSynthetic 318Leu Gly Ser1 31927DNAArtificial SequenceSynthetic 319attcaagttc aacaaactcc gatcacc 273209PRTArtificial SequenceSynthetic 320Ile Gln Val Gln Gln Thr Pro Ile Thr1 5 321337DNAArtificial SequenceSynthetic 321gatattgtga tgactcagac tccactctcc tcacctgtca cccttggaca gccggcctcc 60atctcctgca ggtctagtca aagcctcgta cacagtgatg gaaacaccta cttgagttgg 120cttcagcaga ggccaggcca gcctccaaga ctcctaattt acaagatttc taaccggttc 180tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240agcagggtgg aagctgagga tgtcggggtt tatttctgca tgcaagctac acaatttccg 300tacacttttg gccaggggac caagctggag atcaaag 337322112PRTArtificial SequenceSynthetic 322Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Met Gln Ala 85 90 95 Thr Gln Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 32333DNAArtificial SequenceSynthetic 323caaagcctcg tacacagtga tggaaacacc tac 3332411PRTArtificial SequenceSynthetic 324Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr1 5 10 3259DNAArtificial SequenceSynthetic 325aagatttct 93263PRTArtificial SequenceSynthetic 326Lys Ile Ser1 32727DNAArtificial SequenceSynthetic 327atgcaagcta cacaatttcc gtacact 273289PRTArtificial SequenceSynthetic 328Met Gln Ala Thr Gln Phe Pro Tyr Thr1 5 3298PRTArtificial SequenceSyntheticVARIANT(1)...(1)Xaa = GlyVARIANT(2)...(2)Xaa = PheVARIANT(3)...(3)Xaa = ThrVARIANT(4)...(4)Xaa = PheVARIANT(5)...(5)Xaa = SerVARIANT(6)...(6)Xaa = Ala or SerVARIANT(7)...(7)Xaa = Phe or TyrVARIANT(8)...(8)Xaa = Gly or Ala 329Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3308PRTArtificial SequenceSyntheticVARIANT(1)...(1)Xaa = IleVARIANT(2)...(2)Xaa = Gly or SerVARIANT(3)...(3)Xaa = Tyr or GlyVARIANT(4)...(4)Xaa = Ser or AspVARIANT(5)...(5)Xaa = GlyVARIANT(6)...(6)Xaa = GlyVARIANT(7)...(7)Xaa = Ser or AsnVARIANT(8)...(8)Xaa = Ala or Glu 330Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 33113PRTArtificial SequenceSyntheticVARIANT(1)...(1)Xaa = Ser or AlaVARIANT(2)...(2)Xaa = Thr or LysVARIANT(3)...(3)Xaa = Asp or IleVARIANT(4)...(4)Xaa = Gly or SerVARIANT(5)...(5)Xaa = Ala or HisVARIANT(6)...(6)Xaa = Trp or TyrVARIANT(7)...(7)Xaa = Lys or AspVARIANT(8)...(8)Xaa = Met or IleVARIANT(9)...(9)Xaa = Ser or LeuVARIANT(10)...(10)Xaa = Gly or SerVARIANT(11)...(11)Xaa = Leu or GlyVARIANT(12)...(12)Xaa = Asp or MetVARIANT(13)...(13)Xaa = Val or AspVARIANT(14)...(14)Xaa = Val or absent 331Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 3326PRTArtificial SequenceSyntheticVARIANT(1)...(1)Xaa = GlnVARIANT(2)...(2)Xaa = Asp or GlyVARIANT(3)...(3)Xaa = IleVARIANT(4)...(4)Xaa = SerVARIANT(5)...(5)Xaa = Asp or AsnVARIANT(6)...(6)Xaa = Tyr or Trp 332Xaa Xaa Xaa Xaa Xaa Xaa1 5 3333PRTArtificial SequenceSyntheticVARIANT(1)...(1)Xaa = Thr or AlaVARIANT(2)...(2)Xaa = Thr or AlaVARIANT(3)...(3)Xaa = Ser 333Xaa Xaa Xaa1 3349PRTArtificial SequenceSyntheticVARIANT(1)...(1)Xaa = GlnVARIANT(2)...(2)Xaa = Lys or GlnVARIANT(3)...(3)Xaa = Ala or TyrVARIANT(4)...(4)Xaa = Asp or AsnVARIANT(5)...(5)Xaa = SerVARIANT(6)...(6)Xaa = Ala or PheVARIANT(7)...(7)Xaa = ProVARIANT(8)...(8)Xaa = LeuVARIANT(9)...(9)Xaa = Thr 334Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 335330PRTArtificial SequenceSynthetic 335Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 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 Thr65 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 Trp145 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 Glu225 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 Thr305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 336327PRTArtificial SequenceSynthetic 336Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu Ser 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 Lys Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 337327PRTArtificial SequenceSynthetic 337Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu Ser 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 Lys Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 3381128DNAHomo sapiens 338atgcaaaaac tgcaactctg tgtttatatt tacctgttta tgctgattgt tgctggtcca 60gtggatctaa atgagaacag tgagcaaaaa gaaaatgtgg aaaaagaggg gctgtgtaat 120gcatgtactt ggagacaaaa cactaaatct tcaagaatag aagccattaa gatacaaatc 180ctcagtaaac ttcgtctgga aacagctcct aacatcagca aagatgttat aagacaactt 240ttacccaaag ctcctccact ccgggaactg attgatcagt atgatgtcca gagggatgac 300agcagcgatg gctctttgga agatgacgat tatcacgcta caacggaaac aatcattacc 360atgcctacag agtctgattt tctaatgcaa gtggatggaa aacccaaatg ttgcttcttt 420aaatttagct ctaaaataca atacaataaa gtagtaaagg cccaactatg gatatatttg 480agacccgtcg agactcctac aacagtgttt gtgcaaatcc tgagactcat caaacctatg 540aaagacggta caaggtatac tggaatccga tctctgaaac ttgacatgaa cccaggcact 600ggtatttggc agagcattga tgtgaagaca gtgttgcaaa attggctcaa acaacctgaa 660tccaacttag gcattgaaat aaaagcttta gatgagaatg gtcatgatct tgctgtaacc 720ttcccaggac caggagaaga tgggctgaat ccgtttttag aggtcaaggt aacagacaca 780ccaaaaagat ccagaaggga ttttggtctt gactgtgatg agcactcaac agaatcacga 840tgctgtcgtt accctctaac tgtggatttt gaagcttttg gatgggattg gattatcgct 900cctaaaagat ataaggccaa ttactgctct ggagagtgtg aatttgtatt tttacaaaaa 960tatcctcata ctcatctggt acaccaagca aaccccagag gttcagcagg cccttgctgt 1020actcccacaa agatgtctcc aattaatatg ctatatttta atggcaaaga acaaataata 1080tatgggaaaa ttccagcgat ggtagtagac cgctgtgggt gctcatga 1128339375PRTHomo sapiens 339Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile1 5 10 15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu145 150 155 160 Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr225 230 235 240 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys305 310 315 320 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg Cys Gly Cys Ser 370 375 340109PRTHomo sapiens 340Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 3411224DNAHomo sapiens 341atggtgctcg cggccccgct gctgctgggc ttcctgctcc tcgccctgga gctgcggccc 60cggggggagg cggccgaggg ccccgcggcg gcggcggcgg cggcggcggc ggcggcagcg 120gcgggggtcg ggggggagcg ctccagccgg ccagccccgt ccgtggcgcc cgagccggac 180ggctgccccg tgtgcgtttg gcggcagcac agccgcgagc tgcgcctaga gagcatcaag 240tcgcagatct tgagcaaact gcggctcaag gaggcgccca acatcagccg cgaggtggtg 300aagcagctgc tgcccaaggc gccgccgctg cagcagatcc tggacctaca cgacttccag 360ggcgacgcgc tgcagcccga ggacttcctg gaggaggacg agtaccacgc caccaccgag 420accgtcatta gcatggccca ggagacggac ccagcagtac agacagatgg cagccctctc 480tgctgccatt ttcacttcag ccccaaggtg atgttcacaa aggtactgaa ggcccagctg 540tgggtgtacc tacggcctgt accccgccca gccacagtct acctgcagat cttgcgacta 600aaacccctaa ctggggaagg gaccgcaggg ggagggggcg gaggccggcg tcacatccgt 660atccgctcac tgaagattga gctgcactca cgctcaggcc attggcagag catcgacttc 720aagcaagtgc tacacagctg gttccgccag ccacagagca actggggcat cgagatcaac 780gcctttgatc ccagtggcac agacctggct gtcacctccc tggggccggg agccgagggg 840ctgcatccat tcatggagct tcgagtccta gagaacacaa aacgttcccg gcggaacctg 900ggtctggact gcgacgagca ctcaagcgag tcccgctgct gccgatatcc cctcacagtg 960gactttgagg ctttcggctg ggactggatc atcgcaccta agcgctacaa ggccaactac 1020tgctccggcc agtgcgagta catgttcatg caaaaatatc cgcataccca tttggtgcag 1080caggccaatc caagaggctc tgctgggccc tgttgtaccc ccaccaagat gtccccaatc 1140aacatgctct acttcaatga caagcagcag attatctacg gcaagatccc tggcatggtg 1200gtggatcgct gtggctgctc ttaa 1224342407PRTHomo sapiens 342Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser 35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys65 70 75 80 Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser 85 90 95 Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln 100 105 110 Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115 120 125 Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser 130 135 140 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu145 150 155 160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu 165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe225 230 235 240 Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255 Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 260 265 270 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 275 280 285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys 290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val305 310 315 320 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 340 345 350 Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 355 360 365 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 370 375 380 Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val385 390 395 400 Val Asp Arg Cys Gly Cys Ser 405 343109PRTHomo sapiens 343Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu 35 40 45 Tyr Met Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 100 105 3441503DNAHomo spiens 344atgagactcc ccaaactcct cactttcttg ctttggtacc tggcttggct ggacctggaa 60ttcatctgca ctgtgttggg tgcccctgac ttgggccaga gaccccaggg gaccaggcca 120ggattggcca aagcagaggc caaggagagg ccccccctgg cccggaacgt cttcaggcca 180gggggtcaca gctatggtgg gggggccacc aatgccaatg ccagggcaaa gggaggcacc 240gggcagacag gaggcctgac acagcccaag aaggatgaac ccaaaaagct gccccccaga 300ccgggcggcc ctgaacccaa gccaggacac cctccccaaa caaggcaggc tacagcccgg 360actgtgaccc caaaaggaca gcttcccgga ggcaaggcac ccccaaaagc aggatctgtc 420cccagctcct tcctgctgaa gaaggccagg gagcccgggc ccccacgaga gcccaaggag 480ccgtttcgcc caccccccat cacaccccac gagtacatgc tctcgctgta caggacgctg 540tccgatgctg acagaaaggg aggcaacagc agcgtgaagt tggaggctgg cctggccaac 600accatcacca gctttattga caaagggcaa gatgaccgag gtcccgtggt caggaagcag 660aggtacgtgt ttgacattag tgccctggag aaggatgggc tgctgggggc cgagctgcgg 720atcttgcgga agaagccctc ggacacggcc aagccagcgg cccccggagg cgggcgggct 780gcccagctga agctgtccag ctgccccagc ggccggcagc cggcctcctt gctggatgtg 840cgctccgtgc caggcctgga cggatctggc tgggaggtgt tcgacatctg gaagctcttc 900cgaaacttta agaactcggc ccagctgtgc ctggagctgg aggcctggga acggggcagg 960gccgtggacc tccgtggcct gggcttcgac cgcgccgccc

ggcaggtcca cgagaaggcc 1020ctgttcctgg tgtttggccg caccaagaaa cgggacctgt tctttaatga gattaaggcc 1080cgctctggcc aggacgataa gaccgtgtat gagtacctgt tcagccagcg gcgaaaacgg 1140cgggccccac tggccactcg ccagggcaag cgacccagca agaaccttaa ggctcgctgc 1200agtcggaagg cactgcatgt caacttcaag gacatgggct gggacgactg gatcatcgca 1260ccccttgagt acgaggcttt ccactgcgag gggctgtgcg agttcccatt gcgctcccac 1320ctggagccca cgaatcatgc agtcatccag accctgatga actccatgga ccccgagtcc 1380acaccaccca cctgctgtgt gcccacgcgg ctgagtccca tcagcatcct cttcattgac 1440tctgccaaca acgtggtgta taagcagtat gaggacatgg tcgtggagtc gtgtggctgc 1500agg 1503345501PRTHomo sapiens 345Met Arg Leu Pro Lys Leu Leu Thr Phe Leu Leu Trp Tyr Leu Ala Trp1 5 10 15 Leu Asp Leu Glu Phe Ile Cys Thr Val Leu Gly Ala Pro Asp Leu Gly 20 25 30 Gln Arg Pro Gln Gly Thr Arg Pro Gly Leu Ala Lys Ala Glu Ala Lys 35 40 45 Glu Arg Pro Pro Leu Ala Arg Asn Val Phe Arg Pro Gly Gly His Ser 50 55 60 Tyr Gly Gly Gly Ala Thr Asn Ala Asn Ala Arg Ala Lys Gly Gly Thr65 70 75 80 Gly Gln Thr Gly Gly Leu Thr Gln Pro Lys Lys Asp Glu Pro Lys Lys 85 90 95 Leu Pro Pro Arg Pro Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro 100 105 110 Gln Thr Arg Gln Ala Thr Ala Arg Thr Val Thr Pro Lys Gly Gln Leu 115 120 125 Pro Gly Gly Lys Ala Pro Pro Lys Ala Gly Ser Val Pro Ser Ser Phe 130 135 140 Leu Leu Lys Lys Ala Arg Glu Pro Gly Pro Pro Arg Glu Pro Lys Glu145 150 155 160 Pro Phe Arg Pro Pro Pro Ile Thr Pro His Glu Tyr Met Leu Ser Leu 165 170 175 Tyr Arg Thr Leu Ser Asp Ala Asp Arg Lys Gly Gly Asn Ser Ser Val 180 185 190 Lys Leu Glu Ala Gly Leu Ala Asn Thr Ile Thr Ser Phe Ile Asp Lys 195 200 205 Gly Gln Asp Asp Arg Gly Pro Val Val Arg Lys Gln Arg Tyr Val Phe 210 215 220 Asp Ile Ser Ala Leu Glu Lys Asp Gly Leu Leu Gly Ala Glu Leu Arg225 230 235 240 Ile Leu Arg Lys Lys Pro Ser Asp Thr Ala Lys Pro Ala Ala Pro Gly 245 250 255 Gly Gly Arg Ala Ala Gln Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg 260 265 270 Gln Pro Ala Ser Leu Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly 275 280 285 Ser Gly Trp Glu Val Phe Asp Ile Trp Lys Leu Phe Arg Asn Phe Lys 290 295 300 Asn Ser Ala Gln Leu Cys Leu Glu Leu Glu Ala Trp Glu Arg Gly Arg305 310 315 320 Ala Val Asp Leu Arg Gly Leu Gly Phe Asp Arg Ala Ala Arg Gln Val 325 330 335 His Glu Lys Ala Leu Phe Leu Val Phe Gly Arg Thr Lys Lys Arg Asp 340 345 350 Leu Phe Phe Asn Glu Ile Lys Ala Arg Ser Gly Gln Asp Asp Lys Thr 355 360 365 Val Tyr Glu Tyr Leu Phe Ser Gln Arg Arg Lys Arg Arg Ala Pro Leu 370 375 380 Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu Lys Ala Arg Cys385 390 395 400 Ser Arg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly Trp Asp Asp 405 410 415 Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys Glu Gly Leu 420 425 430 Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Val 435 440 445 Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr Pro Pro Thr 450 455 460 Cys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu Phe Ile Asp465 470 475 480 Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu 485 490 495 Ser Cys Gly Cys Arg 500 346120PRTHomo sapiens 346Ala Pro Leu Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu Lys1 5 10 15 Ala Arg Cys Ser Arg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly 20 25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys 35 40 45 Glu Gly Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 His Ala Val Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr65 70 75 80 Pro Pro Thr Cys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu 85 90 95 Phe Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg 115 120 347107PRTArtificial SequenceSynthetic 347Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Arg Tyr1 5 10 15 Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala 20 25 30 Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val 35 40 45 Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro 50 55 60 Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile65 70 75 80 Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile 85 90 95 Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 348109PRTArtificial SequenceSynthetic 348Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 349111PRTArtificial SequenceSynthetic 349Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp 20 25 30 Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys Ser Gly Glu 35 40 45 Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His 50 55 60 Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys65 70 75 80 Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile 85 90 95 Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 110 350110PRTArtificial SequenceSynthetic 350Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Ser Gly Glu Cys 35 40 45 Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln 50 55 60 Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met65 70 75 80 Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr 85 90 95 Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 110 351114PRTArtificial SequenceSynthetic 351Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Pro 35 40 45 Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Thr65 70 75 80 Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu 85 90 95 Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly 100 105 110 Cys Ser352112PRTArtificial SequenceSynthetic 352Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Pro 35 40 45 Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr 50 55 60 Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Thr Pro Thr65 70 75 80 Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile 85 90 95 Ile Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 110 353109PRTArtificial SequenceSynthetic 353Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Ser Val Asn Ser Lys Ile Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 354109PRTArtificial SequenceSynthetic 354Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105 355109PRTArtificial SequenceSynthetic 355Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Val Pro Thr Glu Leu Ser65 70 75 80 Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys 85 90 95 Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Ser 100 105 356108PRTArtificial SequenceSynthetic 356Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Val Pro Gln Ala Leu Glu65 70 75 80 Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln 85 90 95 Leu Ser Asn Met Ile Val Arg Ser Cys Gly Cys Ser 100 105 357109PRTArtificial SequenceSynthetic 357Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Lys Val Val Leu Lys 85 90 95 Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Ser 100 105 358108PRTArtificial SequenceSynthetic 358Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Lys Pro Lys Val Glu Gln 85 90 95 Leu Ser Asn Met Ile Val Arg Ser Cys Gly Cys Ser 100 105 359360DNAArtificial SequenceSynthetic 359gaggtgcagg tgttggagtc tgggggagac ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt gcctatgcca tgacctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cgcatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacggtatat 240ctgcagatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatggg 300gcctggaaaa tgtccggttt ggacgtctgg ggccaaggga ccacggtcat cgtctcctca 360360120PRTArtificial SequenceSynthetic 360Glu Val Gln Val Leu Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Trp Lys Met Ser Gly Leu Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Ile Val Ser Ser 115 120 36124DNAArtificial SequenceSynthetic 361ggattcacct ttagtgccta tgcc 243628PRTArtificial SequenceSynthetic 362Gly Phe Thr Phe Ser Ala Tyr Ala1 5 36324DNAArtificial SequenceSynthetic 363attagtggta gtggtggtag cgca

243648PRTArtificial SequenceSynthetic 364Ile Ser Gly Ser Gly Gly Ser Ala1 5 36539DNAArtificial SequenceSynthetic 365gcgaaagatg gggcctggaa aatgtccggt ttggacgtc 3936613PRTArtificial SequenceSynthetic 366Ala Lys Asp Gly Ala Trp Lys Met Ser Gly Leu Asp Val1 5 10 367321DNAArtificial SequenceSynthetic 367gacatccaga tgacccagtc tccagcctcc ctgtctgcat ctgttggaga cagagtcacc 60atcacttgtc gggcgagtca ggacattagc gattatttag cctggtatca gcagaaacca 120gggaaaattc ctaggctcct gatctatact acatccactt tgcaatcagg ggtcccatct 180cggttccgtg gcagtgggtc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg caacttatta ctgtcagaag tatgacagtg ccccgctcac tttcggcgga 300gggaccaagg tggagatcaa a 321368107PRTArtificial SequenceSynthetic 368Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asp Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Arg Leu Leu Ile 35 40 45 Tyr Thr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 36918DNAArtificial SequenceSynthetic 369caggacatta gcgattat 183706PRTArtificial SequenceSynthetic 370Gln Asp Ile Ser Asp Tyr1 5 3719DNAArtificial SequenceSynthetic 371actacatcc 93723PRTArtificial SequenceSynthetic 372Thr Thr Ser1 37327DNAArtificial SequenceSynthetic 373cagaagtatg acagtgcccc gctcact 273749PRTArtificial SequenceSynthetic 374Gln Lys Tyr Asp Ser Ala Pro Leu Thr1 5 375363DNAArtificial SequenceSynthetic 375caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaagtc cctgcgactc 60tcctgtgcag cgtctggatt caccttcagt agttttggca tgcattgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt attgggtatg atggaggtaa tgaatactat 180gccgactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgaat 240ctgcaaatga gcagcctgag agccgaagac acggctgtgt attattgttc gactataagt 300cattacgata ttttgagcgg tatggacgtc tggggccgag ggaccacggt caccgtctcc 360tca 363376121PRTArtificial SequenceSynthetic 376Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Lys1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Gly Tyr Asp Gly Gly Asn Glu Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Ile Ser His Tyr Asp Ile Leu Ser Gly Met Asp Val Trp Gly 100 105 110 Arg Gly Thr Thr Val Thr Val Ser Ser 115 120 37724DNAArtificial SequenceSynthetic 377ggattcacct tcagtagttt tggc 243788PRTArtificial SequenceSynthetic 378Gly Phe Thr Phe Ser Ser Phe Gly1 5 37924DNAArtificial SequenceSynthetic 379attgggtatg atggaggtaa tgaa 243808PRTArtificial SequenceSynthetic 380Ile Gly Tyr Asp Gly Gly Asn Glu1 5 38142DNAArtificial SequenceSynthetic 381tcgactataa gtcattacga tattttgagc ggtatggacg tc 4238214PRTArtificial SequenceSynthetic 382Ser Thr Ile Ser His Tyr Asp Ile Leu Ser Gly Met Asp Val1 5 10 383324DNAArtificial SequenceSynthetic 383gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc aactggttag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180cggttcagcg gcagtgcatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgctcac tttcggcgga 300gggaccaagg tggagatcaa acga 324384108PRTArtificial SequenceSynthetic 384Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Ala Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 38518DNAArtificial SequenceSynthetic 385cagggtatta gcaactgg 183866PRTArtificial SequenceSynthetic 386Gln Gly Ile Ser Asn Trp1 5 3879DNAArtificial SequenceSynthetic 387gctgcatcc 93883PRTArtificial SequenceSynthetic 388Ala Ala Ser1 38927DNAArtificial SequenceSynthetic 389caacaggcta acagtttccc gctcact 273909PRTArtificial SequenceSynthetic 390Gln Gln Ala Asn Ser Phe Pro Leu Thr1 5 391340PRTArtificial SequenceSynthetic 391Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu1 5 10 15 Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp 20 25 30 Lys Arg Leu His Cys Tyr Ala Ser Trp Ala Asn Ser Ser Gly Thr Ile 35 40 45 Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp 50 55 60 Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys65 70 75 80 Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu 85 90 95 Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Ser 100 105 110 Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 115 120 125 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 130 135 140 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser145 150 155 160 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 165 170 175 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 180 185 190 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 195 200 205 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 210 215 220 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln225 230 235 240 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 245 250 255 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 260 265 270 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 275 280 285 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 290 295 300 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val305 310 315 320 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 325 330 335 Ser Pro Gly Lys 340

Claims

1-18. (canceled)

19. An antibody or antigen-binding fragment thereof, which specifically binds to, or blocks the biological activity of wild-type mature human growth and differentiation factor-8 (GDF8), comprising: an amino acid sequence comprising three heavy chain complementarity determining region (CDR) sequences which differ by only a single conservative amino acid substitution within only one of the sequences SEQ ID NO: 362, SEQ ID NO:364, and SEQ ID NO: 366; and an amino acid sequence comprising three light chain complementarity determining region (CDR) sequences which differ by only a single conservative amino acid amino acid substitution within only one of the sequences SEQ ID NO: 370, SEQ ID NO: 372, and SEQ ID NO: 374.

20. The antibody or antigen-binding fragment of claim 19, wherein the wild-type mature human growth and differentiation factor-8 (GDF8) comprises an amino acid sequence comprising SEQ ID NO: 340.

21. The antibody or antigen-binding fragment of claim 19, wherein the antibody or antigen-binding fragment does not bind to or block the biological activity of a chimeric GDF8/TGF.beta.1 construct having amino acids 48-72 of mature human GDF8 replaced with the corresponding amino acid sequence of TGF.beta.1.

22. The antibody or antigen-binding fragment of claim 21, wherein the chimeric GDF8/TGF.beta.1 construct comprises the amino acid sequence of SEQ ID NO:352.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 19; and a pharmaceutically acceptable carrier.

24. The antibody or antigen-binding fragment of claim 19, comprising: an amino acid sequence heavy chain variable region (HCVR) of SEQ ID NO: 360 with a single conservative amino acid substitution.

25. The antibody or antigen-binding fragment of claim 19, comprising: an amino acid sequence light chain variable region (LCVR) of SEQ ID NO: 368 with a single conservative amino acid substitution.

26. An antibody or antigen-binding fragment thereof, which specifically binds to, or blocks the biological activity of wild-type mature human growth and differentiation factor-8 (GDF8), comprising: a first amino acid sequence which differs from a heavy chain variable region (HCVR) comprising SEQ ID NO: 360 by only a single conservative amino acid substitution; a second amino acid sequence which differs from a light chain variable region (LCVR) comprising SEQ ID NO: 368 by only a single conservative amino acid substitution.

27. The antibody or antigen-binding fragment of claim 26, wherein the wild-type mature human growth and differentiation factor-8 (GDF8) comprises an amino acid sequence comprising SEQ ID NO: 340.

28. The antibody or antigen-binding fragment of claim 26, wherein the antibody or antigen-binding fragment does not bind to or block the biological activity of a chimeric GDF8/TGF.beta.1 construct having amino acids 48-72 of mature human GDF8 replaced with the corresponding amino acid sequence of TGF.beta.1.

29. The antibody or antigen-binding fragment of claim 28, wherein the chimeric GDF8/TGF.beta.1 construct comprises the amino acid sequence of SEQ ID NO:352.

30. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 26; and a pharmaceutically acceptable carrier.

31. An antibody or antigen-binding fragment thereof, which specifically binds to wild-type mature human growth and differentiation factor-8 (GDF8), wherein the antibody or antigen-binding fragment comprises a heavy chain and a light chain, each comprising CDR1, CDR2 and CDR3 domains, and wherein the heavy chain comprises: an amino acid sequence which differs from SEQ ID NO: 360 by only a single amino acid substitution; and the light chain comprises: an amino acid sequence which differs from SEQ ID NO: 368 by only a single amino acid substitution, wherein the antibody or antigen-binding fragment has at least the same binding affinity and specificity for (GDF8) as an antibody or antigen-binding fragment thereof which comprises SEQ ID NO: 360 and SEQ ID NO: 368.

32. The antibody or antigen-binding fragment of claim 31, wherein the wild-type mature human growth and differentiation factor-8 (GDF8) comprises an amino acid sequence comprising SEQ ID NO: 340.

33. The antibody or antigen-binding fragment of claim 31, wherein the antibody or antigen-binding fragment does not bind to or block the biological activity of a chimeric GDF8/TGF.beta.1 construct having amino acids 48-72 of mature human GDF8 replaced with the corresponding amino acid sequence of TGF.beta.1.

34. The isolated antibody or antigen-binding fragment of claim 33, wherein the chimeric GDF8/TGF.beta.1 construct comprises the amino acid sequence of SEQ ID NO:352.

35. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 31; and a pharmaceutically acceptable carrier.