Human monoclonal antibodies to activin receptor-like kinase 1

1 HUMAN MONOCLONAL ANTIBODIES TO ACTIVIN RECEPTOR-LIKE KINASE 1 Π8ΠΠ 1-TJ’pV 0’ Ό3ΒΖ131Ζ5 Β”ΒΠ3Χ Ο’ΠΙΙ) ϊ’3’ϋ|?Χ 189,642/2 1

It is to be noted that only the subject matter embraced in the scope of the claims appended hereto, whether in the manner defined in the claims or in a manner similar thereto and involving the main features as defined in the claims, is intended to be included in the scope of the present invention, while subject matter described and exemplified to provide background and better understanding of the invention, is not intended for inclusions as part of the present invention.

Field of the Invention

The present invention relates to human monoclonal antibodies and antigen-binding portions thereof that bind to the extracellular domain (ECD) of activin receptor-iike kinase-1 (ALK-1). The invention also relates to nucleic acid molecules encoding such antibodies and antigen-binding portions, methods of making human anti-ALK-1 antibodies and antigen-binding portions, compositions comprising these antibodies and antigen-binding portions and methods of using the antibodies, antigen-binding portions, and compositions.

Background of the invention ALK-1 is a type I cell surface receptor for transforming growth factor beta receptor type 1 (TGF-beta-1). Human ALK-1 is a 503 amino acid polypeptide, which includes a signal sequence (amino acids: 1-21), a N-terminal extracellular TGF-beta-1 ligand binding domain or ECD (amino acids: 22-118), a single transmembrane domain (amino acids: 119-141) a regulatory glycine/serine rich (GS) domain (amino acids: 142-202) and a C-terminal a serine-threonine kinase domain (202-492). The amino acid sequence of human ALK-1 disclosed in Attisano et al. Cell, 1993, vol. 75, pp. 671-680 includes Ser at position 172 (Genbank record L17075), while U.S. Patent 6,316,217 claims the amino acid sequence of human ALK-1 with Thr at position 172 (Genbank record NM_000020). ACVRL1 gene encoding a full-length human ALK-1 disclosed in Attisano et al. is commercially available from Invitrogen Inc., Clone ID IOH21048. Although ALK-1 shares 60-80% overall homology with other type I receptors (ALK-2 through ALK-7), ECD of ALK-1 Is remarkably divergent from ECDs of other ALK family members. For example, in human, only ECD of ALK-2 is significantly related to ECD of ALK-1 (sharing approximately 25 % amino acid identity). U.S. Patent 6,316,217; ten Dijke et al. Oncogene, 1993, vol. 8, pp. 2879-2887; Attisano et al. Cell, 1993, vol. 75, pp. 671-680.

In general, TGF-beta superfamily ligands exert their biological activities via binding to heteromeric receptor complexes of two types (I and II) of serine/threonine kinases. Type II receptors are constitutively active kinases that phosphorylate type I receptor upon ligand binding. In turn, activated type I kinases phosphorylate downstream signaling molecules including the various Smads, which translocate to the nucleus and lead to a transcriptional response. Heldin et al. Nature, 1997, vol. 390, pp. 465-471. In the case of ALK-1, we have shown that Smadl is specifically phosphorylated and translocates to the nucleus where it directly regulates the expression of the Smadl responsive genes Id1 and EphB2. ALK-1 is expressed highly and selectively in endothelial cells and other highly vascularized tissues such as placenta or brain. We have shown by Affymetrix profiling and real-time RT-PCR that the expression of ALK-1 in endothelial cells highly exceeds the expression of its co-receptors activin type II and endoglin, its ligand TGF-beta-1 or ALK-5. Mutations in ALK-1 are associated with heredity hemorrhagic telangiectasia (HHT), suggesting a critical role for ALK-1 in the control of blood vessel 189,642/2 -2- devetopment or repair. Abdalla et al. J. Med. Genet., 2003, vol. 40. pp. 494-502; Sadick et al. Hematologica/The Hematology J., 2005, vol. 90, 818-828. Furthermore, two independent studies of ALK-1 knockout mice provide the key in vivo evidence for ALK-1 function during angiogenesis. Oh et al. Proc Natl Acad Sci USA, 2000, vol. 97, pp. 2626-2631; Urness et al. Nature Genetics , 2000, vol. 26, pp. 328- 5 331.

Angiogenesis is the physiological process involving the formation of new blood vessels from preexisting vessels and/or circulating endothelial stem cells. This is a normal process in growth and development, as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state. Hanahan and Folkman, "Patterns and Emerging 10 Mechanisms of the Angiogenic Switch During Tumorigenesis," Cell, 86(3):353-364, 1996; Carmeliet, “Angiogenesis in Health and Disease,” Nature Medicine, 9(6):653-660, 2003; Bergers and Benjamin, “Tumoreigenesis and the Angiogenic Switch,” Nature Reviews, 3:401-410, 2003. In diseases like cancer, the body loses the ability to maintain balanced angiogenesis. New blood vessels feed diseased tissues, destroy normal tissues, and in the case of some cancers,.the new vessels can allow tumor cells to escape 15 into the- circulation and lodge in other organs (tumor metastases). Angiogenesis inhibitors, including monoclonal antibodies (mAbs), are a very promising class of drugs targeted against this abnormal process to block or slow tumor growth.

In addition to a role in solid tumor growth and metastasis, other notable conditions with an angiogenic component are, for example, arthritis, psoriasis, neovascular age-related macular 20 degeneration and diabetic retinopathy. Bonnet et al. "Osteoarthritis, Angiogenesis and Inflammation,” Rheumatology, 2005, vol. 44, pp. 7-16; Creamer et al. “Angiogenesis in psoriasis,” Angiogenesis, 2002, vol. 5, pp. 231-236; Clavel et al. “Recent data on the role for angiogenesis in rheumatoid arthritis,” Joint Bone Spine, 2003, vol. 70, pp. 321-326; Anandarajah et al. “Pathogenesis of psoriatic arthritis," Curr.

Opin. Rheumatol., 2004, vol. 16, pp. 338-343; Ng et al. “Targeting angiogenesis, the underlying disorder 25 in neovascular age-related macular degeneration," Can. J. Ophthalmol., 2005, vol. 40, pp. 352-368; Witmer et al. “Vascular endothelial growth factors and angiogenesis in eye disease,’ Progress in Retinal &

Eye Research, 2003, vol. 22, pp. 1-29; Adamis et ai. “Angiogenesis and ophthalmic disease," Angiogenesis, 1999, vol. 3, pp. 9-14.

Anti-angiogenic therapies are expected to be chronic in nature. Accordingly, targets with highly 30 selective endothelial function, such as ALK-1, are preferred to reduce attrition resulting from side effects. Furthermore, given the remarkable divergence of the ALK-1 ECD from ECDs of other ALK family members, mAb raised against the human ALK-1 ECD are expected to selectively target ALK-1. Based on these considerations, a monoclonal antibody against the ALK-1 extracellular domain that may inhibit dimerization with the type li receptor and therefore block Smadl phosphorylation and the downstream 35 transcriptional response is highly desirable. R&D Systems, Inc. makes and sells a monoclonal anti-human ALK-1 antibody (Cat. # MAB370) produced from a hybridoma resulting from the fusion of mouse myeloma with B cells obtained from a mouse immunized with purified NSO-derived recombinant human ALK-1 extracellular domain (R&D Systems: “Monoclonal anti-human ALK-1 Antibody,” (downloaded from www.rndsystems.com/pdf/mab370.pdf) May 20, 2007). We have shown that this antibody neither neutralizes the interaction between ALK-1 and TGF-beta-1 nor abrogates Smadl phosporylation. Fernandez-L et al., Cardiovascular Research, 68:235-248 (2005) mentions the R&D Systems anti-ALK-1 189,642/1 -2/A- antibody (Cat. # MAB370) and a Santa Cruz Biotechnology, Inc. rabbit polyclonal antibody (Cat. # sc-402) raised against a peptide mapping within a C-terminal cytoplasmic domain of human TGF-beta receptor type 1. Neither of these publications discloses the antibodies of the present application. Rabbit antisera have been generated against a synthetic peptide corresponding to a part of the intracellular juxtamembrane region of ALK-1 (amino acid residues 145-166), coupled to key-hole limpet haemocyanin (KLH) (U.S. Patent 6,692,925) and against the entire ALK-1 extracellular domain except for the leading sequence (Lux et al., J. Biol. Chem., 1999, vol. 274, pp. 9984-9992). Abdalla et al (Human Mol. Gen., 2000, vol. 9, pp.1227-1237) report generation of a polyclonal antibody to ALK-1 using a recombinant vaccinia virus construct. R&D Systems, Inc. makes and sells a polyclonal anti-human ALK-1 antibody (Cat. # AF370) produced in goats immunized with purified, NSO-derived, recombinant human ALK-1 extracellular domain. 189,642/3 -3-

To date, no fully human monoclonal antibodies the ECD of ALK-1 have been reported, and no-one has demonstrated the efficacy of any monoclonal antibody to the ECD of ALK-1 in abrogating the ALK-1/TGF-beta-1/Smad1 signaling pathway.

Summary of the Invention

This invention provides a neutralizing monoclonal antibody or an antigen-binding portion thereof that binds ALK-1, wherein said antibody comprises the CDR1, CDR2, and CDR3 of the heavy and light chain variable domains SEQ ID NOs: 6 and 8, respectively; SEQ ID NOs: 14 and 16, respectively; SEQ ID NOs: 18 and 20, respectively; SEQ ID NOs: 26 and 28, respectively; SEQ ID NOs: 30 and 32, respectively; SEQ ID NOs: 38 and 40, respectively; SEQ ID NOs: 46 and 48, respectively; SEQ ID NOs: 50 and 52, respectively; SEQ ID NOs: 54 and 56, respectively; SEQ ID NOs: 58 and 60, respectively; SEQ ID NOs: 62 and 64, respectively; SEQ ID NOs: 66 and 68, respectively; SEQ ID NOs: 70 and 72, respectively; or SEQ ID NOs: 104 and 127, respectively. 189,642/3 4

In some embodiments, the heavy and light chain variable domains ofthe antibody comprise the following sequences: SEQ ID NOs: 6 and 8, respectively; EQ ID NOs: 14 and 16, respectively; SEQ ID NOs: 18 and 20, respectively; SEQ ID NOs: 26 and 28, respectively; SEQ ID NOs: 30 and 32, respectively; SEQ ID NOs: 38 and 40, respectively; SEQ ID NOs: 46 and 48, respectively; SEQ ID NOs: 50 and 52, respectively; SEQ ID NOs: 54 and 56, respectively; SEQ ID NOs: 58 and 60, respectively; SEQ ID NOs: 62 and 64, respectively; SEQ ID NOs: 66 and 68, respectively; SEQ ID NOs: 70 and 72, respectively; SEQ ID NOs: 104 and 127, respectively; or the Vh amino acid sequence encoded by the nucleotide sequence of the insert found in the E. coli clone deposited under ATCC accession number PTA-6864 and the VL amino acid sequence encoded by the nucleotide sequence of the insert found in the E. coli clone deposited under ATCC accession number PTA-6865, respectively. 189,642/3 5

In some embodiments, the antibody that binds ALK-1, comprising a heavy chain amino acid sequence of SEQ ID NO: 2 and a light chain amino acid sequence of SEQ ID NO: 4.

In some embodiments, the antibody or antigen-binding portion according to claim 2, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence of SEQ ID NO: 102.

In some embodiments, the antibody or antigen-binding portion as herein described, comprising a heavy chain variable domain of SEQ ID NO: 6 and a light chain variable domain of SEQ ID NO: 8.

In some embodiments, the antibody is an lgG1 or lgG2 molecule. 189,642/2

In some embodiments, the VH domain that is at least 90% identical in amino acid sequence to any one of SEQ ID NOs: 6; 14; 18; 26; 30; 38; 46; 50; 54; 58; 62; 66; 70 and 104.

In some embodiments, the VL domain that is at least 90% identical in amino acid sequence to any one of SEQ ID NOs: 8; 16; 20; 28; 32; 40; 48; 52; 56; 60; 64; 68; 72 and 127. 189,642/3 7

In some embodiments, the wherein the antibody comprises the CDR1, CDR2, and CDR3 of the heavy and light chain variable domains, respectively, of SEQ ID NOs: 6 and 8.

In some embodiments, the antibody is selected from the group consisting of:

a) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 6, or differs from SEQ ID NO: 6 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 8, or differs from SEQ ID NO: 8 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

b) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 14, or differs from SEQ ID NO: 14 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 16, or differs from SEQ ID NO: 16 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

c) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 18, or differs from SEQ ID NO: 18 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 20, or differs from SEQ ID NO: 20 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

d) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 26, or differs from SEQ ID NO: 26 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 28, or differs from SEQ ID NO: 28 by at least one conservative amino acid 189,642/3 8 substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

e) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 30, or differs from SEQ ID NO: 30 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 32, or differs from SEQ ID NO: 32 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

f) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 38, or differs from SEQ ID NO: 38 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at ieast 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 40, or differs from SEQ ID NO: 40 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

g) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 46, or differs from SEQ ID NO: 46 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 48, or differs from SEQ ID NO: 48 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

h) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 50, or differs from SEQ ID NO: 50 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 52, or differs from SEQ ID NO: 52 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

i) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 54, or differs from SEQ ID NO: 54 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% 189,642/3 9

j) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 58, or differs from SEQ ID NO: 58 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 60, or differs from SEQ ID NO: 60 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

k) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 62, or differs from SEQ ID NO: 62 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 64, or differs from SEQ ID NO: 64 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

l) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 66, or differs from SEQ ID NO: 66 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 68, or differs from SEQ ID NO: 68 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence; m) an antibody or antigen-binding portion thereof that comprises a VH domain as set forth in SEQ ID NO: 70, or differs from SEQ ID NO: 70 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 72, or differs from SEQ ID NO: 72 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence; 189,642/3 10 n) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 104, or differs from SEQ ID NO: 104 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 127, or differs from SEQ ID NO: 127 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence;

o) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 6, or differs from SEQ ID NO: 6 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 127, or differs from SEQ ID NO: 127 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence; and

p) an antibody or portion thereof that comprises a VH domain as set forth in SEQ ID NO: 104, or differs from SEQ ID NO: 104 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence, and a VL domain as set forth in SEQ ID NO: 8, or differs from SEQ ID NO: 8 by at least one conservative amino acid substitution, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence. 189,642/3 11

In some embodiments, the antibody or antigen-binding portion comprises a heavy chain that utilizes a human VH 4-31, VH 3-11, VH 4-61 or VH 4-59 gene, or wherein at least one conservative amino acid substitution occurs in the human VH 4-31, VH 3-11, VH 4-61 or VH 4-59 gene, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence.

In some embodiments, the antibody or antigen-binding portion comprises a light chain that utilizes a human VK A27, VK A2, VK B3, VK B2, or VK L2 gene, or wherein at least one conservative amino acid substitution occurs in the human VK A27, VK A2, VK B3, VK B2, or VK L2 gene, wherein said sequence with the at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence.

In some embodiments, this invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain or an antigen binding portion thereof, or the light chain or an antigen binding portion thereof, or both, of the monoclonal antibody as herein described. 189,642/2 12

Brief Description of the Drawings

Figure 1 shows an example of epitope binding data. The 1.12.1(M291/D19A) antibody was injected for 10 minutes followed by a second 10 minute injection of the 1.12.1 (M29I/D19A) antibody. This defines the maximum response for a 20 minute injection of that antibody. The 20 minute injection maximum response was similarly determined for the 1.27,1 antibody. The 1.12.1(M29I/D19A) antibody was injected for 10 minutes followed by a 10 minute Injection of the 1.27.1 antibody. If the total response falls between the defined maximum responses then the two antibodies must bind to the same epitope. If 1 -13- the total response exceeds the highest maximum response then the antibodies must bind to different epitopes. The experiment was repeated with the order of injections reversed as described in Example 9.

Figure 2 shows sequence alignment of human and Cyno ALK-1 proteins.

Figure 3 shows Ko determination of the recombinant 1.12.1 antibody binding to cell surface ALK-5 1. (a) Human, (b) Cyno. 1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressed recombinant mAb. 1.12.1(M29I/D19A) refers to the mAb 1,12.1 variant that was expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced with isoieucine and aspartic acid at position 19 in the light chain replaced with alanine). 10 1.12.1 (M29I) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific singte amino acid mutation where the methionine at position 29 in the heavy chain was replaced with isoleucine. 1.12.1 (D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the aspartic acid at position 19 in the light chain was replaced 15 with alanine.

Figure 4 shows examples of ID1 titrations using ID1 Taqman Assay for the 1.12.1 antibody variants. 1.12.1 refers to the mAb 1.12.1 variant that was Isolated from the hybridoma. 1.12.1 (rWT) refers to the mAb 1.12.1 variant that was expressed recombinant mAb. 20 1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing two specific amino acid mutations (methionine-at· position 29 in the heavy chain replaced with isoleucine and aspartic acid at position 19 In the light chain replaced with alanine). 1.12.1 (M29I) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the methionine at position 29 in theheavy chain was replaced 25 with isoleucine. 1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the aspartic acid at position 19 in the light chain was replaced with alanine.

Figure 5 shows examples of ID1 titrations using ID1 Taqman Assay for the 1.12.1 antibody 30 sequence variants and the Fab derivative. 1.12.1 refers to the mAb 1.12.1 variant that was Isolated from the hybridoma. 1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressed recombinant mAb. 1.12.1 (M29I) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the methionine at position 29 in the heavy chain was replaced 35 with Isoleucine. 1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the aspartic acid at position 19 in the tight chain was replaced with alanine. -14- 1.12.1 (M29I/D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced with isoleucine and aspartic acid at position 1Θ in the light chain replaced with alanine).

Fab 1.12.1(M29I/D19A) refers to the Fab fragment of mAb 1.12.1(M29I/D19A) prepared by 5 digesting 1.12.1(M29I/D19A) lgG1 using papain.

Figure 6 shows ALK-1 internalization, (a) Monitor neutralizing antibody remaining on cell surface, (b) Monitor remaining cell surface receptor ALK-1.

Figure 7A shows alignment of variable domain sequences for anti-ALK-1 antibodies of the invention, to germline sequences. Mutations compared to germline are in bold. CDR sequences are 10 underlined. Figure 7B shows alignment of the predicted amino acid sequences of light chain variable domains for anti-ALK-1 antibodies 1.12.1, 1.14.1, 1.162.1, 1.31.1, 4.62.1 and 4,72.1 to the human germline A27 Vk sequence. Figures 7C and 7D show alignment ofthe predicted amino acid sequences of heavy tight chain variable domains for anti-ALK-1 antibodies 1.12.1, 1.151.1, 1.162.1, 1.8.1, 4.24.1, 4.38.1, 4.58.1, 4.62.1, 4.68.1, 4.72.1, 5.13.1 and 5.34.1 tothe human germline4-31 VHsequence. 15 Figure 8 shows an example of the histological (Η & E Staining) analysis of a section of the engrafted human skin post surgery.

Figure 9 (A) shows the trichrome staining of collagen In a human skin chimera mouse.

Figure 9 (B) shows detecting human vessels in the collagen gel implanted in a human foreskin chimera mouse. Tex-red: human vessels. FITC: mouse vessels. Yellow: co-stalning. 20 Figure 10 shows an immunofluorescent image of human (red) and mouse (green) vessels of the M24met tumor in the human foreskin SCID chimera mouse.

Figure 11 shows the IHC Image of human vessels (brown) of the M24met tumor tn the human foreskin SCID chimera mouse.

Figure 12 shows the representative immunofluorescent images of human (red) and mouse 25 (green) vessels of the control and the 1.12.1(M29I/D19A) antibody treated (10 mg/kg) M24met tumors in the human foreskin SCID chimera mouse.

Figure 13 shows dose-dependent inhibition of human tumor vessel growth by the 1.12.1(M29I/D19A) antibody in the human foreskin SCID chimera mouse model.

Figure 14 shows the SCID mouse plasma concentration of the 1.12.1(M291/D19A) antibody. 30 Figure 15 shows the estimated ECm for the 1.12,1 (M29I/D19A) antibody in the M24met foreskin SClD-chimera model. The control value at 100% was given an artificial serum concentration of 0.1 nM for graphing purposes. This does not alter the apparent EC50.

Detailed Description of the Invention 35 Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used In connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art Further, unless otherwise required by context, singular terms shall include pluralities and plural terms -15- shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to 5 conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook J. & Russell D.. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols In Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. 10 (2002); Harlow and Lane Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wley, John & Sons, Inc. (2003), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and 15 techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

The following terms, unless otherwise Indicated, shall be understood to have the following meanings:

As used herein, the term “ALK-1” refers to mammalian activin receptor-like kinase-1. The term 20 ALK-1 is intended to include recombinant ALK-1 and recombinant chimeric forms of ALK-1, which can be prepared by standard recombinant expression methods. '

As used herein, the acronym "mAh" refers to a monoclonal antibody.

As used herein, an antibody that is referred to by number is a monoclonal antibody (mAb) that is obtained from the hybridoma of the same number. For example, monoclonal antibody 1,12.1 is obtained 25 from hybridoma 1.12:1. 1.12.1 refers to the mAb 1.12.1 variant that was isolated from the hybridoma. 1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressed recombinant mAb. 1.12.1(M29I/ D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced with 30 isoleucine and aspartic acid at position 19 in the light chain replaced with alanine). 1.12.1 (M291) refers to the mAb 1:12,1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the methionine at position 29 in the heavy chain was replaced with isoleucine. 1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a 35 specific single amino acid mutation where the aspartic acid at position 19 in the light chain was replaced with alanine.

As used herein, "abnormal cell growth", unless otherwise indicated, refers to cell growth that is independent of norma! regulatory mechanisms (e.g., loss of contact inhibition). -16 ·

As used herein, the term "adjacent" is used to refer to nucleotide sequences which are directly attached to one another, having no intervening nucleotides. By way of example, the pentanucleotide 5'-AAAAA-3' is adjacent to the trinucleotide 5'-TTT-3' when the two are connected thus: 5'-AAAAATTT-3' oi 5-TTTAAAAA-3', but not when the two are connected thus: 5’-AAAAACTTT-3'. 5 The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, to "alleviate" a disease, disorder or condition means reducing the severity of the symptoms of the disease, disorder, or condition. This includes, but is not limited to, affecting the size, growth and/or mass of a tumor, the extent or progression of metastasis, and the like, in a patienl 10 compared with these same parameters In the patient prior to or in the absence of the method of treatment.

As used herein, the acronym Mld1" refers to a specific downstream target gene of ALK-1, the Id1 gene, which is important for angiogenesis. The Id1 gene has been reported to control the angiogenesis pathway in certain cancers by turning off the production of a protein, thrombospondin-1 (TSP-1), a naturaliy occurring angiogenesis suppressor. For example, it has been reported that the Id 1 gene, which 15 is highly expressed in melanoma, breast, head and neck, brain, cervical, prostate, pancreatic and testicular cancers, results in decreased expression of TSP-1 and increased tumor blood vessel formation. Volpert, Olga V. et al, "Id1 regulates angiogenesis through transcriptional repression of thrombospondin-1," Cancer Cell, Dec. 2002, Vol. 2, pp. 473-483.

As used herein, the term "Smad" refers to Smad domain proteins found in a range of species from 20 nematodes to humans. These highly conserved proteins contain an N-terminal MH1 domain that contacts DNA, and is separated by a short tinker region from the C-terminal MH2 domain, the latter showing a striking similarity to fbrkhead-assoclated (FHA) domains. FHA and Smad (MH2) domains share a common structure consisting of a sandwich of eleven beta strands in two sheets with Greek key topology. Smad proteins mediate signalling by the TGF-beta/activin/BMP-2/4 cytokines from receptor. Ser/Thr 25 protein kinases at the cell surface to the nucleus. Smad proteins fall into three functional classes: the receptor-regulated Smads (R-Smads), including Smadl, -2, -3, -5, and -8, each of which is involved in a ligand-specific signalling pathway; the comediator Smads (co-Smads), including Smad4, which interact with R-Smads to participate in signalling; and the inhibitory Smads (l-Smads), including Smad-6 and -7, which block the activation of R-Smads and Co-Smads, thereby negatively regulating signalling pathways, 30 As used herein, the term “TGF-beta" refers to the transforming growth factors-beta, which constitutes a . family of multi-functional cytokines (TGF-beta 1-5) that regulate cell growth and differentiation. Transforming growth factor (TGF) Is one of many characterized growth factors that exist In nature. It plays crucial roles in “SCID" mice with severe combined Immunodeficiency. Many cells synthesize TGF-beta, and essentially all have specific receptors for this peptide. TGF-beta regulates the 35 actions of many other peptide growth factors and determines a positive or negative direction of their effects. TGF-beta is a tumor suppressing cytokine with growth inhibitory effects in epithelial cells. TGF-β may also act as a tumor promoter by eliciting an eplthelial-to-mesenchymal transition. TGF-β inactivates several proteins Involved in cell cycle progression and thereby exerts its growth-inhibitory effects on epithelial cells by causing them to arrest in the G1 phase of the cell cycle. The protein functions as a -17- disulphide-linked homodimer. Its sequence is characterised by the presence of several C-terminal cysteine residues, which form interlocking disulphide links arranged in a knot-like topology. A similar "cystine-knot" arrangement has been noted in the structures of some enzyme inhibitors and neurotoxins . that bind to voltage-gated Ca2+ channels, although the precise topology differs. TGF-beta genes are 5 expressed differentially, suggesting that the various TGF- beta species may have distinct physiological roles in vivo.

As used herein, the term "TGF-beta Γ refers to transforming growth factor beta receptor type 1, which Is a peptide of 112 amino acid residues derived by proteolytic cleavage from the C-terminal of a precursor protein. Examination of TGF-beta 1 mRNA levels in adult murine tissues indicates that 10 expression is predominant in spleen, lung and placenta. TGF-beta 1 is believed to play important roles in pathologic processes.

As used herein, the term “SCID" refers to mice with severe combined immunodeficiency.

As used herein, the term “HUVEC" refers to human umbilical vein endothelial cells.

As used herein, "amino acids" are represented by the full name thereof, by the three letter code 15 corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code

Aspartic Acid Asp D Glutamic Acid Gtu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine - Tyr- Y Cysteine Cys C Asparagine Asn N Glutamine Gin Q Serine Ser S Threonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine - Leu L Isoleucine lie I Methionine Met M Proline . Pro P Phenylalanine Phe.. F Tryptophan Trp W

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See lmmunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. -18 A "conservative amino acid substitution" is one in which an amino acid residue is substituted bj another amino acid residue having a side chain R group) with simitar chemical properties (e.g., charge o hydrophobicity). In general, a conservative amino acid substitution will not substantially change tht functional properties of a protein. In cases where two or more amino acid sequences differ from eaci 5 other by conservative substitutions, the percent sequence identity or degree of similarity may be adjuster 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, Methods Mot. Biol. 243:307-31 (1994).

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: 10 . 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; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucfne, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. .1.5 Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et ai., Science 256:1443-45 (1992), herein incorporated by . reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

In certain embodiments, amino acid substitutions to an anti-ALK-1 antibody or antigen-binding 20 portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation,· (3) alter binding affinity for forming protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogs, but still retain specific binding to ALK-1. Analogs can include various substitutions to the normally-occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made 25 in the normally-occurring sequence, for example In the portion of the polypeptide outside the domaln(s) forming intermolecular contacts. Amino acid substitutions can also be made in the domain(s) that form intermolecular contacts that can improve the activity of the polypeptide. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not .alter the anti-parallel β-sheet that makes up the Immunoglobulin 30 binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that . characterizes the parent sequence; in general, glycine and proline would not be used in an anti-parallel β-sheet Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, 35 N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), incorporated herein by reference.

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 -19- 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, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). Another preferred algorithm when comparing a sequence ofthe 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., J. Mol. Biol. 215:403-410 (1990); Altschul et al.r Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable.to compare amino acid sequences. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced naturally-occurring amino acid sequence and which has at least one of the properties of the naturally-occuring polypeoptide. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-iength naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling.

Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: —CH2NH—, —CH2S—, —CH?—CHr-, —CH=CH— (cis and trans), —COCH2—, —CH(OH)CHz—, and CH2 SO—, by methods welt known in the art Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-tysine in place of L-lysine) may 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 and Gierasch Ann. Rev. Biochem. 61:387 '-20- (1992), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

An intact "antibody" or "immunoglobuiin" (Ig) comprises at least two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) inter-connected by disulfide bonds. There are only two '5 types of light chain: λ and k. In humans they are similar, but only one type is present in each antibody. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes), in ,a preferred embodiment, the antibody is an IgG and is an lgG1, lgG2, lgG3 or lgG4 subtype. In a more 10 preferred embodiment, the anti-ALK-1 antibody is subclass lgG2.

Each heavy chain is comprised of a heavy chain variable domain (Vh) and a heavy chain constant region (Ch). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable domain (Vt) and a light chain constant region. The light chain constant region is comprised of one domain, CL, Within light and heavy chains, the variable and 15 constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. The VH and VL regions can be further subdivided Into regions of hypervariabillty, termed "complementarity determining regions" (CDR), interspersed with regions that are more conserved, termed "framework regions" (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order FR1, .20 CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain Es in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), or Chothia & Lesk, J. Moi. Biol. 196:901^917 (1987); Chothia et al., Nature 342:878-883 (1989).

The variable domains of each heavy/llght chain pair (VH and VL) form the antibody binding site 25 that interacts with an antigen. Thus, an intact IgG antibody, for example, has two binding sites. Except in bifunctional or bispeclfic antibodies, the two binding sites are the same. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, Including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. 30 Antibodies must· have enough antigen-binding diversity to recognize every possible pathogen .. (many V regions) while maintaining the biological effectiveness of. their C regions (few C regions), lg genes are randomly spliced together from gene segments that allow many V regions to be used with a few C regions. Gene segments encoding Ig H, kappa and lambda chains are found on three different chromosomes. During B cell development, recombinase enzymes remove introns and some exons from 35 the DNA and splice segments into functional Ig genes.

Ig gene segments in mammals are arranged in groups of "variable" (V), "diversity" (D), "joining"

(J), and "constant" (C) exons. V kappa (Vk) segments each encode the first two CDR and three FR of the .kappa chain V region, plus a few residues of CDR3; J kappa (Jk) segments each encode the remainder of CDR3 and the fourth FR. C kappa (Ck) encodes the complete C region of the kappa light chain. DNA -21-

encoding human kappa chain includes approximately 40 functional V kappa (Vk) segments, five J kappa (Jk) segments, and one C kappa (Ck) gene segment, as well as some gene segments which contain stop codons (“pseudogenes"). Human lambda (λ) chain DNA contains approximately 30 functional V lambda (VA) segments and four functional sets of J lambda (JA) and C lambda (CA) segments. A particular J

5 lambda (JA) always pairs with Its corresponding C lambda (CA), unlike J kappa (Jk) which all pair with the same C kappa (Ck). DNA for human H chain includes approximately 50 functional VH segments, 30 DH segments, and six JH segments. The first, two CDR and three FR of the heavy chain variable domain are encoded by VH. CDR3 is encoded by a few nucleotides of VHl all of DH, and part of JHt while FR4 is encoded by the remainder of the JH gene segment. There are also individual gene segments in the DNA 10 for each heavy chain domain and membrane region of each isotype, arranged in the order in which they are expressed by B cells.

The term “polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence, A polypeptide may be monomeric or polymeric.

The term “isolated protein", “isolated polypeptide" or “isolated antibody" is a protein, polypeptide 15 or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native .state, (2) is free of other proteins from the, same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized In a cellular system, different from the cell from which it naturally •originates will be “isolated” from its naturaliy associated components. A protein may also be rendered .20 substantially free, of naturally associated components by isolation, using protein purification techniques well known in the art

Examples of isolated antibodies include, but not limited to, an anti-ALK-1 antibody that has been affinity purified using ALK-1, and an anti-ALK-1 antibody that has been synthesized by a cell line in vitro. . A protein, or polypeptide is “substantially pure," “substantially homogeneous," or "substantially 25 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 can typically comprise about 50%, 60%, 70%, 80% or 90% w/w of a protein sample, more usually about 95%, and preferably can be 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 30 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 of· purification.

The term “polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining, amino acid sequence is . 35 identical, to the corresponding positions in the naturally-occurring .sequence.. in some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80,90,100,150 or 200 amino acids long.

The term "analog" or "polypeptide analogies used herein refers to a polypeptide that comprises a segment that has substantial identity to some reference amino acid sequence and has substantially the

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-22 same function or activity as the reference amino acid sequence. Typically, polypeptide analogs comprise a conservative amino acid substitution (or insertion or deletion) with respect to the reference sequence. Analogs can be at least 20 or 25 amino acids long, or can be at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as the full-length polypeptide, Some embodiments 5 of the invention include polypeptide fragments or polypeptide analog antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from the germline amino acid sequence. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art following the teachings of this specification.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, 10 refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e:g., ALK-1 or ECD of ALK-1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl and Cr1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab 15 fragments linked by a disulfide bridge at the hinge region; (Hi) a Fd fragment consisting of the VH and CK1 domains; (iv) a Fvfragment consisting of the Vl and VH domains of a single arm of an antibody, (v) a dAb .fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vl) an isolated complementarity determining region (CDR). Furthermore, although the two domains ofthe Fv fragment, Vl and VHl are coded for by separate genes, they can be Joined, using recombinant methods, by a 20 synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair •to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, 25 bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al. Structure 2:1121-1123 (1994)). 30 Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion molecules, formed by covalent or noncovaient association of the antibody or antibody portion with one or ·. more other proteins or peptides. Examples of such immunoadhesion molecules Include use of the streptavidin core.region to.make a tetrameric scFv molecule (Kipriyanov et al; Human Antibodies and Hybridomas 6:93-101 (1995)) and use . of a cysteine residue, a marker peptide and a C-terminal 35 polyhistidine tag. to make bivalent and biotinylated scFv molecules (Kipriyanov et al. Moi. Immunol. 31:1047-1058 (1994)). Other'examples include where one or more CDRs from, an antibody are incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that .specifically binds ,to an antigen of interest, such as ALK-1 or ECD of ALK-1. in such embodiments, the -23- CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain; or may be incorporated noncovalently.

Antibody portions, such as Fab and F(ab')2· fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. 5 Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

As used herein, the term "human antibody" means any antibody in which the variable and constant domain sequences are human sequences. The term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g. to decrease possible immunogenicity, 10 increase affinity, eliminate cysteines that might cause undesirable folding, etc.· The term also encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells. These antibodies may be prepared in a variety of ways, as described below.

As used herein, the term "neutralizing antibody," "an Inhibitory antibody" or antagonist antibody 15 means an antibody that inhibits the ALK-1/TGF-beta-1/Smad1 signaling pathway, in a preferred embodiment, the antibody inhibits the ALK-1/TGF-beta-1/Smadl signaling pathway by at least about 20%, preferably 40%, more preferably 60%, even more preferably 80%, or even more preferably 85%. Neutralizing or inhibiting potential of human anti-ALK-1 antibodies may be determined, for example, by their ability to inhibit up-regulation of a specific downstream target gene of ALK-1, Id1, as presented in 20 Example 12; to inhibit Smadl phosphorylation determined by Western Blotting using Odyssey Infrared Imaging System from LI-COR Biosciences as presented in Example 13.

The term "chimeric antibody" as used herein means an antibody that comprises regions from two or more different antibodies. For example, one or more of the CDRs of a chimeric antibody can be derived from a human anti-ALK-1 antibody. In another example, all of the CDRs can be derived from 25 human antl-ALK-1 antibodies. In another example, the CDRs from more than one human anti-ALK-1 antibody can be combined In a chimeric antibody. For.instance, a chimeric antibody may comprise a CDR1 from the light chain of a first human antl-ALK-1 antibody, a CDR2 from the light chain of a second human anti-ALK-1 antibody and a CDR3 from the light chain of a third human antl-ALK-1 antibody, and CDRs from the heavy chain may be derived from one or more other anti-ALK-1 antibodies. Further, the 30 framework regions may be derived from one of the anti-ALK-1 antibodies from which one or more of the CDRs are taken or from one or more different human antibodies. Moreover, as discussed previously herein, chimeric antibody includes an antibody comprising a portion derived from the germiine sequences of more than one species.

In some embodiments, a chimeric antibody of the invention is a humanized anti-ALK-1 antibody. . 35 A humanized antkALK-1 antibody of- the invention comprises the amino acid sequence of one or more framework regions and/or the amino acid sequence from at least a portion of the constant region of one or more human anti-ALK-1 antibodies of the invention and further comprises sequences derived from a non-. human anti-ALK-1 antibody, for example CDR sequences. -24

As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay. This assa) is well known to those of skill in the art. Examples of this assay can be found in Vaughan, T.J. et ai.

Nature Biotech. 14:309-314 (1996), as well as in Example 2 of the present application.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon tha*· 5 allows for the analysis of real-time biospecific interactions by detection of alterations in proteir concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacie Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al., Ann. Biot. Clin. 51:19-26 (1993); Jonsson etal., Biotechniques 11:620-627 (1991); Jonsson etal., J. Moi.

Recognit. 8:125-131 (1995); and Johnsson etal., Anal. Biochem. 198:268-277 (1991). 10 The term "affinity" refers to a measure of the attraction between an antigen and an antibody. The intrinsic attractiveness of the antibody for the antigen is typically expressed as the binding affinity equilibrium constant (Kd) of a particular antibody-antigen interaction. An antibody is said to specifically bind an antigen when the KD Is s 1 mM, preferably s 100 nM. A Kd binding affinity constant can be measured by surface plasmon resonance, for example using the BIACORE™ system as discussed in 15 Examples 7 and 8.

The term “koff" refers to the dissociation rate constant of a particular antibody-antigen interaction. A kOff dissociation rate constant can be measured by surface plasmon resonance, for example using the BlAcore system as discussed in Examples 7 and 8.

The term “avidity" refers to the functional combining strength of an antibody with its antigen which 20 . is based on both affinity, and valences of the antibody. As used herein, this term describes the increased affinity that occurs as result of multiple antigen binding sites on an immunoglobulin.

As used herein, the term “molecular selectivity" refers to the binding affinity of an antibody for a specific antigen being greater than for other antigens. For example; the antibodies of the present invention can be selective for ALK-1 over ALK-2 through ALK-7, meaning that the binding affinity of the 25 antibody for ALK-1 is at feast 2-fold greater, for example 4-fold, or 10-fold, or 50-fold, or 100-fold or more, than for ALK-2 through ALK-7. Such binding affinities can be measured using standard techniques known to those of skill in the art

The term “epitope" Includes any protein determinant capable · of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epltopic determinants 30 generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." (n a linear epitope, all of the points of interaction between the protein and the Interacting molecule (such as an antibody) occur linearally along the primary amino acid sequence of the protein. In a conformational 35 epitope, the points of interaction occur across amino acid residues on the protein that are separated from . one another. Once a desired epitope on an antigen Is determined, it is possible to generate antibodies to that epitope, e.g,, using the techniques described in the present invention. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding -25- to the same epitope. An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, i.e. the antibodies compete for binding to the antigen. A high throughput process for “binning" antibodies based upon their cross-competition is described in International Patent Application No. WO 03/48731. 5 As used herein, the term "binning” refers to a method to group antibodies based on their antigen binding characteristics. The assignment of bins is somewhat arbitrary, depending on how different are the observed binding patterns for all the antibodies tested. Therefore, bins do not always correlate with epitopes determined.by other means.and should not be used to define epitopes.

The term "compete", as used herein with regard to an antibody, means that a first antibody, or an 10 antigen-binding portion thereof, competes for binding to the antigen with a second antibody, or an antigenbinding portion thereof, where binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, 15 a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, .where each antibody detectably Inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for. binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the 20 present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g:, steric hindrance, conformational change, or binding to a common epitope, or portion thereof, and the like), the skilled artisan would appreciate,, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

The term “polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 25 10 bases in length, either ribonucleotides or deoxy nucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

The term "isolated polynucleotide" as used herein means a polynucleotide, of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of Its origin the "isolated polynucleotide" (1) is not associated with all or a portion of polynucleotides with which the "isolated polynucleotide" is found in 30 nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

The-term “naturally occurring nucleotides'1 as used herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages” referred to herein includes 35 oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, . phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche etal, Nucl. Acids Res. 14:9081 (1986); Stec etal., J. Am. Chem. Soc. 106:6077 (1984); Stein etal., Nucl. Acids Res. 16:3209 (1988); Zon etal., Anti-Cancer Drug Design 6:539 (1991); Zon etal, Oligonucleotides and Analogues: A Practical Approach, pp.87-108 (F. .Eckstein, Ed., Oxford -26-

University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired.

Operably linked" sequences Include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression .control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.. The nature of such control sequences .differs, depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal.binding site, and transcription termination sequence; in eukaryotes,, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, .at a minimum, all. components whose preserice is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. „ .. .

The term "vector", as used herein, means a nucleic acid molecule capable of transporting· another nucleic acid to which it has been linked. In some embodiments, the vector is. a plasmid, i.e., a circular double stranded . piece of DNA into which additional DNA segments may be ligated.. In some embodiments, the. vector Is a viral vector, wherein additional DNA segments may be ligated Into the viral genome.· In some embodiments, the vectors are capable of autonomous replication in a host ceil into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated Into the genome of a.host celt upon Introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of. directing the expression of genes to which. they are operatively linked. Such .vectors are referred to herein as “recombinant expression vectors" (or simply, "expression vectors"). .

The term “recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host ceil" and “host ceil" mean not only the particular subject cell but also the progeny of.such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such, progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.

As used herein, the term “germline". refers to the nucleotide sequences and amino acid sequences of the antibody genes and gene segments as they are passed from parents to offspring via the germ cells. This germline sequence is distinguished from the nucleotide sequences encoding antibodies -27 · in mature B celis which have been altered by recombination and hypermutation events during the course of B cell maturation. An antibody that "utilizes" a particular germline has a nucleotide or amino acid sequence that most closely aligns with that germline nucleotide sequence or with the amino acid sequence that it specifies. Such antibodies frequently are mutated compared with the germline sequence. 5 The term "percent sequence identity" in the context of nucleic acid sequences means the residues in two sequences that 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, 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 10 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, Wisconsin. 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 15 (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson; Methods Mot. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated .. . herein 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 20 using Gap with its default parameters as provided In GCG Version 6.1, incorporated herein by reference. A reference to a nucleotide sequence encompasses its complement unless otherwise specified.

Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with Its complementary sequence.

The term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic 25 acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions wtth another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence Identity, such as FASTA, BLAST or Gap, as discussed above. 30 The term "percent sequence identity" in the context of amino acid sequences means the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about five amino acids, usually at least about 20 amino acids, more usually at least about 30 amino acids, typically at least about 50 amino acids, more typically at least about 100 amino acids, and even more typically about 150, 200 or more amino acids. 35 There are a number of different .algorithms known ip the. art that can be used to measure amino acid sequence identity. For instance, amino acid sequences can be compared using FASTA Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wsconsln. -28-

As applied to polypeptides, the term "substantial identity" or "substantial similarity" means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. 5 in certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

The term "signal sequence," also called signal peptide, leader peptide, refers to a segment of about 15 to .30 amino acids at the N terminus of a protein that enables the protein to be secreted (pass through a cell membrane). The signal sequence is removed as the protein is secreted. 10 As used herein, the terms "label" or "labeled" refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug 15 conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited, to, the following: radioisotopes or radionuclides (e.g., 3H, :14C, 1δΝ, MS, B0Y, MTc, l11ln, 12δΙ, 131i), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, 20 predetermined polypeptide epitopes recognized by a secondary-reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposlde, vincristine’ vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 25 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments^ labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The term "primate" refers to a mammal of the order primates, which Includes the anthropoids and prosimians, characterized by refined development of the hands and feet, a shortened snouts and a large 30 brain. The mammalian order Primates includes humans, apes, monkeys, and prosimians, or lower primates. 'Therapeutically effective amount" refers to that amount of the· therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has 35 at least one of the following effects: reducing the size of the tumor; inhibiting (that is, stowing to some extent, preferably stopping) tumor metastasis; inhibiting to some extent (that is, slowing to some extent, : preferably stopping) tumor growth, and relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. --29- "Treat", "treating" and "treatment" refer to a method of alleviating or abrogating a biological disorder and/or its attendant symptoms. With regard to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced. 5 "Contacting" refers to bringing an antibody or antigen binding portion thereof of the present invention and a target ALK-1, or epitiope thereof, together In such a manner that the antibody can affect the biological activity of the ALK-1. Such "contacting" can be accomplished "/n vitro," i.e., in a test tube, a petri dish, or the like. In a test tube, contacting may involve only an antibody or antigen binding portion thereof and ALK-1 or epitope thereof or It may involve whole cells. Cells may also be maintained or .10 grown in cell culture dishes and contacted with antibodies or antigen binding portions thereof in that environment. In this context, the ability of a particular antibody or antigen binding portion thereof to affect a ALK-1-related disorder, I.e., the IC50 of the antibody, can be determined before use of the antibody in vivo with more complex living organisms is attempted. For cells outside the organism, multiple methods ' exist, and are well-known to those skilled in the art, to contact ALK-1 with the antibodies or antigen- 15 . binding portions thereof.

The acronym "FACS" refers to Fluorescence Activated Cell Sorting. The acronym FACS and flow cytometry are used interchangeably. Fluorescent labeling allows investigation of cell structure and . function. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin. This method is often 20 used to label.molecules on the cel) surface, but antibodies can be directed at targets in cytoplasm, ih direct immunofluorescence an antibody to a molecule is directly conjugated to a fluorescent dye, and cells are stained in one step. In indirect immunofluorescence the primary antibody is not labeled, and a second fluorescently conjugated antibody is added which Is specific for the first antibody.

Antl-ALK-1 Antibodies 25 This invention pertains to isolated neutralizing anti-ALK-1 monoclonal antibodies or antigen binding portions thereof that bind to primate ALK-1, preferably the ECD of primate ALK-1, more preferably the ECD of human ALK-1. In a preferred embodiment, the invention pertains to isolated neutralizing antibodies that are fuily human monoclonal antibodies or antigen-binding portions thereof. Preferably, the human antibodies are recombinant human anti-ALK-1 antibodies that have greater affinity for ALK-1 than 30 for ALK-2 through ALK-7. in some embodiments, human anti-ALK-1 antibodies are produced by immunizing a non-human transgenic animal,' e.g., · a rodent, whose genome comprises human immunoglobulin genes so that the transgenic animal produces human antibodies. Various aspects of the invention relate to such antibodies and antigen-binding portions, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such 35 antibodies and antigen-binding portions. Methods of using the antibodies and antigen-binding portions of the present Invention to abrogate the ALK-1/ TGF-beta-1 /Smadl signaling pathway or to detect ALK-1, . either in vitro or in vivo, are also encompassed by the invention. . -30 10 15 - . 20 25 30

An anti-ALK-1 antibody of the invention can comprise a human kappa or a human lambda ligh chain or an amino acid sequence derived therefrom. In some embodiments comprising a kappa ligh chain, the light chain variable domain (VJ utilizes a human A27, A2, A1, A3, B3, B2, L1 or L2 VKgene. Ii some embodiments, the light chain utilizes a human VK L1 gene and a human JK 4 gene; a human Vk A2‘ gene and a human Jk 5 gene or a human JK 4 gene; a human VK B3 gene and a human JK 1 gene; &lt; human VK L2 gene and a human JK 3 gene; a human VK A2 gene and a human JK 1 gene; a human VK A: gene and a human JK 4 gene; a human VK A1 gene and a human JK 1 gene; a human VK B2 gene and ί human JK 4 gene; or a human Vk A2 gene and a human Jk 1 gene.

In some embodiments, the VL of the anti-ALK-1 antibody comprises one or more amino ack substitutions, deletions or insertions (additions) relative to the germiine VK amino acid sequence. In some embodiments, the VL of the anti-ALK-1 antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11, 12, 13,14 or 1£ amino acid substitutions relative to the. germiine VK amino acid, sequence. In some embodiments, one oi more of the substitutions from germiine is in the CDR regions of the light chain. In some embodiments, the VK amino acid substitutions relative to germiine are at one or more of the same positions as the substitutions relative to germiine found in any one or more of the VL of the antibodies provided herein as shown, for example, at Figure 7. In some embodiments, the amino acid changes are at one or more ol the same positions, but involve a different substitution than in the reference antibody. in some embodiments, amino acid substitutions relative to germiine occur at one or more of the same positions as substitutions from germiine in any of the VL of antibodies 1.11.1; 1.12.1; 1.124(rWT); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1,183.1; 1.274; 1:294; 1.-314; 1.8.1; 1.9.1; 4.10.1; 4.244 ; 4.384; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5:564; 5.57.1; and 5.59.1, but the substitutions may represent conservative amino acid substitutions at such position(s) relative to the amino acid in the reference antibody. For example, if a particular position In one of these antibodies is changed relative to germiine and is glutamate, one may substitute aspartate at that position. Similarly, if an amino acid substitution compared to germiine In an exemplified antibody is serine, one may conservatively substitute threonine for serine at that position. Conservative amino acid substitutions are discussed supra.

In some embodiments, the anti-ALK-1 antibody comprises a light chain amino acid sequence of SEQ ID NO: 4. In other embodiments, the light chain comprises the light chain .amino acid sequence of antibody 1.11.1; 1.12.1; 1424(rWT); 1.12.1(M29I/D19A); 1.124(M29I); 1.124(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183,1; 1.27.1; 1.29.1; ,1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4:38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1.

In some embodiments, the light chain of the human anti-ALK-1 antibody comprises the VL amino acid sequence of antibody 1.12.1 (SEQ ID NO: 8); 1.11.1 (SEQ ID NO: 42); 1.13.1 (SEQ ID NO: 16); 144.1 (SEQ ID NO: 20); 14514 (SEQ ID NO: 24); 14624 (SEQ ID NO: 28); 14834 (SEQ ID NO: 32); 1.84(SEQ ID NO: 36); 1.9.1(SEQ ID NO: 40); 4404 (SEQ ID NO: 44); 4.244 (SEQ ID NO: 48); 4.38,1(SEQ ID NO: 52); 4.584 (SEQ ID NO: 56); 4.624 (SEQ ID NO: 60); 4.684 (SEQ ID NO: 64); 4.724 (SEQ ID NO: 68); 5434 (SEQ ID NO: 72); 5.344 (SEQ ID NO; 76); 5.534 (SEQ ID NO: 80); 5.564 (SEQ ID NO: 84); 5.574 (SEQ ID NO: 88); or 5.594 (SEQ (D NO: 92); or said amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid 35 -31 - substitutions and/or a total of up to 3 non-conservative amino acid substitutions, in other embodiments the light chain of the human anti-ALK-1 antibody comprises the VL amino acid sequence of antibody 1.27.1; 1.29.1 or 1.31.1. In some embodiments, the light chain comprises the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any one of the foregoing antibodies. 5 In some embodiments, the light chain may comprise the amino acid sequences of CDR1, CDR2 and CDR3 regions independently selected from the light chain CDR1, CDR2 and CDR3 regions, respectively, of two or more monoclonal antibodies selected from 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1,12.1 &lt;D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 10 5.57.1; or 5.59.1, or said CDR regions each having less than 3 or less than 2 conservative amino acid substitutions and/or a total of three or fewer non-conservative amino acid substitutions. in certain embodiments, the light chain of the anti-ALK-1 antibody comprises the . amino acid sequences ofthe light chain CDR1, CDR2 and CDR3 regions of an antibody selected from 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1{M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 15 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9,1; 4.10.1; 4.24.1; 4,38.1; 4.58.1; 4.62.1;. 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1, or said CDR regions each having less than 3 or less than 2 conservative amino acid substitutions and/or a total of three or fewer non-conservative amino acid substitutions.

With regard to the heavy chain, in some embodiments, the variable domain (VH) utilizes a human VH 4-31, VH 3-11, Vh 3-15, VH 3-33, VH 4-61 or Vh 4-59 gene, in some embodiments, the VH sequence of

20 the anti-ALK-1 antibody contains one or more amino acid substitutions, deletions or insertions (additions), collectively "mutations", relative to the germline VH amino acid sequence. In some embodiments, the variable domain of the heavy chain comprises 1,2, 3, 4, 5, 6, 7, 8, 9,10 or 11 mutations from the germline Vh amino acid sequence. In some embodiments, the mutation(s) are non-conservative substitutions compared to the germline amino acid sequence. In some embodiments^. the mutations are in the CDR 25 regions of the heavy chain. In some embodiments, the heavy chain utilizes a human VH 3-33 gene, a human D 6-19 gene and a human JH 3B gene; a human VH 4-31 gene, a human D 6-19 gene and a human JH 4B gene; a human VH 4-61 gene, a human D 6-19 gene and a human JH 4B gene; a human VH 4-31 gene, a human D 3-3 gene and a human JH 3B gene; a human VH 4-31 gene and a human JH 3B gene; a human VH 4-59 gene, a human D 6-19 gene and a human JH 4B gene; a human VH 3-11 gene, a

30 human D 3-22 gene and a human JH 6B gene; a human VH 3-15 gene, a human D 3-22 gene and a human JH 4B gene; a human Vh.4-31 gene, a human D 5-12 gene and a.human JH 6B gene; a human VH . . 4-3.1 gene, a human D 4-23 gene, and a human Jh 4B.gene; a human VH 4-31 gene, a human D 2-2 gene and a human JH 5B gene; a human VH 4-31 gene and a human Jh 6B gene; human VH 3-15 gene, a human D 1 -1 gene and a human Jh 4B gene; a human VH 3-11 gene, a human D 6-19 gene and a human 35 Jh 6B gene; a human VH 3-11 gene, a human D 3-10 gene and a human JH 6B gene; or a human Vh 3-11 gene, a human D 6-6 gene and a human JH 6B gene, .

In some embodiments, amino.acid substitutions are at one or more of the same positions as the substitutions from germline in any one or more of the VH of antibodies 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1(M29i/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; -32- 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1. In other embodiments, the amino acid changes are at one or more of the same positions but involve a different substitution than in the reference antibody.

In some embodiments, the heavy chain comprises an amino acid sequence-of SEQ ID NO: 2. In other embodiments, the heavy chain comprises the heavy chain amino acid sequence of antibody 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1 (D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; .1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53,1; 5.56.1; 5.57.1; or 5.59.1. In some embodiments, the heavy chain comprises the VH amino acid sequence of antibody 1.12.1 (SEQ ID NOS: 6); 1.11.1 (SEQ ID NO: 10); 1.13:1 (SEQ ID NO: 14); 10 15 20 1.14.1 (SEQ ID NO: 18); 1.151.1 (SEQ ID NO: 22); 1.162.1 (SEQ ID NO: 26); 1.183.1 (SEQ ID NO: 30); 1.8.1 (SEQ-ID NO: 34); 1.9.1 (SEQ ID NO: 38); 4.10.1 (SEQ ID NO: 42); 4.24.1 (SEQ ID NO: 46); 4.38.1(SEQ ID NO: 50); 4.58.1 (SEQ ID NO: 54); 4.62.1 (SEQ ID NO: 58); 4.68.1 (SEQ ID NO: 62); 4.72.1 (SEQ ID NO: 66); 5.13.1 (SEQ ID'NO: 70); 5.34.1 (SEQ ID NO: 74):-5.53.1 (SEQ ID NO: 78); 5.56.1 (SEQ ID NO: 82); 5.57.1 (SEQ ID NO: 86); or 5.59.1 (SEQ ID NO: 90); or said VH amino acid sequence having up to 1, 2, 3, 4, 6, 8; 9, 10 or 11 conservative amino acid substitutions and/or a total of up to 3 non-conservative amino acid substitutions. In other embodiments, the heavy chain comprises the Vh amino acid sequence of antibody 1.27.1; 1.29.1 or 1.31.1. In some embodiments, the heavy chain comprises the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any one of the foregoing antibodies.

In some embodiments, .the heavy chain comprises the heavy chain CDR1, CDR2 and CDR3 regions of antibody 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 25 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1, or said CDR regions each having less than 8, less than 6, less than 4, or less than 3 conservative amino' acid substitutions and/or a total of three or fewer non-conservative amino acid substitutions.

In some embodiments, the heavy chain CDR regions are independently selected from the CDR regions of two or more antibodies selected .from antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12,1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68,1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 30 5.57.1; or 5.59.1. In another embodiment; the antibody comprises a light chain as disclosed above and a heavy chain as disclosed above. In a further embodiment, the light chain CDRs and the heavy chain CDRs are from the same antibody.

In various embodiments, the anti-ALK-1 antibodies have the full-length heavy chain and full length light chain amino acid sequence(s), the Vh and V l .amino acid sequences, the heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 amino acid sequences or the heavy chain amino acid sequence from the beginning of the CDR1 to the end of the CDR3 and the light chain amino acid sequence from the beginning of the CDR1 to the end of the CDR3 ofan anti-ALK-1 antibody provided herein. 35 -33- 10 15 . 20 25

One type of amino acid substitution that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. In one embodiment, there is a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonical.

Another type of amino acid substitution that may be made is to remove potential proteolytic sites in the antibody. Such sites may. occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by altering one or both of the residues.

In some embodiments, the C-terminal lysine of the heavy chain of the anti ALK-1 antibody of the invention is cleaved. In various embodiments of the invention, the heavy and light chains of the anti-ALK-1 antibodies may optionally include a signal sequence.

In one aspect, the invention provides twenty five inhibitory human anti-ALK-1 monoclonal antibodies and the hybridoma celt lines that produce them, in certain embodiments, antibodies of the present invention are IgGs designated as: 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29t/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162,1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1, In preferred embodiments, the human anti-ALK-1 antibody is antibody 1.12.1, 1.12.1(M29I/D19A), 1.12.1(M29I), 1.12.1(D19A), 1.27.1, 1.14.1, 1.162.1,1:31.1,4.62.1 or 4.72.1.

Antibodies recognize surface-exposed epitopes on antigens as regions of linear (primary) sequence or structural (secondary) sequence. BIAcore was used in order to define the functional epitope landscape and determine the epitope exclusivity of the anti-ALK-1 antibodies exemplified by this invention.

Table .1 lists the sequence identifiers (SEQ ID NO) of the nucleic acids encoding the full-length heavy and light chains of 1.12.1 antibody variants and variable domain-containing.portions of antl-ALK-1 antibodies of the invention, and the corresponding deduced amino acid sequences. -34·

Table 1 SEQUENCE IDENTIFIERS (SEQ ID NO)

Antibody FULL LENGTH V DOMAIN CONTAINING PORTION Heavy Light Heavy Light DNA PROTEIN DNA PROTEIN DNA . PROTEIN DNA . PROTEIN 1.11.1 9 10 11 12 1.12.1(M29I/D10A) 1 2. 3 4 5 6 7 8 1.12.1 95 100 101 102 103 104 126 127 1.12.1(rWT) 128 100 101 102 129 104 126 127 1.13.1 13 14 15 16 1.14.1 17 18 19 20 1.151.1 21 22 23 24 1.162.1 25 26 27 28 1.183.1 29 30 31 32 1.8.1 33 34 35 ' 36 1.9.1 37 38 39 40 4.10.1 41 42 43 44 4.24.1 45 46 47 48 4.38.1 49 50 51 52 4.58.1 53 54 55 56 4.62.1 57 58 59 60 4.68.1 61 62 63 . 64 4.72,1 65 66 67 68 5.13.1 6? 70 71 72 5.34.1 73 74 75 78 5.53.1 . . . 77 78 79 80 5.56.1 81 82 83 €4 5.57.1 85 86 87 88 5.59.1 89 90 91 92 1.12.1(M291/D19A) refers to the anti-ALK-1 antibody containing a specific single amino acid 5 mutation In the heavy chain where the methionine at position 29 was replaced with isoleucine and a specific single amino acid mutation in the light chain where the aspartic acid at position 19 was replaced with alanine as described in Example 4. 1.12.1 refers to the mAb 1.12.1 variant that was isolated from the hybridoma. 1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressed as a recombinant mAb 10 described in Example 3. -35-

The invention further provides heavy and/or fight chain variants of certain of the above-listed human anti-ALK-1 antibodies, comprising one or more amino acid modifications. To designate the variants, the first letter is the one letter symbol for the amino acid of the naturally-occurring antibody chain, the number refers to the position of the amino acid (wherein position one is the N-terminal amino acid of 5 the FR1), and the second letter is the one letter symbol for the variant amino acid.

In still further embodiments, the invention includes antibodies comprising variable domain amino acid sequences with more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 97%, more than 98% or more than 99% sequence identity to a variable domain amino acid sequence of any of the above-listed human anti-ALK-1 antibodies. 10 Class and Subclass of Anti-ALK-1 Antibodies

The class and subclass of anti-ALK-1 antibodies may be determined by any method known in the art In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA, Western Blot as well as other techniques. Alternatively, 15 the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

The class of an anti-ALK-1 antibody obtained as described above may be switched with another. 20 In one aspect of the invention, a nucleic acid molecule encoding VL or VH Is isolated using methods well-known in the art such that It does not include nucleic acid sequences encoding CL or CH. “Antibody Engineering" (Kontermann &amp;amp; Dubel, Eds., Springer-Verlag, Berlin .(2001)). The.nucleic acid molecules . encoding VL or VH are then operatively linked to. a nucleic, acid sequence encoding a CL. or Ch, respectively, from a different class of immunoglobulin molecule. This may be achieved using a vector or 25 nucleic acid molecule that comprises a CL or CH chain, as described above. For example, an anti-ALK-1 antibody that was originally IgM may be class switched to an IgG. Further, the class switching may be . used to convert one IgG subclass to another, e.g,, from lgG1 to lgG2. A preferred method for producing an antibody of the invention comprising a desired isotypes comprises the steps of isolating a nucleic acid molecule encoding the heavy chain of an anti-ALK-1 antibody and a nucleic acid molecule encoding the 30 light chain of an anti-ALK-1 antibody, obtaining the variable domain of the heavy chain, ligating the variable domain of the heavy chain with the constant domain of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and collecting the anti-ALK-1 antibody with the desired isotype.

In some embodiments, the anti-ALK-1 antibody is a monoclonal antibody. The anti-ALK-1 35 antibody can be an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In a preferred embodiment, the anti-ALK-1 antibody is an IgG and is an lgG1, lgG2, JgG3, lgG4 subclass. In another preferred embodiment, the antibody Is subclass l’gG2. :36-

Identification of ALK-1 Epitopes Recognized bv Antt- ALK-1 Antibodies

The invention provides a human anti-ALK-1 monoclonal antibody that binds to ALK-1 and competes or cross-competes with and/or binds the same epitope as: (a) an antibody selected from 1.11.1; 1.12.1; 1.12.1((WT); 1.12.1 (M29I/D19A); 1.12.1 (M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 5 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1; (b) an antibody that comprises a heavy chain variable domain having the amino acid sequence of the VH domain in any one of SEQ ID NOS: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104, (c) ah antibody that comprises a light chain variable domain having the amino acid sequence of the Vl domain in any one of SEQ ID NOS: and 10 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 127, (d) an antibody that comprises both a heavy chain variable domain as defined in (b) and a light chain variable domain as defined in (c).

One can determine whether an antibody binds to the same epitope or cross competes for binding with an anti-ALK-1 antibody by using methods known in the art In one embodiment, ond allows the anti- 15 ALK-1 antibody of the invention to bind to ALK-1 under saturating conditions and then measures the ability of the test antibody to bind to ALK-1, If the test antibody is able to bind to ALK-1 at the same time as the reference anti-ALK-1 antibody, then the test antibody binds to'a different epitope than the reference anti-ALK-1 antibody. However, If the test antibody is not able to bind to ALK-1 at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to 20 the epitope bound by the anti-ALK-1 antibody of the invention. This experiment can be performed using ELISA, RIA, BIACORE™, or flow cytometry. To test whether an anti-ALK-1 antibody cross-competes with another anti-ALK-1 antibody, one may use the competition method described above in two directions, i.e. determining if the known antibody blocks the test antibody and vice versa. In a preferred embodiment, the experiment Is performed using BIACORE™. 25 Binding Affinity of Anti-ALK-1 Antibodies to ALK-1

The binding affinity (KD) and dissociation rate (kofr) of an anti-ALK-1 antibody or antigen-binding portion thereof to ALK-1 can be determined by methods known in the art The binding affinity can be measured by ELISAs, RIAs, flow' cytometry, or surface plasmon resonance, such as BIACORE™. The dissociation rate can be measured by surface plasmon resonance. Preferably, the binding affinity and 30 dissociation rate is measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE™. One can determine whether an antibody has substantially the same KD as an anti-ALK-1 antibody by using methods known in the art Such methods, of determining Ko and korr can be used during the initial screening stage, as well as during subsequent optimization stages.

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-37- inhibition of ALK-1 Activity bv Antl-ALK-1 Antibody

Anti-ALK-1 monoclonal antibodies that inhibit ALK-1 binding can be identified using a number of assays. For example, neutralizing anti-ALK-1 antibodies can be identified by their inhibition of upregulation of a specific downstream target gene of ALK-1, Id1, as described in Example 12. Preferred 5 anti-ALK-1 antibodies have an ICgo of no more than 500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 1 nM,

One also can determine the ability of an anti-ALK-1 antibody to inhibit Smadl phosphorylation determined by Western Blotting using Odyssey Infrared Imaging System, as described in Example 13. In various embodiments, the anti-ALK-1 antibody has an IC50 in this .assay of no more than 250 nM, 200 nM, 10 150 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 1nM.

Inhibition of Angiogenesis bv Anti-ALK-1 Antibody

In another embodiment, the anti-ALK-1 antibody or portion thereof inhibits human vessel, angiogenesis as demonstrated In a SCID mouse engrafted with human foreskin tissue, in which human melanoma M24met tumor cells are Intradermally Implanted as determined by IHC analysis of human CD- 15 31 signal assay by a factor of at least 40% as compared to a control sample as as described in Example 17 and shown in Table 13.

In another embodiment, the antl-ALK-1 antibody or portion thereof inhibits human vessel angiogenesis as demonstrated in a SCID mouse engrafted with human foreskin tissue, in which collagen is intradermally implanted as determined by IHC analysis of human CD-31 signal assay by a factor of at 20 least 50% as compared to a control sample as described In Example 16 and shown In Table 12.

Species and Molecular Selectivity

In another aspect of the Invention, the anti-ALK-1 antibodies demonstrate both species and molecular selectivity. Following the teachings of the specification, one may determine the species or molecular selectivity for the anti-ALK-1 antibody using methods well known in the art For instance, one 25 may determine the species selectivity using Western blot, surface plasmon resonance, e.g., BIAcore', ELISA, immunoprecipitation or RIA.

In some embodiments, the anti-ALK-1 antibody binds to primate ALK-1 with a Ko that is at least two times smaller than its Ko for rodent ALK-1. In a further embodiment the Ko for primate ALK-1 is at least 3-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at 30 least 1000-fold smaller than its Ko for rodent ALK-1 as measured by flow cytometry.

In other embodiments, the anti-ALK-1 antibody has a selectivity for ALK-1 over ALK-2 through ALK-7. In some embodiments, the antl-ALK-1 antibody does not exhibit any appreciable specific binding to any other protein other than ALK-1. Preferably, the anti-ALK-1 antibody binds to the ECD of human ALK-1. -38-

Methods of Producing Antibodies and Antibody Producing Cell Lines ALK-1 Immunogen

In some embodiments, the ALK-1 immunogen or antigen is isolated and/or purified ALK-1. in some embodiments, the ALK-1 immunogen is human ALK-1. In preferred embodiments, the ALK-1 5 immunogen is the ECD of human ALK-1. Human ALK-1, or antigenic portions thereof, can be prepared according to methods well known to those in the art, or can be purchased from commercial vendors. The human ALK-1 amino acid and nucleotide sequences are known (see e.g. Genbank record Accession No. L17075). ACVRL1 gene encoding a full-lentgh ALK-1 is commercially available from Invitrogen inc., Clone ID IOH21048. For example, R&amp;amp;D Systems, Inc. sells the recombinant human ALK-1/Fc chimera 10 (Catalog Number 370-AL) prepared by expression of a DNA sequence encoding the ECD amino acid residues 1-118 of ALK-1, which DNA sequence was fused to a DNA sequence encoding the Fc region of human JgG via a DNA sequence encoding a polypeptide linker in a mouse myeloma cell line. The recombinant mature human ALK-1/Fc chimera Is a dtsulfide-iinked homodimeric protein having Asp 22 at the amino-terminus. In addition, Example 1 describes preparation of ALK-1 ECD His-Tag protein which 15 has been used for generation of hybridomas producing an anti-ALK-1 antibody according to the present invention.

In other embodiments, the ALK-1 antigen is a cell that expresses or overexpresses ALK-1. In other embodiments, the ALK-1 antigen is a recombinant protein expressed from yeast, insect cells, bacteria such as E. coli, or other resources by recombinant technology. 20 Immunization

In some embodiments, human antibodies are produced by immunizing a non-human, transgenic animal comprising within its genome some or all of human immunoglobulin heavy chain and light chain loci with a ALK-1 antigen, in a preferred embodiment, the non-human animal is a XENOMOUSE® animat. (Abgenix, Inc., Fremont, CA). 25 XENOMOUSE® mice are engineered mouse strains that comprise large fragments of human

immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green etal., Nature Genetics 7:13-21 (1994) and U.S. Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 30 99/45031, WO 99/53049, WO 00/09560, and WO 00/037504.

In another aspect, the invention provides a method for making anti-ALK-1 antibodies from non human, non-mouse animals by immunizing non-human transgenic animals that comprise human immunoglobuiin loci with an ALK-1 antigen. One can produce such animals using the methods described In the above-cited documents. The.methods disclosed in these documents can be modified as described 35 in U.S. Patent 5,994,619, which is hereby incorporated by reference. U.S. Patent 5,994,619 describes methods for producing novel cultured inner cell mass (CICM) ceils and cell lines, derived from pigs and cows, and transgenic CICM cells into which heterologous DNA has been inserted. CICM transgenic cells -39- can be used to produce cloned transgenic embryos, fetuses, and offspring. The ‘619 patent also describes methods of producing transgenic animals that are capable of transmitting the heterologous DNA to their progeny, in preferred embodiments of the current invention, the non-human animals are mammals, particularly rats, sheep, pigs, goats, cattle, horses or chickens. 5 XENOMOUSE® mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies, in some embodiments, the XENOMOUSE® mice contain approximately 80% of the human antibody V gene repertoire through introduction, of megabase sized, germiine configuration fragments of the human heavy chain loci and kappa light chain loci in yeast artificial chromosome (YAC). In other embodiments, XENOMOUSE® mice further contain approximately 10 all of the human lambda light chain locus. See Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovlts, J. Exp. Med. 188:483-495 (1998), and WO 98/24893, the disclosures of which are hereby incorporated by reference. in some embodiments, the non-human animal comprising human immunoglobulin genes are animals that have a.human immunoglobulin “minilocus". In the minilocus approach, an exogenous lg 15 locus is mimicked through the inclusion of individual genes from the lg locus. Thus, one or more Vh genes, one or more DH genes, one or more JH genes, a mu constant domain, and a second constant domain (preferably a gamma constant domain) are formed into a construct for insertion into an animal. This approach is described, inter aiia, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and 20 5,643,763, hereby incorporated by reference.

In another aspect, the Invention provides a method for making humanized anti-ALK-1 antibodies.

In some embodiments, non-human animals are immunized with a ALK-1 antigen as described below under conditions that permit antibody production. Antibody-producing cells are isolated from the animals, and nucleic acids encoding the heavy and light chains of an anti-ALK-1 antibody of interest are isolated 25 from the isolated antibody-producing celis or from an immortalized celt line produced from such cells. These nucleic acids are subsequently engineered using techniques known to those of skill in the art and as described further below to reduce the amount of non-human sequence, I.e., to humanize the antibody to reduce the Immune response in humans;

Immunization of animals can be by any method known in the art See, e.g., Harlow and Lane, 30 Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the ait See, e.g., Harlow and Lane, supra, and U.S. Patent 5,994,619. In a preferred embodiment, the ALK-1 antigen is administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating 35 complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, no spread out over several weeks. Example 2 exemplifies a method for producing anti-ALK-1 monoclonal antibodies in XENOMOUSE® mice.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a ALK-1 antigen, antibodies and/or antibody-producing celts 5 can be obtained from the animal. In some embodiments, anti-ALK-1 antibody-containing serum is obtained from the animal by bleeding or sacrificing the animal. The serum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-ALK-1 antibodies may be purified from the serum. -

In some embodiments, antibody-producing cell lines are prepared from cells isolated from the 10 immunized animal. After Immunization, the animal is sacrificed and lymph node and/or splenic B cells are immortalized by any means known in the art Methods of immortalizing cells include, but are not limited to, transfecting them with oncogenes, infecting them with an oncogenic virus and cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized celt, e.g., a myeloma cell, and inactivating a tumor suppressor gene, 15 See, e.g., Harlow and Lane, supra. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line). Immortalized cells are screened .using ALK-1, or a portion thereof. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay. An example of ELISA screening is provided in WO 00/37504, incorporated herein by reference. 20 . Anti-ALK-1 antibody-producing cells, e.g., hybridomas,,are selected, cloned and further screened for desirable characteristics, including robust growth, high antibody production and desirable antibody characteristics, as discussed further below. Hybridomas can be expanded in-vivo in syngeneic animals, in animals that lack an Immune-system, e.g., nude mice, or in cell culture in vitro.. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art 25 In a preferred embodiment, the immunized animal is a non-human animal that expresses human immunoglobulin genes and the splenic B cells are fused to a myeloma cell line from the same species as the non-human animal. In a more preferred embodiment, the immunized animal is a XENOMOUSE® mouse and the myeloma cell line is a non-secretory mouse myeloma. In an even more preferred embodiment, the myeloma cell line is P3-X63-Ag8.653 (American Type Culture Collection). See, e.g., 30 Example 2.

Thus, In one embodiment, the invention provides methods for producing a cell line that produces a human monoclonal antibody or a fragment thereof directed to ALK-1 comprising (a) Immunizing a nonhuman transgenic animal described herein with ALK-1, a portion of ALK-1 or a cell or tissue expressing ALK-1; (b) allowing the transgenic animal to mount an Immune response to ALK-1; (c) isolating antibody- 35 producing cells from transgenic animal; (d) immortalizing the antibody-producing cells; (e) creating individual monoclonal populations of the Immortalized antibody-producing cells; and (f) screening the immortalized antibody-producing cells to identify an antibody directed to ALK-1. -41 -

In another aspect, the invention provides a cell line that produces a human anti-ALK-1 antibody. In some embodiments the ceil fine is a hybridoma cell line. In some embodiments, the hybridomas are mouse hybridomas, as described, above. In other embodiments, the hybridomas are produced in a nonhuman, non-mouse species such as rats, sheep, pigs, goats, cattle or horses, in another embodiment, 5 the hybridomas are human hybridomas.

In another embodiment, a transgenic animal is immunized with an ALK-1 antigen, primary cells, e.g., spleen or peripheral blood B cells, are isolated from an immunized transgenic animat and individual ceils producing antibodies specific for the desired antigen are identified. Polyadenylated mRNA from each individual cell is isolated and reverse transcription polymerase chain reaction (RT-PCR) is performed 10 using sense primers that anneal to variable domain sequences, e.g., degenerate primers that recognize most or all of the FR1 regions of human heavy and light chain variable domain genes and anti-sense primers that anneal to constant or joining region sequences. cDNAs ofthe heavy and light chain variable domains are then cloned and expressed in any suitable host cell, e.g., a myeloma cell, as chimeric antibodies with respective immunoglobulin constant regions, such as the heavy chain and κ or λ constant 15 domains. See.Babcook, J.S. etal., Proc. Natl. Acad Sci. USA 93:7843-48, 1996, incorporated herein by reference. Anti ALK-1 antibodies may then be identified and isolated as described herein.

In another embodiment, phage display techniques can be used to provide libraries containing a repertoire , of. antibodies with varying affinities for ALK-1. For production of such repertoires, it is unnecessary to. immortalize the B cells from the immunized animal. Rather, the primary B cells can be 20 used directly as a source of DNA. The mixture of cDNAs obtained from B cell, e.g., derived from spleens, is used to prepare an expression library, for example, a phage display library transfected into Ecoli. The resulting cells are tested for immunoreactivity to ALK-1; Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths etal,, EMBO J., 13:3245-3260 (1994); Nlssim etal,, ibid, pp. 692-698 and by Griffiths etal., Ibid, 12:725-734, which are incorporated by 25 reference. Ultimately, clones from the library are identified that produce binding affinities of a desired . magnitude for the antigen and the DNA encoding the product responsible for such binding is recovered and manipulated for standard recombinant expression. Phage display libraries may also be constructed using previously manipulated nucleotide sequences and screened in a similar .fashion. In general, the cDNAs encoding heavy and light chains are independently supplied or linked to form Fv analogs for 30 production in the phage library. . .

The phage library is then screened for the antibodies with the highest affinities for ALK-1 and the genetic material recovered from the appropriate clone. Further rounds of screening can increase affinity of the original antibody Isolated. . . . .

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of Making Antibodies 35 Nucleic Adds

The present invention also encompasses nucleic acid molecules encoding anti-ALK-1 antibodies or an antigen-binding fragments thereof. In some embodiments, different nucleic acid molecules encode -42 10 15 .20 25 30 a heavy chain and a light chain of an anti-ALK-1 immunoglobulin. In other embodiments, the.same nucleicacid molecule encodes a heavy chain and a light chain of an anti-ALK-1 immunoglobulin.

In some embodiments, the nucleic acid molecule encoding the variable domain of the light chair (Vu) utilizes a human A27, A2, A1, A3, B3, B2, L1 or L2 V* gene, and a human Jk5, Jk1, Jk3 or Jk4 gene. In some embodiments the nucleic acid molecule utilizes a human A27 Vk gene and a human Jk5 gene. In other embodiments, the nucleic acid molecule utilizes a human A2 gene and a human.JKl gene. In some embodiments, the nucleic acid molecule encoding the light chain encodes an amino acid sequence comprising'1, 2, 3, 4, 5, 6, 7, 8,.9,40, 11, 12; 13, 14 or 15 substitutions from the germiine amino acid sequence(s). tn some embodiments, the nucleic acid molecule comprises, a nucleotide sequence that encodes a VL amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions and/or. 1, 2, or 3 non-conservative substitutions compared· to germiine VK and JK sequences. Substitutions may .be in the CDR regions, the framework regions, or in the constant domain.

In some embodiments, the nucleic acid molecule encodes, a VL amino acid sequence comprising one or more mutations compared to the germiine sequence that are identical, to the mutations from germiine found in the VL of any one of antibodies 1:11.1; 1.12.1; 44-24(rWT); 1.12.1 (M291/D19A); 1424(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 14624; 1.183.1 ;.-1.274;. 1.294; 1.314; 1.84; 1.94; 4404; 4.244; 4.384; 4.584; 4:624; 4.68.1; 4.724;·5434; 5.344; 5.534; 5.564 ; 5.574; or 5.594. ..

In some embodiments, the-nucleic acid molecule encodes at least three amino acid substitutions compared to the germiine sequence.that are identical to the mutations from germiine found in the VL of any one of antibodies 1414; 1.124; 1424(rWT); 142.1(M29I/D19A); 1.124(M29I); 142.1(D19A); 1434; 1444; 14514; 14624; 14834; 1.274; 1.294; 1.314; 1.84; 4.94.;,4.104; 4.244; 4.384; 4.584; 4.624; 4.68.1; 4.724; 543.1; 5.344; 5.534;5:564; 5.57.1; or 5.594.

In some embodiments, the nucleic acid molecule comprises a.nucleotide sequence selected, from the group consisting of SEQ ID NOs: 7, 11, 15,19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91 or 126, which encodes· the VL amino acid sequence of monoclonal antibody 1424(M29I/D19A), 1.114, 1434; 1444; 14514; 14624; 14834; 1.274; 1.29.1- 1.31.1; 1.84; 1.94; 4404; 4.244; 4.384; 4.584 ; 4.624; 4.684; 4.724;· 5434; 5.344; 5.534; 5.564; 5.574 ; 5.594 or 1424.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68;. 72; 76; 80; 84; 88; 92 or427. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ· ID NO: 3 or a portion thereof. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain of one, two or all three CDRs of said antibody. In some embodiments, said portion encodes a contiguous region from CDR1-CDR3 of the light chain of an anti-ALK-1 antibody.. .

In some embodiments, the nucleic acid molecule encodes a .Vl amino acid sequence that is at least 70%, 75%, 50%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VL amino, acid sequence of any one of antibodies 1.114; 1.124; 1.12.1(rWT); 1.124(M29I/D19A); 1424(171291); 1.12.1(D19A); 1.134; 35 -43- 10 15 • 20 25 30 35 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34,1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1, or to the amino acid sequence of the VL region of SEQ ID NO: 4. Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, or that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a nucleic acid encoding the amino acid sequence the VL region of SEQ ID NOs: 8;. 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; .88; 92 or 126 or to a nucleic acid comprising the VL region nucleotide sequence of SEQ ID NO: 4.

In other preferred embodiments, the nucleic acid molecule encodes the variable domain of a heavy chain (VH) that utilizes a human Vh 4-31, VH 3-11, Vh 3-15, VH 3-33, VH 4-61 or VH 4-59 gene sequence or a sequence derived therefrom. In some embodiments, the nucleic acid molecule utilizes a human VH 4-31 gene, a DH6-19 gene and a human JH4B gene.

In some embodiments, the nucleic acid molecule encodes an amino acid sequence comprising 1, '2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations compared to the germline amino acid sequence of the human V, D or J genes. In some embodiments, said mutations are In the VH region. In some embodiments, said mutations are in the CDR regions.

In some embodiments, the nucleic acid molecule encodes a VH sequence comprising one or more amino acid mutations compared to the germline Vh sequence that are identical to amino acid mutations found In the VH of any one of monoclonal antibody 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1(M29I/D19A); 1.12.1(M29i); 1.12.-1 (D19A); .1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; Ί.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; .5.13.1; 5.34.1; 5.53.1;, 5.56.1; 5.57.1; or 5.59.1. In some embodiments, the nucleic acid encodes at least three amino acid mutations compared to the germline sequences that are identical to at least three amino acid mutations found in one of the above-listed monoclonal antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85* 89, or· 103, which encodes the VH amino acid sequence of monoclonal, antibody 1;12,1(M29i/D19A), 1.11.1, 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; 5.59.1 or 1.12.1. in some embodiments, the nucleic acid, molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of SEQ ID NOs: SEQ ID NOs: 2; 6; 10;. 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104. In various preferred embodiments, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of SEQ ID NOS: 1 or 95. In some embodiments, said portion encodes the VH region, a CDR3 region, all three CDR regions, or a contiguous region including CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the Vh amino acid sequence in any one of SEQ ID NOS: SEQ ID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104. Nucleic acid molecules of the invention include nucleic acids that hybridize under -44- highly stringent conditions, such as those described above, or that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90, 100 or 104, or to a VH region thereof, or to a nucleic acid comprising the nucleotide sequence of SEQ ID NOs: 1, 5, 9, 5 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 95, 103, 128, or 129, or the nucleotide sequence that encodes a VH region thereof

In another embodiment, the nucleic acid encodes a full-length heavy chain of an antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29)/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 10 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13:1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1, or a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. Further, the nucleic acid may comprise the nucleotide sequence Of SEQ ID NOs: 1 or 95, A nucleic acid molecule encoding the heavy or light chain of an anti-ALK-1 antibody or portions thereof can be isolated from any source that produces such antibody. In various embodiments, the 15 nucleic acid molecules are isolated from a B cell that expresses an anti-ALK-1 antibody isolated from an animal immunized with ALK-1 or from an immortalized cell derived from such a B ceil. Methods of . isolating nucleic acids encoding an antibody are well-known in the art. See, e.g., Sambrook J. &amp;amp; Russell . D„ Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000). mRNA may be isolated and used to produce cDNA for use in the polymerase chain 20 reaction (PCR) or cDNA cloning of antibody genes. In a preferred embodiment, the nucleic acid molecule ‘ · is isolated from a hybridoma that has as one of its fusion partners a cell from a non-human transgenic animal, said cell producing a human immunoglobulin, in an even more preferred embodiment, the cell producing human immunoglobuiin is Isolated from a XENOMOUSE® animal. In another embodiment, the celt producing the-human immunoglobuiin is isolated from a non-human, non-mouse transgenic animal, as 25 described above. In another embodiment, the nucleic acid Is isolated from a non-human, non-transgenic animal. The nucleic acid molecules isolated from a non-human, non-transgenic. animal may be used, e.g., for humanized antibodies that comprise one or more amino acid sequences from a human anti-ALK-1 antibody of the present invention.

In some embodiments, a nucleic acid encoding a heavy chain of an anti-ALK-1 antibody of the 30 invention can comprise a nucleotide sequence encoding a Vh domain of the invention joined in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding a light chain of an anti-ALK-1 antibody of the invention can comprise a nucleotide sequence encoding a VL domain of the invention joined in-frame to a nucleotide sequence encoding a light chain constant domain from any source. 35 in a further aspect of the Invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and/or light (VL) chains are “converted" to full-length antibody genes. In one embodiment, nucleic acid molecules encoding the Vh or VL domains are converted to full-length antibody genes by insertion into an expression- vector already encoding heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operatively linked to the CH segments) within the -45- 10 15 20 vector, and/or the VL segment is operatively finked to the Cl segment within the vector. In another embodiment, nucleic acid molecules encoding the Vh and/or Vl domains are converted into full-length antibody genes by linking, e.g., ligating, a nucleic acid molecule encoding a VH and/or VL domains to a nucleic acid molecule encoding a Ch and/or CL domain using standard molecular biological techniques. Nucleic acid sequences of human heavy and fight chain immunoglobulin constant domain genes are known in the art. See, e.g., Kabat etai., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. Νό. 91-3242, 1991. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they have been introduced and the anti-ALK-1 antibody isolated.

The nucleic acid molecules may be used to recombinantly express large quantities of anti-ALK-1 antibodies. The nucleic acid molecules also may be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described further below. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization, also as described below.

In another embodiment, a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence. For instance, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify regions of DNA that could be used, inter alia, to isolate additional nucleic acid molecules encoding variable domains of anti-ALK-1 antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains of the heavy and fight chains of the antibody of interest. In some embodiments, the oligonucleotides encode all or a part of one or more of the CDRs of antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151J; 1.162.1; 1.183.1; 1.27,1; 1.29,1; 1.31.1; 1.8.1; 1.9,1; 4.10.1; 4.24.1; 4.38:1; 4.58.1; 4.62,1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1 or variants thereof as described herein. 25 Vectors

The invention provides vectors comprising nucleic acid molecules that encode the heavy chain of an anti-ALK-1 antibody of the invention or an antigen-binding portion thereof. The invention also provides vectors comprising nucleic acid molecules that encode the light chain of such antibodies or antigenbinding portion thereof. The invention further provides vectors comprising nucleic acid molecules 30 encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

In some embodiments, the anti-ALK-1 antibodies of the Invention or antigen-binding portions are expressed by inserting DNAs encoding partial or full-length tight and heavy chains, obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include 35 plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), -plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The -46- expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody tight chain gene and the antibody heavy chain gene can be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of 5 ' complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). A convenient vector is one that encodes a functionally complete human Ch or Cu immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or Vl sequence can easily be inserted and expressed, as described above,. In such vectors, splicing usually occurs between the splice 10 donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice regions that occur within the human Ch exons. Polyadenylation and transcription termination . occur at native chromosomal sites downstream ofthe coding regions. The recombinant expression vector also can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned Into the vector such that the signal peptide Is linked in-frame to the 15 amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). in addition to the antibody chain genes,, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a . host cell. It will -be

- -appreciated by those skilled in the art that the design of the expression vector,-including the selection of 20 regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host ceil expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV . promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., 25 the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245 and U.S. Patent No. 4,968,615. Methods for expressing antibodies in plants, Including a description of promoters and vectors, as well as transformation of plants is known in the art See, e.g., United States Patent 6,517,529, incorporated 30 herein by. reference. Methods of. expressing .polypeptides in bacterial -cells or fungal cells, e.g., yeast cells, are also well known in the art..

In addition to .the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker 35 gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, incorporated .herein by reference). For example, typically the selectable marker gene confers resistance to drugs; such as G418, hygromycin or methotrexate, , on a host cell into which the vector has been introduced. For example, selectable marker genes include the -47- dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding anti-ALK-1 antibodies and vectors comprising these nucleic acid 5 molecules can be used for transfection of a suitable mammalian, plant, bacterial or yeast host cell. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct 10 microinjection of the DNA Into nuclei. In-addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art See, e.g.,. U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference). Methods of transforming plant cells are well known in the art, including, e.g., Agrobacterlum-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods 15 of transforming bacterial and yeast cells are also well known in the art

Mammalian cell lines available as hosts for expression are well known in the art and include many

Immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells . (Invitrogen), NIH-3T3 cells, HeLa cells,' baby hamster kidney (BHK) cells, African green monkey kidney 20 cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell tines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of 25 the antibody in the host ceils or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nlcotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coil and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris. 30· - Further, expression of antibodies of the invention from production cell lines can be enhanced . using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS . system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841. 35 It is likely that antibodies expressed by different cell lines or in transgenic animals will have : different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies. -48-

Transgenic Animals and Plants

Anti-ALK-1 antibodies of the invention also can be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic 5 production in mammals, anti-ALK-1 antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference. In some embodiments, non-human transgenic animats that comprise human immunoglobulin loci are immunized with ALK-1 or an immunogenic portion thereof, as described above. Methods for making antibodies in plants are described, e.g., In U.S. patents 6,046,037 and 10 5,959,177, incorporated herein by reference.

In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding an antl-ALK-1 antibody of the invention into the animal or piant by standard transgenic techniques. See Hogan and United States Patent 6,417,429, supra. The transgenic cells used for making the transgenic animal can be embryonic stem cells or somatic cells or a 15 fertilized egg. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1999); Jackson etal., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook. Academic Press (1999), all incorporated herein by reference. In some 20 embodiments, the transgenic non-human animals have a targeted disruption and replacement by a targeting construct that encodes a heavy chain and/or a light chain of interest In a preferred embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy" and light chains that specifically bind to ALK-1, preferably human ALK-ί. In some embodiments, the transgenic animals comprise nucleic acid molecules encoding a modified antibody such as a single-chain 25 antibody, a chimeric antibody or a humanized antibody. The anti-ALK-1 antibodies may be made in any transgenic animal, in a preferred embodiment, the non-human animals are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal expresses said encoded polypeptides In blood, milk, urine, saliva, tears, mucus and other bodily fluids.

Phage Display Libraries 30 The invention provides a method for producing an anti-ALK-1 antibody or antigen-binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with ALK-1 or an antibody-binding portion thereof, isolating phage that bind ALK-1, and obtaining the antibody from the phage. By way of example, one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human 35 immunoglobulin loci with ALK-1 or an antigenic portion thereof to create an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the invention from the extracted cells, reverse transcribing the RNA to produce cDNA, -49- amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage. Recombinant anti-ALK-1 antibodies of the invention may be obtained in this way.

Recombinant human anti-ALK-1 antibodies of the invention can be isolated by screening a 5 recombinant combinatorial antibody library. Preferably the library is a scFv phage display library, generated using human VL and Vh cDNAs prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612). There also are other 10 methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., U.S. Patent No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology 9:1370-1372 (1991); Hay etal., Hum. Antibod. Hybridomas 3:81-85 (1992); Huse etal., Science 246:1275-1281 (1989); McCafferty et al., Nature 348:552-554 (1,990); Griffiths et al., EMBO J. 12:725-734 (1993); Hawkins et al., 15 J. Mol. Biol. 226:889-896 (1992); Clackson etal., Nature 352:624-628 (1991); Gram etal., Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992); Garrad etal., Bio/Technology 9:1373-1377 (1991); Hoogenboom etal., Nuc. Acid Res. 19:4133-4137 (1991); and Barbas etal., Proc. Natl. Acad. Sci. USA 88:7978-7982 ¢1991), all incorporated herein by reference.

In one embodiment, to isolate and produce human anti-ALK-1 antibodies with the desired 20 characteristics, a human anti-ALK-1 antibody as described herein is first used to select human heavy and tight chain sequences having similar binding activity toward ALK-1, using the epitope imprinting methods described in PCT Publication No. WO 93/06213, incorporated herein by reference. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described in PCT Publication No. WO 92/01047, McCafferty et al., Nature 348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734 25 . (1993), all Incorporated herein by reference. The scFv antibody libraries preferably are screened using human ALK-1 as the antigen.

Once Initial human VL and VH domains are selected, "mix and match" experiments are performed, in which different pairs of the initially selected VL and VH segments are screened for ALK-1 binding to select preferred VL/VH pair combinations. Additionally, to further improve the quality of the antibody, the 30 . Vl and Vh segments of the preferred VL/VH pair(s) can be randomly mutated, preferably within the CDR3 region of VH and/or VL( in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VH and VL domains using PCR primers complimentary to the VH CDR3 or VL CDR3, respectively, which primers have been "spiked" with a random mixture of the four nucleotide bases 35 at certain positions such that the resultant PCR products encode Vh and VL segments into which random mutations have been introduced Into the VH and/or VL CDR3 regions. These randomly mutated Vh and VL segments can be re-screened for binding to ALK-1. .... . Following screening and isolation of an anti-ALK-1 antibody of the invention from a recombinant immunoglobulin display library, nucleic acids encoding the selected antibody can be recovered from the -50- display package (e.g,, from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic, acid can further be manipulated to create other antibody forms of the invention, as described below. To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant 5 expression vector and introduced into a mammalian host cells, as described above.

Deimmunized Antibodies

In another aspect ..of the invention, the antibody may be deimmunized to reduce its immunogenicity using the techniques described in, e.g., PCT Publication Nos. WO98/52976 and WOOO/34317 (incorporated herein by reference). 10 Mutated Antibodies in another embodiment, the nucleic acid molecules, vectors and host cells may be used to make mutated anti-ALK-1 antibodies. The antibodies may be mutated In the variable domains of the heavy and/or light chains, e.g., to alter a binding property of the antibody. For example, a mutation may be .made in one or more of the CDR regions to increase or decrease the KD of the antibody for ALK-1, to 15 increase or decrease kon, or to alter the binding specificity of the antibody. Techniques in site-directed mutagenesis are well-known In the art. See, e.g., Sambrook etal. and Ausubel etal., supra. In another embodiment, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in monoclonal antibody 1.11.1; 1.12.1; 1.12.1{rWT); 1.12.1(M29I/D19A); 1.12.1(M29t); 1.12.1 (D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9,1; 20 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56,1; 5.57.1; or 5.59.1. The mutations may be made in a CDR region or framework region of a variable domain, or In a constant domain. In a preferred embodiment, the mutations are made in a variable domain. In some embodiments, one or more mutations are made at -an amino acid residue that is known to be changed . compared to the germline in a CDR region or framework region of a variable domain of an amino acid 25 sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20,22, 24,.26, 28, 30, 32,.34, 36, 38, 40, 42, 44, 46,-48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 92 or 127, or whose nucleic acid sequence is presented in SEQ ID NO: 1, 3, 5, 7, 9,11,13,15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79; 81, 83, 85, 87, 89, 91,95,102 or 126. 30 In another embodiment, the framework region is mutated so that the resulting framework region(s) have the amino acid sequence of the corresponding germline gene. A mutation may be made in a framework region or constant domain to increase the half-life of the anti-ALK-1 antibody. See, e.g., PCT Publication No. WO 00/09560, incorporated herein by reference. A mutation in a framework region or constant domain also can be made to alter the immunogenicity of the antibody, to provide a site for 35 covalent or non-covalent binding to another molecule, or to alter such properties as complement fixation, FcR binding and antibody-dependent cell-mediated cytotoxicity (ADCC). According to the invention, a -51 - single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant domain.

In some embodiments, there are from 1 to 13, including any number in between, amino acid mutations in either'the VH or VL domains of the mutated anti-ALK-1 antibody compared to the anti-ALK-1 5 antibody prior to mutation, in any of the above, the mutations may occur in one or more CDR regions. Further, any of the mutations can be conservative amino acid substitutions, in some embodiments, there are no more than 5, 4, 3, 2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be made that comprises all or a 10 portion of an anti-ALK-1 antibody of the invention linked to another polypeptide, in a preferred embodiment, only the variable domains of the anti-ALK-1 antibody are linked to the polypeptide, in another preferred embodiment, the VH domain of an anti-ALK-1 antibody is linked to a first polypeptide, . while the Vl domain of an anti-ALK-1 antibody is linked to a second polypeptide that associates with the first polypeptide In a manner such that the Vh and Vl domains can interact with one another to form an 15 antigen, binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker such that the VH and Vl domains can interact with one another (see below under Single Chain Antibodies). The VH-linker-VL antibody is then linked to the polypeptide of interest. In addition, fusion antibodies can be created in which two (or more) single-chain, antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one 20 wants to create a bispecific antibody.

To create a single chain antibody, (scFv) the Vh- and VL-encodtng DNAfragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker. See, e.g., Bird et al., Science 242:423-426 (1988); Huston 25 etal., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); McCafferty etal., Nature 348:552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two Vh and VL are used, or polyvalent, if more than two VH and VL. are used. Bispecific or polyvalent antibodies may be generated that bind specifically to ALK-1 and to another molecule. . In other embodiments,. other modified antibodies may be prepared using anti-ALK-1'antibody 30 encoding nucleic acid molecules.. For instance, “Kappa bodies" (III et al., Protein Eng. 10: 949-57 (1997)), “Minibodies" (Martin et ai., EMBO J. 13: 5303-9 (1994)), "Diabodies" (Holliger etal., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or "Januslns" (Traunecker etal., EMBO J. 10:3655-3659 (1991) and Traunecker etal., Int J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standard.molecular biological techniques following the teachings ofthe specification. 35 Blspecific antibodies or antigen-binding fragments, can be produced by a variety of methods including fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songslviiai &amp;amp; Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). In addition, blspecific .antibodies may be formed as “diabodies" or "Janusins,"· In some embodiments, the blspecific antibody -52- binds to two different epitopes of ALK-1. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibody 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29i); 1.12.1 (D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1 and an additional antibody heavy chain and light chain. In some embodiments, the additional light chain and heavy chain also are from one of the above-identified monoclonal antibodies, but are different from the first heavy and light chains.

In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions from a human anti-ALK-1 monoclonal antibody provided herein. 10 Derivatized and Labeled Antibodies

An anti-ALK-1 antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein).. In general, the antibodies or portion thereof are derivatized such that the ALK-1 binding is not affected adversely by the. derivatization or labeling.. Accordingly, the antibodies and antibody portions of the invention are intended to include both intact and 15 modified forms of the human anti-ALK-1. antibodies described herein.. For examplej an antibody.or. antibody portion of the invention can be. functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent,.a pharmaceutical agent, and/or a protein or • peptide that can mediate association of the antibody or antibody portion with another molecule (such as a 20 streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same . type .or .of different types, e.g., to create bispeclfic antibodies), Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups, separated, by an appropriate spacer (e.g., m-maleimldobenzoyl-N-hydroxysucdnimide ester) or homobifunctional (e.g., disuccinimidyl. suberate). 25 Such linkers are available from Pierce Chemical Company, Rockford, II. - - Another type of derivatized antibody is a labeled antibody. Useful detection agents with which an antibody or antigen-binding portion of the invention may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. An antibody can also be labeled with enzymes 30 that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labeled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen . peroxide and diaminobenzidine leads to a colored reaction product,, which is detectable. An antibody can.: 35 also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. An antibody can also be labeled with a. predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding -53- domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

An anti-ALK-1 antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, -or a carbohydrate group. These groups are useful to improve the 5 biological characteristics of the antibody, e.g., to increase serum half-life.

Pharmaceutical Compositions and Administration

This invention also relates to a pharmaceutical composition for the treatment of conditions associated with undesirable increased angiogenesis in a mammal, Including a human, comprising an 10 amount of an anti-ALK-1 antibody or antigen binding portion thereof, as described herein, that is effective in treating such conditions, and a pharmaceutically acceptable carrier.

The antibodies and antigen-binding portions of the present invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antigen-binding portion of the invention and a pharmaceutically 15 acceptable carrier. As used herein, “pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like thatare physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, .as well as. combinationsthereof. In many cases, It will be preferable to include isotonic agents, for example, sugars, 20 polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. . The compositions of this invention may be In a variety of forms,: for example, liquid, semi-solid and 25 solid dosage forms, such as liquid, solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are In the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is .parenteral (e.g., intravenous, 30 subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection; In another-preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection. Formulations for injection may be presented in unit dosage form, e.g., In ampoules or in multi-dose containers, with or without an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions In oily or aqueous vehicles, 35 and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the. active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. - Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or -54- other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the anti-ALK-1 antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic 5 dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper, fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required 10 particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

The antibodies or antibody portions of the present invention can be administered by a variety of methods known In the art, although for many therapeutic applications, the preferred route/mode of 15 administration is subcutaneous, intramuscular, or intravenous Infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

In certain embodiments, the antibody compositions of the present invention may.be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and - microencapsulated delivery systems. Biodegradable, 20 biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are generally known to those skilled In the art. See, e.g., Sustained arid Controlled Release Drug Delivery Systems J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, which Is incorporated herein by reference. . 25 , Additional active compounds also can be incorporated into the compositions, . In certain embodiments, an inhibitory anti-ALK-1 antibody of the Invention is co-formulated with and/or coadministered with one or more additional therapeutic agents. These agents include, without limitation, antibodies that bind other targets, anti-tumor agents, anti-angiogenesis agents, signal transduction inhibitors, anti-proliferative agents, chemotherapeutic agents, or peptide analogues that inhibit anti-ALK-1. 30 Such combination therapies may require lower dosages of the inhibitory anti-ALK-1 antibody as well as . the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies. ' As rioted above, the. compositions of the present Invention optionally may further comprise a pharmaceutically acceptable antioxidant in addition to a chelating agent Suitable antioxidants, include, 35 but are not limited to, methionine, sodium thiosulfate, catalase, and platinum.

For example, the composition may contain methionine in a concentration that ranges from 1 mM to.about 100 mM, and in particular, is about 27. mM. For example, an aqueous formulation may be: 10 mg/mL anti-ALK-1 antibody, 20 mM Histidine, pH 5.5, 84 mg/mL Trehalose dihydrate, 0.2 mg/mL Polysorbate 80, 0.05 mg/mL disodium EDTA, 0.1 mg/mL L-Methionlne. -55-

The compositions of the invention may include a "therapeutically effective amount" or a “prophylactically effective amount" of an antibody or antigen-binding portion of the invention. A “therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or 5 antigen-binding portion may vary according to factors such as the disease state, age, sex, and weight of the Individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antigen-binding portion are outweighed by the therapeutically beneficial effects.. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time 10 necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can 15 be administered over time or-the dose can be. proportionally reduced or increased as indicated by the . exigencies of. the therapeutic situation, lt is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound, calculated to produce the desired 20 therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage ......unit forms of the invention, are dictated by.and.directly dependent on (a) the unique characteristics of the- anti-ALK-1 antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent In the art of compounding such-an antibody for the treatment of sensitivity in individuals. 25 ,...... An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.025 to 50 mg/kg, more preferably 0.1 to· 50 mg/kg, more preferably 0.1-25, 0.1 to 10 or 0,1 to 3 mg/kg.· In some embodiments, a formulation contains 5 mg/mL of antibody in a buffer of 20mM sodium citrate, pH 5.5,140mM NaCl, and 0.2mg/mL polysorbate 80. It is to. be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to 30 be further understood that for any particular subject, specific dosage regimens should, be .adjusted over time according to the individual .need and the professional judgment of the person, administering or supervising the administration ofthe compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

Another aspect of the present invention provides kits comprising an anti-ALK-1. antibody or 35 antigen-binding portion of the invention or a composition comprising such an antibody or portion, A kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents. A kit can also include instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit includes the antibody or a composition comprising it and a diagnostic agent that can be used in a method. I -;56 - described below. In another preferred embodiment, the kit includes the antibody or a composition comprising it and one or more therapeutic agents that can be used in a method described below. Diagnostic Methods of Use

The antl-ALK-1 antibodies or antigen-binding portions thereof can be used in diagnostic methods 5 to detect ALK-1 in a biological sample in vitro or in vivo. For example, the anti-ALK-1 antibodies can be used In a conventional immunoassay, including, without limitation, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot or immunoprecipitation. . The anti-ALK-1 antibodies of the invention can be used to detect ALK-1 from humans. The anti-ALK-1 antibodies can also be used to detect ALK-1 from other primates, e.g. cynomolgus monkeys. 10 . The invention provides a.method for detecting ALK-1 in a biological sample comprising contacting the biological sample with an antl-ALK-1 antibody of the invention and detecting the bound antibody. In one embodiment, the antl-ALK-1 antibody is directly labeled with a detectable label, in another embodiment, the anti-ALK-1 antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the anti-ALK-T antibody is labeled. As is well known to one of skill in the.art, a 15 second antibody is chosen that is able to specifically bind the particular species and class of the first antibody. For example, if the antl-ALK-1 antibody is a human IgG, then the secondary ,antibody could be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially, e.g., from Pierce Chemical Co;

Suitable labels for the antibody, or secondary antibody have been discussed previously, and .20 include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples, of suitable enzymes. include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable , prosthetic, group complexes include streptavidin/blotin and avidin/biotin; examples of suitable fluorescent materials include umbelilferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichiorotriazinylamine fluorescein, dansyl chloride or 25 phycoerythrin; an example of a luminescent material includes luminol; and examples. of .suitable radioactive material include 126l, 131l, 35S or3H. in other embodiments, ALK-1 can be assayed In a biological sample by a competition immunoassay utilizing ALK-1 standards labeled with a detectable substance and an.unlabeled anti-ALK-1 antibody. In this assay, the biological sample, the labeled ALK-1 standards and the anti-ALK-1 antibody 30 are combined and the amount of.labeled ALK-1 standard bound.to the unlabeled antibody is determined. The amount of ALK-1 in the biological sample is inversely proportional to the amount of labeled ALK-1 standard bound to the anti-ALK-1 antibody.

One can use the immunoassays disclosed above, for a number of purposes. For example, the anti-ALK-1 antibodies can be used to detect ALK-1 In cultured cells. In a preferred embodimenL the anti- 35 . ALK-ί antibodies are used to determine the amount of ALK-1 produced by cells that have been treated with various compounds. This method can be used to identify compounds that modulate ALK-1 protein levels. According to this method, one sample of cells is treated with a test compound for a period of time while another sample is left untreated, if the total level of ALK-1 is . to be measured, the cells are lysed -57- and the total ALK-1 level is measured using one of the immunoassays described above. The total level of ALK-1 in the treated versus the untreated celis Is compared to determine the effect of the test compound. A preferred immunoassay for measuring total ALK-1 levels is flow cytometry or immunohistochemistry. Methods such as ELISA, RIA, flow cytometry, Western blot, 5 immunohistochemistry, cell surface labeling of integral membrane proteins and immunoprecipitation are welt known in the art. See, e.g., Harlow and Lane, supra, in addition, the immunoassays can be scaled . up for high throughput screening in order to test a large number of compounds for either activation or inhibition of ALK-1 expression.

The anti-ALK-1 antibodies of the invention also can be used to determine the levels of ALK-1 in a 10 tissue or in cells derived from the tissue. In some embodiments, the tissue is a diseased tissue, in some embodiments of the method, a tissue or a biopsy thereof is excised from a patient. The tissue or biopsy is then used in an immunoassay to determine, e.g., total ALK-1 levels or localization of ALK-1 by the methods discussed above.

The antibodies of the present invention also can be used in vivo to identify tissues and organs 15 that express ALK-1. One advantage of using the human anti-ALK-1 antibodies of the present invention is that they may safely be used in vivo without eliciting a substantial immune response to the antibody upon ... administration, unlike antibodies of non-human origin or with humanized or chimeric antibodies.

The method comprises the steps of administering a detectably labeled antl-ALK-1 antibody or a composition comprising them to a patient in need of such a diagnostic test and subjecting the patient to 20 Imaging.analysis to determine the location of the.ALK-1-expressing tissues. Imaging analysis is well, known in the medical art, and Includes, without limitation, x-ray analysis, magnetic resonance imaging (MRI) or computed tomography (CT). The antibody can be labeled ..with any agent suitable for in vivo imaging, for example a contrast agent, such as barium, which can be used for x-ray analysis, or a magnetic contrast agent, such as a gadolinium chelate, which can be used for MRI or CT. Other labeling 25 agents include, without limitation, radioisotopes, such as “Tc. In another embodiment, the anti-ALK-1 antibody will be unlabeled and will be imaged by administering a second antibody or other molecule that is detectable and that can bind the anti-ALK-1 antibody. In one embodiment, a biopsy is obtained from the patient to determine whether the tissue of interest expresses ALK-1.

Therapeutic Methods of Use · 30 In another embodiment, the invention provides a method for inhibiting ALK-1 activity by administering an anti-ALK-1 antibody to 8 patient in need thereof. Any of the antibodies or antigenbinding portions thereof described herein may be used therapeutically. In a preferred embodiment, the anti-ALK-1 antibody is a human, chimeric or humanized antibody. In another preferred embodiment, the anti-ALK-1 antibody is human antibody, and the patient is a human patient Alternatively, the patient may 35 be a mammal that expresses ALK-1 .that the anti-ALK-1 antibody cross-reacts with. The antibody may be administered to a non-human mammal expressing ALK-1 with which the· antibody cross-reacts (e.g. a cynomolgus monkey) for veterinary purposes or as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of this invention. -58-

In another embodiment, an anti-ALK-1 antibody or antibody portion thereof may be administered to a patient who expresses inappropriately high levels of ALK-1. The antibody may be administered once, but more preferably is administered multiple times. The antibody may be administered from three times daily to once every six months or longer. The administering may be on a schedule such as three times 5 daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months. The antibody may also be administered continuously via a minipump. The antibody may be administered via a mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, or intratumor route. The antibody may be administeredonce, at least twice or for at least the period of time 10 until the condition is treated, palliated or cured. The antibody generally wilt be administered for as long as the condition is present The antibody will generally be administered as part of a pharmaceutical composition as described supra. The dosage of antibody will generally be in the range of 0.1 to 100 mg/kg, more preferably 0.5 to 50 mg/kg, more preferably 1 to 20 mg/kg, and even more preferably 1 to 10 mg/kg.. The serum concentration of the antibody may be. measured by any method known in the art 15 In one embodiment, the antibody is administered in a formulation as a sterile aqueous solution having a pH that ranges from about 5.0 to about 6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody, from about 1 millimolar to about 100 millimolar of histidine buffer, from about 0.01 mg/m! to about 10 mg/mi of polysorbate 80, from about 100 millimolar to about 400 millimolar of trehalose, and from about 0.01 millimolar to about 1.0 millimolar of disodium EDTA dihydrate. 20 It is further contemplated by the present invention that any of the compositions herein may be administered to a patient susceptible to or suffering from a condition associated with increased angiogenesis ("an angiogenic condition").

Examples of angiogenic conditions that may be treated/prevented by the compositions/methods of the present invention include, but are not limited to, cancer (both solid and hematologic), age-related 25 macular degeneration (AMD), developmental abnormalities (organogenesis), diabetic blindness, endometriosis, ocular neovascularization, psoriasis, rheumatoid arthritis (RA), and skin disclolorations (e.g., hemangioma, nevus flammeus, or nevus simplex).

For example, the present Invention relates to methods for treating or preventing conditions associated with ocular neovascularization using any of the compositions/methods herein. Conditions 30 associated with ocular neovascularization include, but are not limited to, diabetic retinopathy, age related macular degeneration ("ARMD"), rubeotic· glaucoma, interstitial keratitis, retinopathy of prematurity, ischemic retinopathy (e.g., sickle, cell), pathological myopic, ocular histoplasmosis, pterygia, punitiate inner choroidopathy, and the like.

Treatment of Abnormal Cell Growth . 35 , This Invention also relates to a method for the treatment of abnormal cell growth in a mammal, including a human, comprising administering to said mammal a therapeutically effective amount of an anti-ALK-1 antibody or antigen binding portion thereof, as described herein, that is effective in treating abnormal cell growth. -59-

In one embodiment of this method, the abnormal cell growth is cancer, including, but not limited to, mesothelioma, hepatobilliary (hepatic and bltiiary duct), a primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal - 5 cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal), breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, 10 cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, non hodgkins's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gait bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, 15 neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

In a preferred embodiment of the present Invention the cancer is selected from lung cancer " (NSCLC and SCLC), cancer of the head or neck, ovarian'cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, breast cancer, cancer of the kidney or ureter, renal· ceil carcinoma, carcinoma of the renal pelvis, neoplasms of tha central nervous system (CNS); primary CNS lymphoma, 20 non hodgkins's lymphoma, spinal axis tumors, or a combination of one or more of the foregoing cancers.

In another preferred embodiment of the present invention the cancer is selected from lung cancer · (NSCLC and SCLC), ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, or a combination of one or more of the foregoing cancers.

In another embodiment of said method, said abnormal· cell growth is a benign proliferative disease, 25 including, but not limited to, psoriasis, benign prostatic hypertrophy or restinosis.

This Invention also relates to a method for the treatment of abnormal cell growth in a mammal which comprises administering to said mammal an amount of an anti-ALK-1 antibody or antigen binding portion thereof, as described herein, that is effective in treating abnormal cell growth in combination with an antitumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, 30 intercalating antibiotics, growth factor Inhibitors; cell cycle inhibitors, enzymes,'topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics, anti-hormones, and anti-androgens;

The invention also relates to a pharmaceutical composition for the treatment of abnormal celt growth in a mammal, including a human; which comprises an amount of an anti-ALK-1 antibody or antigen binding portion thereof, as described herein, that is effective in treating abnormal ceil growth in combination with a 35.....pharmaceutically acceptable carrier and an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, ahti-nietabolltes, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, and antiandrogens. 189,642/2 -60-

The invention also relates to a method for the treatment of a hyperproliferative disorder in a mammal which comprises administering to said mammal a therapeutically effective amount of an anti-ALK-1 antibody or antigen binding portion thereof, as described herein, in combination with an anti-tumor-agent selected from the group consisting antiproliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor 5 inhibitors, cox-l inhibitors, cox-ll inhibitors, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics, anti-hormones, statins, and antiandrogens.

In one embodiment of the present invention the anti-tumor agent used in conjunction with an anti-10 ALK-1 antibody or antigen binding portion thereof, and pharmaceutical compositions described herein, is an anti-angiogenesis agent kinase inhibitor, pan kinase inhibitor or growth factor inhibitor. Preferred pan kinase inhibitors include Sutent (Pfizer Inc,, SU-11248), described in U.S. Patent No. 6,573,293 (Pfizer,

Inc, NY, USA).

Anti-angiogenesis agents, include but are not limited to the following agents, such as EGF 15 inhibitor, EGFR inhibitors, VEGF inhibitors, VEGFR inhibitors, TIE2 inhibitors, JGF1.R inhibitors, COX-II (cyclooxygenase II) inhibitors, MMP-2 (matrix-metailoprotienase 2) inhibitors, and MMP-9 (matrix-metailoprotienase-9) inhibitors. Preferred VEGF inhibitors, include for example, Avastin (bevacizumab), an anti-VEGF monoclonal antibody of Genentech, Inc. of South San Francisco, California.

Additional VEGF inhibitors include CP-547,632 (Pfizer Inc., NY, USA), Axitinib (Pfizer inc.; AG-20 013736), ZD-6474 (AstraZeneca), AEE788 (Novartis), AZD-2171), VEGF Trap (Regeneron,/Aventis),

Vatalanib (also known as PTK-787, ZK-222584: Novartis &amp;amp; Schering AG), Macugen (pegaptanib octasodium, NX-1838, EYE-001, Pfizer lnc./Gilead/Eyetech), IM862 (Cytran Inc. of Kirkland, Washington, USA); and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville,

California,) and combinations thereof. VEGF inhibitors useful in the practice of the present invention are disclosed in US Patent No. 6,534,524 and 6,235,764. Particularly preferred VEGF inhibitors include CP-547,632, AG 13736, Vetalanib, Macugen and combinationsthereof.

Additional VEGF inhibitors are described in, for example in WO 99/24440 (published May 20, 1999), WO 99/62890 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995), WO 99/61422 (published December 2, 1999), United States Patent 6, 534,524 (discloses AG13736), United States Patent 5,834,504 (issued November 10, 1998), WO 98/50356 (published November 12, 1998), United States Patent 5,883,113 (issued March 16, 1999), United States Patent 5,886,020 (issued March 23, 1999), United States Patent 5,792,783 (issued August 11, 1998), U.S. Patent No. US 6,653,308 (issued November 25, 2003), WO 99/10349 (published March 4, 1999), WO 97/32856 (published September 12, 1997), WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 3, 1998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8, 1999), and WO 98/02437 (published January 22, 1998). 189,642/2 -61-

Other antiproliferative agents that may be used with the antibodies, or antigen-binding portions thereof, of the present invention include inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFr, including the compounds disclosed and claimed in the following United States Patent Nos. 6,080,769 (filed December 28, 1998); 6,194,438 (filed December 2, 1999); 6,258,824 (filed February 9, 2000); 6,586,447 (filed March 31,2000); 6,071,935 (filed May 22, 1997); 6,495,564 (filed August 26, 1999); and 6,150,377 (filed August 26, 1999); and the compounds disclosed and claimed in the following United States Patent Nos. 6,596,735; 6,479,513; and 6,844,357.

For additional PDGRr inhibitors, see W001/40217, published July 7, 2001 and W02004/020431, published March 11, 2004. Preferred PDGRr inhibitors include Pfizer’s CP-868,596 and its pharmaceutically acceptable salts.

Preferred GARF inhibitors include Pfizer’s AG-2037 (pelitrexol and its pharmaceutically acceptable salts), GARF inhibitors useful in the practice ofthe present invention are disclosed in US Patent No. 5,608,082.

Examples of useful COX-II inhibitors which can be used in conjunction with a Anti-ALK-1 antibody or antigen binding portion thereof, as described herein, and pharmaceutical compositions described herein include CELEBREX™ (celecoxib), parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189 (Lumiracoxib), BMS 347070, RS 57067, NS-398, Bextra (valdecoxib), paracoxib, Vioxx (rofecoxib), SD-8381, 4-Methyl-2-(3,4-dimethylphenyl)-1 -(4-sulfamoyl-phenyl)-1 H-pyrrole, 2-(4-Ethoxyphenyl)-4-methyl-1 -(4-sulfamoylphenyl)-1 H-pyrrole, T-614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125 and Arcoxia (etoricoxib). For additonal COX-II inhibitors, see U.S. Patent Application Publication No. US 2005/0148627 and U.S. Patent No. 7,259,266. 25 In one preferred embodiment the anti-tumor agent is celecoxib, see U.S. Patent No. 5,466,823, the contents of which are incorporated by reference in its entirety for all purposes. The structure for Celecoxib is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00023.tif" id="idf0003" />

30 In one preferred embodiment the anti-tumor agent is valecoxib, see U.S. Patent No. 5,633,272, the contents of which are incorporated by reference in its entirety for ail purposes. The structure for valdecoxib is shown below: -62-

V :h3 7^N valdecoxib /—&lt; CAS Mo. 101695-72-7 f \ 5,633,272 \^/ C-2065 SC-65072

In one preferred embodiment the anti-tumor agent is parecoxib, see U.S. Patent No. 5,932,598, the contents of which are incorporated by reference in its entirety for aii purposes. The structure for paracoxib is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00024.tif" id="idf0004" />

In one preferred embodiment the anti-tumor agent is deracoxib, see U.S. Patent No. 5,521,207, the contents of which are incorporated by reference in its entirety for all purposes. The structure for deracoxib is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00025.tif" id="idf0005" />

10 In one preferred embodiment the anti-tumor agent is SD-8381, see U;S. Patent No. 6,034,256, the dontents of which are incorporated by reference in its entirety for all purposes. The structure for SD-8381 is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00026.tif" id="idf0006" />

In one preferred embodiment the anti-tumor agent is ABT-963, see International Publication 15 Number WO 2002/24719, the contents of which are Incorporated by reference in Its entirety for all purposes. The structure for ABT-963 is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00027.tif" id="idf0007" />

In one preferred embodiment the anti-tumor ageint is rofecoxib as shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00028.tif" id="idf0008" />

rofecoxib CAS Nd, 162011-90-7 -63-

In one preferred embodiment the anti-tumor agent is MK-663 (etoricoxib), see International

Publication Number WO 1998/03484, the contents of which are incorporated by reference in its entirety for ail purposes. The structure for etoricoxib is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD00029.tif" id="idf0009" />

In one preferred embodiment the anti-tumor agent is COX-189 (Lumiracoxib), see International

Publication Number WO 1999/11605, the contents of which are incorporated by reference in its entirety for all purposes. The structure for Lumiracoxib is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD000210.tif" id="idf0010" />

10

Lumiracoxib CAS Ho. 220991-20-B Novartis WO 99/11605

In one preferred embodiment the anti-tumor agent is BMS-347070, see United States Patent No. 6,180,651, the contents of which are incorporated by reference in its entirety for all purposes. The structure for BMS-347070 is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD000211.tif" id="idf0011" />

BUS 347070 CAS No, 197438-4B-5 6,180,651

In one preferred embodiment the anti-tumor agent is NS-398 (CAS 123653-11-2). The structure for NS-398 (CAS 123653-11-2) Is shown below:

<img img-format="tif" img-content="drawing" file="IL189642AD000212.tif" id="idf0012" />

NS-398 CAS HO. 123653-11-2

In one preferred embodiment the anti-tumor agent is RS 57067 (CAS 17932-91-3). The structure for RS-57067 (CAS 17932-91-3) is shown below: 15 -64-

<img img-format="tif" img-content="drawing" file="IL189642AD000213.tif" id="idf0013" />

11 RS 57067 CAS No. 17932-91-3

In one preferred embodiment the anti-tumor agent is 4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1 H-pyrrole. The structure for 4-Methyi-2-(3,4-dimethylphenyI)-1-(4-sulfamoyl-phenyl)-1 H-pyrrole is shown below: HH.

<img img-format="tif" img-content="drawing" file="IL189642AD000214.tif" id="idf0014" />

In one preferred embodiment the anti-tumor agent is 2-(4-Ethoxyphenyl)-4-methyl-1 -(4-sulfamoylpheny[)-1 H-pyrrole. The structure for 2-(4-Ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole is shown below: so2nh2 10 In one preferred embodiment the anti-tumor agent Is meloxlcam. The structure for meloxicam is shown below:

Other useful inhibitors as anti-tumor agents used in conjunction with antibodies of the present invention and pharmaceutical compositions described herein include aspirin, and non-steroidal anti- 15 inflammatory drugs (NSAIDs) which inhibit the enzyme that makes prostaglandins (cyclooxygenase I and II), resulting in lower levels of prostaglandins, include but are not limited to the following, Salsalate (Amigeslc), Diflunisal (Dolobid), Ibuprofen (Motrin), Ketoprofen (Orudis), Nabumetone (Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn), Diclofenac (Voltaren), Indomethacin (Indocln), Sulindac (Cllnorll), Tolmetin (Tolectln), Etodolac (Lodlne), Ketorolac (Toradol), Oxaprozin (Daypro) and 20 combinations thereof. Preferred COX-I inhibitors include ibuprofen (Motrin), nuprin, naproxen (Aleve), indomethacin (Indocin), nabumetone (Relafen) and combinations thereof.

Targeted agents used in conjunction with an anti-ALK-1 antibody or antigen binding portion thereof, as described herein, and pharmaceutical compositions thereof as described herein, include EGFr inhibitors such as Iressa (gefitinib, AstraZeneca), Tarceva (erlotinib or OSI-774, OSI Pharmaceuticals Inc.), Erbitux 25 (cetuximab, Imclone Pharmaceuticals, Inc.), EMD-7200 (Merck AG), ABX-EGF (Amgen Inc. and Abgenix -65-

Inc.), HR3 (Cuban Government), IgA antibodies (University of Erlangen-Nuremberg), TP-38 (IVAX), EGFR fusion protein, EGF-vaccine, anti-EGFr immunoliposomes (Hermes Biosciences Inc.) and combinations thereof.

Preferred EGFr inhibitors include Iressa, Erbitux, Tarceva and combinations thereof. 5 The present invention also relates to anti-tumor agents selected from pan erb receptor inhibitors or ErbB2 receptor inhibitors, such as CP-724,714 (Pfizer, Inc.), CI-1033 (canertinib, Pfizer, Inc.), Herceptin (trastuzumab, Genentech Inc.), Omitarg (2C4, pertuzumab, Genentech Inc,), TAK-165 (Takeda), GW-572016 (Ionafamib, GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth), PK1-166 (Novartis), dHER2 (HER2 Vaccine, Corixa and GlaxoSmithKline), APC8024 (HER2 Vaccine, 10 Dendreon), anti-HER2/neu bispecific antibody (Decof Cancer Center), B7.her2.lgG3 (Agensys), AS HER2 (Research Institute for Rad Biology &amp;amp; Medicine), trifuntional bispeclfic antibodies (University of Munich) and mAB AR-209 (Aronex Pharmaceuticals Inc) and mAB 2B-1 (Chiron) and combinations thereof. Preferred erb selective anti-tumor agents include Herceptin, TAK-165, CP-724,714, ABX-EGF, HER3 and combinations thereof. Preferred pan erbb receptor Inhibitors include GW572016, CI-1033, EKB-569, and 15 Omitarg and combinations thereof.

Additional erbB2 Inhibitors Include those in WO 98/02434 (published January 22, 1998), WO 99/35146 (published July 15, 1999), WO 99/35132 (published July 15, 1999), WO 98/02437 (published

January 22, 1998), WO. 97/13760. (published April, 17, 1997), WO 95/19970 (published July 27, 1995), United States Patent 5,587,458 (issued December 24,1996), and United States Patent 5,877,305 (issued 20 . March 2, .1999), each of which is herein incorporated by reference in its entirety. For additional ErbB2 receptor inhibitors useful In the present invention, see United States Patent Nos. 6,465,449, and 6,284,764, and international Application No. WO 2001/98277 each of which are herein incorporated by reference in theirentirety.

Additionally, other anti-tumor agents may be selected from the following agents, Sorafenib (Onyx 25 . Pharmaceuticals .Inc.; BAY-43-9006), Genasense (augmerosen, Genta), Panftumumab (Abgenix/Amgen), Zevalin (Schering), Bexxar (Corixa/GlaxoSmlthKline), Abarelix, Alimta, EPO 906 (Novartis), discodermolide (XAA-296), ABT-510 (Abbott), Neovastat (Aetema), enzastaurin (Eli Lilly), Combrestatin A4P (Oxigene), ZD-6126 (AstraZeneca), flavopiridol (Aventis), CYC-202 (Cyclacel), AVE-8062 (Aventis), DMXAA (Roche/Antisoma), Thymitaq (Eximias), Temodar (temozolomide, Schering Plough) and Revilimd 30 (Celegene) and combinations thereof.

Other anti-tumor agents may be selected from the following agents, CyPat (cyproterone acetate), Histerelin (histrelin acetate), Plenalxis (abarelix depot), Atrasentan (ABT-627), Satraplatin (JM-216), thalomid (Thalidomide), Theratope, Temllifene (DPPE), ABI-007 (paclitaxel), Evista (raloxifene), Atamestane (Biomed-777), Xyotax (polyglutamate paclitaxel), Targetin (bexarotine) and combinations 35 thereof.

Additionally, other anti-tumor agents may be selected from the following agents, Trizaone (tirapazamine), Aposyn (exisulind), Nevastat (AE-941), Ceplene (histamine dihydrochloride), Orathecin (rubitecan), Virulizin, Gastrimmune (G17DT), DX-8951f (exatecan mesylate), Onconase (ranpirnase), BEC2 (mltumoab), Xcytrin (motexafin gadolinium) and combinations thereof. -66-

Further anti-tumor agents may be selected from the following agents, CeaVac (CEA), NeuTrexin (trimetresate glucuronate) and combinations thereof. Additional anti-tumor agents may selected from the following agents, OvaRex (oregovomab), Osidem (IDM-1), and combinations thereof. Additional antitumor agents may be selected from the following agents, Advexin (ING 201), Tirazone (tirapazamine), and combinations thereof. Additional anti-tumor agents may selected from the following agents, RSR13 (efaproxiral), Cotara (1311 chTNT 1/b), NBI-3001 (IL-4) and combinations thereof. Additional anti-tumor agents may be selected from the following agents, Canvaxin, GMK vaccine, PEG Interon A, Taxoprexin (DHA/paciltaxel) and combinations thereof.. Other preferred anti-tumor agents include Pfizer's MEK1/2 inhibitor PD325901, Array Biopharm's MEK inhibitor ARRY-142886, Bristol Myers' CDK2 Inhibitor BMS-387,032, Pfizer's CDK inhibitor PD0332991 and AstraZeneca's AXD-5438 and combinations thereof. Additionally, mTOR inhibitors may also be utilized such as CCI-779 (Wyeth) and rapamycin derivatives RAD001 (Novartis) and AP-23573 (Ariad), HDAC inhibitors SAHA (Merck Inc./Aton Pharmaceuticals) and combinations thereof. Additional anti-tumor agents include aurora 2 inhibitor VX-680 (Vertex), Chk1/2 inhibitor XL844 (Exilixis).

The following cytotoxic agents, e.g., one or more selected from the group consisting of epirubicin (Ellence), docetaxel (Taxotere), paclitaxel, Zinecard (dexrazoxane), rituximab (Rituxan) imatinib mesylate (Gleevec), and combinations thereof, may be used in conjunction with a Anti-ALK-1 antibody or antigen binding portion thereof, as described herein, and pharmaceutical compositions thereof , as described herein.

The invention also contemplates the use of the antibodies and antigen-binding portions thereof of the present invention together with hormonal therapy, including but not limited to, exemestane (Aromasin, Pfizer Inc.), leuprorelin (Lupron or Leuplin, TAP/Abbott/Takeda), anastrozole (Arimidex, Astrazeneca), gosreiln (Zoladex, AstraZeneca), doxercalciferol, fadrozole, formestane, tamoxifen citrate (tamoxifen, Nolvadex, AstraZeneca), Casodex (AstraZeneca), Abarelix (Praecis), Trelstar, and combinations thereof... .

The invention also relates to hormonal therapy agents such as. anti-estrogens Including, but not limited to fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole (Femara, Novartis), anti-androgens such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex®(4'-cyano-3-(4-fluorophenylsulphonyI)-2-hydroxy-2-methyl-3'-(trifiuoromethyl) , propionaniiide, bicalutamide) and combinations thereof. . ,

Further, the invention provides antibodies of the present Invention alone or in combination with one or more supportive care products, e.g., a product-selected from the group consisting of Filgrastim (Neupogen), ondansetron (Zofran), Fragmin, Procrit, Aloxl, Emend, or combinations thereof.

Particularly preferred cytotoxic agents include Camptosar, Erbltux, Iressa, Gleevec, Taxotere and combinations thereof.

The following topoisomerase I inhibitors may be utilized as anti-tumor agents camptothecin, irinotecan HCI (Camptosar), edotecarin, orathecin (Supergen), exatecan (Daiichi), BN-80915 (Roche) and combinations thereof. Particularly preferred toposimerase II Inhibitors include epirubicin (Eilence). -67-

The antibodies of the invention may be used with antitumor agents, alkylating agents, antimetabolites, antibiotics, plant-derived antitumor agents, camptothecin derivatives, tyrosine kinase inhibitors, other antibodies, interferons, and/or biological response modifiers.

Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, 5 Ifosfamide, melphalan, busulfan, mltobronltol, carboquone, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, altretamine, AP-5280, apazlquone, brostallicln, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, mafosfamide, and mitolactol; platinum-coordinated alkylating compounds include but are not limited to, cisplatin, Paraplatln (carboplatin), eptaplatin, lobaplatin, nedaplatin, Eloxatin (oxaliplatin, Sanofi) or satrplatin and combinations thereof. 10 -Particularly preferred alkylating agents include Eloxatin (oxaliplatin).

Antimetabolites include but are not limited to, methotrexate, 6-mercaptopurine -riboside, mercaptopurine, 5-fluorouracil (5-FU) alone orin combination with leucovorin, tegafur, U FT,, doxifluridine, carmofur, cytarabine, cytarabine ocfosfate; enocitablne, S-1, Alimta (premetrexed disodium, LY231514, MTA), Gemzar (gemcitabine, Ell Lilly), fludarabln, 5-azacitidine, capecitabine, cladribine, clofarabine, 15 decitabine, eflomithine, ethynylcytidine, cytosine arabinoside, hydroxyurea, TS-1, melphalan, nelarabine, nolatrexed, ocfosfate, dfsodium premetrexed, pentostatin, pelftrexol, raltitrexed, triapine,- trimetrexate, vidarabine, vincristine, vinorelbine; or for example, one of the preferred anti-metabolites disclosed In European Patent Application No. 239362 such as N-(5-[N~(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamIno]-2-then0yi)-L-glutamic acid and combinations thereof. 20 Antibiotics include intercalating antibiotics but are not limited to: aclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, bleomycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, galarubicln, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin, stimalamer, streptozocin, valrubicin, zinostatin and combinations thereof. . Plant derived anti-tumor substances include for example those selected from mitotic inhibitors, for 25 example vinblastine, .docetaxel (Taxotere), paclitaxel and combinations thereof. &lt;

Cytotoxic topoisomerase inhibiting agents include one or more agents selected from-the group consisting of actarubicn, -amonafide, belotecan, camptothecin, -1 Orhydroxycamptothecin, ·· 9-aminocamptothecin, dlflomotecan,· irinotecan HCI (Camptosar), edotecarin, epirubicin (Ellence), etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirarubicin, pixantrone, rubitecan, sobuzoxane, 30 . SN-3.8, tafluposide, topotecan, and combinations thereof:· · ·

Preferred cytotoxic topoisomerase inhibiting agents include one or more agents selected from the group consisting of camptothecin, 10-hydroxycamptothecln, 9-aminocamptothecin, . irinotecan HCI (Camptosar), edotecarin, epirubicin (Ellence), etoposide, SN-38, topotecan, and combinations thereof..

Immunologicals include interferons and numerous other immune enhancing agents. Interferons 35 Include interferon alpha, interferon aipha-2a, interferon, alpha-2b, interferon beta, interferon gamma-1a, Interferon gamma-1 b (Actimmune), or Interferon gamma-n1 and combinations thereof. Other agents include filgrastim, lentinan, sizofilan, TheraCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa), molgramostim, OncoVAX-CL, sargramostim, tasonermin, tecleukin, 189,642/2 -68- thymatasin, tositumomab, Virulizin, Z-100, epratuzumab, mitumomab, oregovomab, pemtumomab (Y-muHMFGI), Provenge (Dendreon) and combinations thereof.

Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses, such as survival, growth, or differentiation of tissue cells to direct them to have anti- 5 tumor activity. Such agents include krestin, lentinan, sizofiran, picibanil, ubenimex and combinations thereof.

Other anticancer agents include aiitretinoin, ampligen, atrasentan bexarotene, bortezomib. Bosentan, calcitriol, exisulind, finasteride,fotemustine, ibandronic acid, miltefosine, mitoxantrone, I-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazarotne, 10 Telcyta (TLK-286, Telik Inc.), Velcade (bortemazib, Millenium), tretinoin, and combinations thereof.

Other anti-angiogenic compounds include acitretin, fenretinide, thalidomide, zoledronic acid, angiostatin, apiidine, cilengtide, combretastatin A-4, endostatin, halofuginone, rebimastat, removab, Revlimid, squalamine, ukrain, Vitaxin and combinations thereof.

Platinum-coordinated compounds include but are not limited to, cisplatin, carboplatin, nedaplatin, 15 oxaliplatin, and combinations thereof.

Camptothecin derivatives include but are not limited to camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan and combinations thereof.

Other antitumor agents include mitoxantrone, l-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pentostatin, tretinoin and combinations thereof.

Anti-tumor agents capable of enhancing antitumor immune responses, such as CTLA-4 (cytotoxic lymphocyte antigen 4) antibodies, and other agents capable of blocking CTLA-4 may also be utilized, such as MDX-010 (Medarex) and CTLA-4 compounds disclosed in United States Patent No. 6,682,736; and anti-proliferative agents such as other farnesyl protein transferase inhibitors, for example the farnesyl protein transferase inhibitors. For additional, specific CTLA-4 antibodies that can be used in the present invention see United States Patent No. 7,109,003, United States Patent No. 6,682,736 both of which are herein incorporated by reference in their entirety. For example, another anti-CTLA-4 antibody that can be used in accordance with the present invention is ticilimumab, which has the sequence of monoclonal antibody 11.2.1 in US Patent 6,682,736.

For specific IGF1R antibodies that can be used in the present invention, see international Patent Application No. WO 2002/053596.

For specific CD40 antibodies that can be used in the present invention, see international Patent Application No. WO 2003/040170.

Gene therapy agents may also be employed as anti-tumor agents such as TNFerade (GeneVec), which express TNFalpha in response to radiotherapy.

In one embodiment of the present invention, statins may be used, in conjunction with a Anti-ALK-1 antibody or antigen binding portion thereof, as described herein, and pharmaceutical compositions thereof.

Statins (HMG-CoA reducatase inhibitors) may be selected^ from the group consisting of Atorvastatin (Lipitor, Pfizer Inc.), Pravastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.),

Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin -69- (Crestor, AstraZeneca), Lovostatin and Niacin (Advicor, Kos Pharmaceuticals),, derivatives and combinations thereof.

In a preferred embodiment the statin is selected from the group consisting of Atovorstatin and Lovastatin, derivatives and combinations thereof. 5 Other agents useful as anti-tumor agents include Caduet

For any of the methods of treating a hyperproliferative disorder or abnormal cell growth as described herein using a combination of an anti-ALK-1 antibody or antigen binding portion with at least one additional therapeutic agent, the anti-ALK-1 antibody can be conjugated, or derivatized, with the additional therapeutic agent The at least one additional therapeutic agent can also be administered 10 separately, or in a non-derivatized or non-conjugated mariner. When the at least one additional therapeutic agent is not derivatized or conjugated to the antibody, it can be administered within· the same • pharmaceutical formulation as the antibody, or it can be administered in a separate formulation.

Treatment of Vision Loss

The inventive compounds and pharmaceutical compositions containing them, are useful for 15 treating severe vision loss from age-related macular degeneration and other diseases affecting the posterior segment of the eye, such as choroidal neovascularization, diabetic retinopathy, glaucoma, retinitis pigmentosa, and the like;

For example, the inventive compositions may be used to form a drug depot behind the eye and may include one or more pharmaceutically active agents, in addition to one or more non-active excipients 20 as described herein. Examples of pharmaceutically active agents useful in the inventive compositions includes anti-infectives, including, without limitation, antibiotics, antivirals, and antifungals; antiailergenic agents and mast cell stabilizers; steroidal and nonsteroidal anti-inflammatory agents (such-asnepafenac); cyclooxygenase inhibitors, including, without limitation, Cox I and Cox II Inhibitors; combinations of anti- . infective and anti-inflammatory agents; decongestants; anti-glaucoma agents, including, without limitation, 25 adrenergics, beta-adrenergic blocking agents,, alpha-adrenergic agonists, parasypathomimetlc agents, cholinesterase inhibitors, carbonic anhydrase inhibitors, and prostaglandins; combinations of antl- . .glaucoma agents; antioxidants; nutritional supplements; drugs for the treatment of cystoid macular edema including, without limitation, non-steroidal anti-inflammatory agents; drugs for the treatment of age related macular degeneration (AMD) including nonexudative (dry) and exudative.(wet) AMD, including, without 30 limitation, angiogenesis inhibitors, including angiogenesis inhibitors that inhibit protein kinase receptors, including protein kinase-receptors that are-VEGF receptors; and nutritional supplements; drugs for the treatment of herpetic infections and CMV ocular infections; drugs for the treatment of proliferative vitreoretinopathy including, without limitation, antimetabolites and fibrinolytics; wound modulating agents, including, without limitation, .growth factors; antimetabolites; neuroprotecttve drugs, including, without 35 limitation, .eliprodil;. and angiostatic steroids .for the treatment of diseases or conditions of posterior segment 26, including, without limitation; age related macular degeneration (AMD) including nonexudative (dry) and exudative (wet) AMD, choroidal neovascularization, retinopathies, retinitis, uveitis, macular edema, and glaucoma. For additional information about such angiostatic steroids see U.S. Patent Nos. -70- 5,679,666 and 5,770,592. A non-steroidal anti-inflammatory for the treatment of cystoid macular edema is nepafenac.

For administration to the eye, a compound of the present invention is delivered in a pharmaceutically acceptable ophthalmic vehicle such that the compound is maintained in contact with the . 5 ocular surface for a sufficient time period to allow the compound to penetrate the cornea and/or sclera and internal regions of the eye, Including, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary's, lens, choroid/retina and sclera.. The pharmaceutically acceptable ophthalmic vehicle may be an ointment, vegetable oil, or an encapsulating, material. A compound of the invention may also be injected directly into the vitreous humor or aqueous 10 humor.

Further, a compound may be also be administered by well known, acceptable methods, such as sub-Tenon and/or subconjunctival injections. As is well known in the ophthalmic art, the macula is comprised primarily of retinal cones and is the region of maximum visual acuity in the retina. A Tenon's capsule or Tenon's membrane is disposed on the sclera. A conjunctiva covers a short area of the globe of 15 the eyeposterior to the limbus (the bulbar conjunctiva) and folds up (the upper cui-de-sac) or down-(the lower cul-de-sac) to cover the inner areas of. the upper eyelid and lower eyelid,. respectively. The conjunctiva is disposed on top of Tenon's capsule. The sclera and Tenon's capsule define the exterior . surface of the globe of the eye. For treatment of ocular diseases such as age related macular degeneration (AMD) including nonexudative (dry) and exudative (wet) AMD, choroidal neovascularization, 20 retinopathies (such as diabetic retinopathy, retinopathy of prematurity), diabetic macular edema, retinitis, ' uveitis, cystoid macular edema (CME), glaucoma, and other diseases or conditions of the posterior segment of the eye, it is preferable to dispose a depot of a specific quantity of an ophthalmically acceptable pharmaceutically active agent directly on the outer surface of the sclera and below Tenon's ... capsule. Iri addition, in cases of age related macular degeneration (AMD) including nonexudative (dry) 25 and exudative (wet) AMD and CME it is most preferable to dispose the depot directly on the outer surface of the sclera, below Tenon's capsule, and generally above the macula.

The compounds may. be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or Intramuscularly) intramuscular injection or by the above mentioned sub-Tenon or intravitreal injection. Alternatively, the active ingredient may be in 30 powder form for constitution with a suitable.vehicle, e.g.,.sterile pyrogenrfree water, before use.·

Within particularly preferred embodiments ofthe invention, the compounds may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, the present 35 " compositions, prepared as described above, may also be administered directly to the. cornea.

Within preferred embodiments, the composition is prepared with a muco-adhesive polymer that binds to cornea. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt -71 - A pharmaceutical carrier for hydrophobic compounds is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be a VPD co-solvent system. VPD Is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute 5 ethanol. The VPD co-solvent system (VPD:5W) contains VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent, system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example,, other low-toxicity nonpolar surfactants may be used 10 instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e;g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic 15 drugs. Gertain organic solvents such as dimethylsulfoxide also may be.employed, although usually at the cost of greater toxicity. Additionally, the compounds may. be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent Various sustained-release materials have been established and are known by those skilled In the art. Sustained-,release capsules may, depending on their chemical nature, release the compounds for a few weeks up to 20 over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, ,. sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. 25 Any of the compositions can be formulated for administration to an individual. An individual of the present Invention is preferably a mammal, or more preferably a human.

The pharmaceutical formulations herein can further include a therapeutic agent selected from the group consisting of: an antineoplastic agent, an anti-infiammatory agent; an antibacterial agent, an antiviral agent, an angiogenic agent, and an anti-angiogenic agent Examples of such agents are 30 . disclosed herein. .

For example, an antineoplastic agent may be selected from the group consisting of Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin ; Azacitidine; Azetepa; -Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; 35 Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;. Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin ; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide ; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; -72-

Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride ; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbuiozoie; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; 5 Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198 ; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofoslne; Interferon Alfa-2a; Interferon Alfa-2b ; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-la; Interferon Gamma-lb; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone 10 Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate;. Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; - Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; 15 Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomyclnl, Spirogermanium. Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; 20 Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine;. Topotecan Hydrochloride; Toremifene . Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepldlne Sulfate; Vlnglycinate Sulfate; Vlnleurosjne Sulfate; Vinorelbine 25 Tartrate; Vinrosldlne Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.

An anti-anglogenic agents are any agents that inhibit angiogenesis, whether disclosed herein or known tn the art In preferred embodiments, an anti-angiogenic agent is an anti-VEGF agent, such as

Macugen™ (Eyetech, New York, NY); or anti-VEGF antibody.

Pharmaceutical compositions can be formulated by standard techniques using one or more 30 suitable carriers, excipients, and dilutents. See, e.g., Remington's Pharmaceutical Sciences, (19°1 Ed. Williams &amp;amp; Wilkins, 1995) (incorporated herein by reference for all purposes).

Formulations suitable for parenteral administration include aqueous and non-aqueous formulations isotonic with the blood of the intended recipient; and aqueous and non-aqueous .sterile suspensions which may incltide suspending -systems designed to target the compound to blood 35 components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules or vials. For Intraocular formulations, unit dosages are preferred because no preservatives are in the formulation. For other parenteral formulations, preservative may be used, which would allow formulti dose containers -73-

Extemporaneous injections solutions and suspensions may be prepared, for example, from sterile powders. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Particular parenteral administrations contemplated by the present invention include intraocular and intravitreous administrations to the eye. Pharmaceutical formulations for intraocular and intravitreous administrations include phosphate buffered saline (PBS) and balanced isotonic salt solution (BSS) with or without excipients such as mannitol or sorbitol as protein stabilizers. in general, water, suitable oil, saline, aqueous dextrose (glucose), or related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions tor parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents, such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid salts thereof, or sodium EDTA. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl- or propyi-paraben, dr chlorobutanol. Suitable pharmaceutical carriers are described in Remington, cited supra.

In any of the embodiments herein, a composition or pharmaceutical formulation herein may by lypholized.

In any of the embodiments herein, the pharmaceutical formulations preferable have-less than - about 10, more preferably less than about 5, more preferably less than about 3, or more preferably less than about 1 endotoxin unit(s) per milligram of therapeutic agents. tn some embodiments, the methods of treatment disclosed herein further include administering to an individual suffering from an angiogenic condition one or more therapeutic agents selected from the group consisting of antineoplastic agents, antiviral agents, anti-inflammatory agents, antibacterial agents, anti-angiogenic agents,, or antl-angiogenic agents.

Such combination treatments can be achieved by-.either administering to an individual, a. coformulating of the compositions herein with the additional therapeutic agent(s) or by administering the compositions herein and the-therapeutic agent(s)· as-two separate ·pharmaceutical formulations, tn embodiments wherein more than one composition/the'rapeutic agent is administered to an Individual, lower dosages of the compositions and/or therapeutic agent(s) may be utilized as a resuit of the synergistic effect of both active ingredients.

Antineoplastic agents' that may be administered to an individual include, but are not limited to, Aclarubicin; Acodazole Hydrochloride; Acronlne; Adozelesln; Aldesleukin; Altretamine; Ambomycln; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperiin ; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequlnar Sodium; Bropirimine; Busulfan; Cactinomycin;-Calusterone; Caracemlde; Carbetimer; Carboplatin; Carmustine; Carubicln Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin ; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide ; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitablne; Dexormaplatin; Dezaguanlne; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin -74-

Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate;

Eflomithine Hydrochloride ; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride;

Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole;

Ethiodized Oil 1131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride;. Fazarabine; 5 Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198 ; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofosine; Interferon Alfa-2a; Interferon Alfa-2b ; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-la; Interferon Gamma-lb; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone 10 Hydrochloride; Masoprocol; Maytansfne; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate .Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;. Ormaplatin; Oxlsuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustlne; Peplomycin Sulfate; Perfosfamide; 15 Pipobroman; Piposulfan; Plroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium;

Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurln; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl, Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; 20 Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxlrone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tlrapazamine; Topotecan Hydrochloride;. Toremifene Citrate; Trestolone Acetate; Tricirlbine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole . Hydrochloride; Uracil Mustard; Ufodepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine 25 . Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.

Antibacterial agents that may be administered to an individual include, but are not limited to, penicillins, aminoglycosides, macroiides, monobactams, rifamycins, tetracyclines, chloramphenicol, clindamycin, lincomycin, imipenem, fusldic acid, novobiocin, fosfomycln, fusidate sodium, neomycin, polymyxin, capreomycin, colistimethate, colistin, gramicidin, minocycline, doxycycline, vanomycin, 30. bacitracin, kanamycin,, gentamycin, erythromicin and cephalosporins.

Anti-infiammatory agents that may be administered to an individual include, but are not limited to, NSAIDS (e.g., aspirin (salicy(amide), sodium sallcylamide, indoprofen, indomethacin; sodium Indomethacin trihydrate, Bayer™, Bufferin'™, Celebrex™, diclofenac,· Ecotrin™, diflunisal,. fenoprofen, naproxen, sulindac, Vioxx™), corticosteroids or corticotropin (ACTH), colchicine, and anecortave acetate. 35 Antiviral agents that may be administered to an individual include, but are not limited to, a-methyl- p-adamantane methylamine, 1,-D-ribofuranosy|-1,2,4-triazole-3 carboxamide, 9-[2-hydroxy-ethoxyjmethylguanlne, adamantanamine,· 5-lodo-2’-deoxyuridine, trifluorothymidine, Interferon, adenine arabinoside, CD4, 3’-azldo-3'-deoxythymidlne (AZT), 9-{2-hydroxyethoxymethyl)-guanine (acyclovir), phosphonoformlc acid, 1-adamantanamine, peptide T, and 2’,3'dideoxycy.tidine. -75-

Administration of a composition of the present invention to a target cell in vivo can be accomplished using any of a variety of techniques well known to those skilled in the ait

For example, compositions of the present invention can be administered systemically or locally by any means known in the art (e.g., orally, intraocularly, intravascularly (i.v.), intradermally, intramuscularly, 5 transdermally, transmucosally, enterically, parentafiy, by inhalation spray, rectally, or topically) in dosage unit formulations and containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.

As used herein the term intraocularly includes intravitreal, sub-retinal, and the like.

As used herein the term parenteral as used herein includes, subcutaneous, intravenous, 10 intramuscular, intrasternal, infusion techniques or intraperitoneally. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-kritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid atthe recta) temperature and will therefore melt in the rectum and release the drug. : .

The dosage regimen for treating a disorder or a disease with the· compositions of this invention is 15 based on a variety of factors, including the type of disease, the age, weight, sex, medical condition ofthe patient, the severity..of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods.

For systemic administration, the anti-ALK-1 antibody or antigen-binding portion thereof of the present invention and/or one or more additional therapeutic agents are preferably administered at a dose 20 . of at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 46, 50, 75, 100, or 150 mg/kg body weight In other embodiments, the polypeptides (preferably dimers or homodimers) and/or small molecules herein are administered systemically at a dose of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-30 mg/kg.body weight, or more preferably 5-20 mg/kg.

For localized administration, the anti-ALK-1 antibody or antigen-binding portion thereof of the 25 present invention and/or one or more additional therapeutic agents are preferably administered at a dose .. of at least 50 pg, 100 pg; 150 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg,· 550 pg, 600 pg, 650 pg, or 700 pg. In other embodiments, the polypeptides (preferably dimers or homodlmers) and/or small molecules herein are administered locally at a dose of 50-1000 pg, more .preferably 100-800 pg, ‘ more preferably 200-500 pg, or more preferably 300-400 pg per site. 30 For example, for dermal administration the anti-ALK-1 antibody-or antigen-binding portion thereof of the present invention and/or peptidomimetics and/or one or more additional therapeutic agents are . . administered at a dose of 50-1000 pg/cm2, more preferably 100-800 pg/cm2, or more preferably 200-500 pg/cm2. In another example, for ocular administration,· the polypeptides and/or peptidomimetics and/or shnall molecules of the present invention are administered at a dose of 50-1000 pg/eye, more preferably 35 100-800 pg/eye, or more preferably 200-500 pg/eye.

The pharmaceutical compositions preferably include the active ingredient (e.g., an antl-ALK-1 antibody) in an effective amount, I.e., in an amount effective to achieve therapeutic or prophylactic benefit The actual amount effective for a particular application will depend on the condition being treated and the -76- route of administration. Determination of an effective amount is well within the capabilities of those skilled in the art, especially In light ofthe disclosure herein.

Preferably, the effective amount of the active ingredient, e.g., an anti-ALK-1 antibody, is from about 0.0001 mg to about 500 mg active agent per kilogram body weight of a patient, more preferably 5 from about 0.001 to about 250 mg active agent per kilogram body weight of the patient, still more preferably from about 0.01 mg to about 100 mg active agent per kilogram body weight of the patient, yet still more preferably from about 0,5 mg to about 50 mg active agent per kilogram body weight of the patient, and most preferably fromabout 1 mg to about 15 mg active agent per kilogram body weight of the patient. - 10 In terms of weight percentage, the formulations of the .present invention will preferably comprise the active agent, e.g., an anti-ALK-1 antibody, in an amount of from about 0.0001 to about 10 wt. %, more preferably from about 0.001 to about 1 wt. %, more preferably from about 0.05 to about 1 wt %, or more preferably about 0.1 wt. to about 0.5 wt. %.

Gene Therapy 15 The nucleic acid molecules that encode the antibodies and antibody portions of the present invention can be administered to a patient in need thereof via gene therapy. The therapy may be either in vivo or ex vivo, in a preferred embodiment, nucleic acid molecules encoding both a heavy chain and a Tight chain are administered to a patient In a more preferred embodiment the nucleic acid molecules are administered such that they are stably integrated into chromosomes of 6 cells because these cells are 20 specialized for producing antibodies. In a preferred embodiment, precursor B ceils are transfected or infected ex vivo and re-transplanted into a patient In need thereof, in anotherembodiment, precursor B cells or other cells are· infected in vivo using a virus known to infect the ceil type of interest Typical .. vectors used for gene therapy include liposomes, plasmids, and viral vectors. Exemplary viral vectors are retroviruses, adenoviruses and adeno-associated viruses. After infection either in vivo or ex vivo, levels of 25 ' antibody expression can be monitored by taking a sample from the treated patient and using any immunoassay known in the art or discussed herein. .

In a preferred embodiment, the gene therapy method comprises the steps, of .administering an isolated nucleic acid molecule encoding the heavy chain or an antigen-binding portion thereof of an anti-ALK-1 antibody and expressing the nucleic acid molecule, in another embodiment, the gene therapy 30 method comprises the steps of administering an isolated nucleic acid molecule encoding the light chain or an antigen-binding portion thereof of an anti-ALK-1 antibody and expressing the nucleic acid molecule. In a more preferred method, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy .chain or an antigen-binding portion thereof and an isolated nucleic acid molecule encoding the light chain or the antigen-binding portion thereof of an anti-ALK-1 35 antibody of the invention and'expressing the nucleic acid molecules. The gene therapy method may also comprise the step of administering another therapeutic agent, such as any of the agents discussed previously in connection with combination therapy. -77-

Method for Screening ALK-1 Antagonists or Agonists

In one embodiment, the present invention provides a method for determining if a substance . . inhibits up-regulation of a.specific downstream target gene of ALK-1, Id1, such as, for example, the 5 Taqman Assay for IdT desribed in Example 12. The method comprises contacting a sample of cells that express Id1 with the substance and determining if IdT expression is inhibited, wherein a reduced level of Id1 expression in the sample of cells contacted with the substance as compared to a control sample of ' cells is indicative of said substance inhibiting Id1 expression, in one specific embodiment, the substance is an antibody that binds to the extracellular domain of ALK-1. In another embodiment, the substance is a 10 . small molecule. According to the invention, the cells can Inherently express both ALK-1 and Id1, such as HUVECs desribed in Example 12, or which have been transformed or transfected with DNA encoding one or both of these. One can determine the expression of Id1 via, e.g., the use of Taqman Assay for Id1 desribed in Example 12.

Conversely, activators or agonists can also be tested for, or utilized, following the same type of 15 procedures.

In order .that this invention may be better understood, the following examples are set forth. These examples are for purposes of Illustration only and are not to be construed as limiting the scope of the invention in any manner. . · 20 . Examples

In the following examples and preparations, “MW" means molecular weight; “His-Tag" means C-terminai polyhistidine (6xHls) tag for rapid purification with nickel-chelating resin and detection with an • anti-His (C-term) antibody; . "BSA" means bovine serum .· albumin; . "EDTA" means ethylenediaminetetraacetic acid; "DMSO" means dimethyl sulfoxide; “MOPS" means 3-(N-morpholino) 25 propanesuifonic acid; "MES" means 2-(N-Morpholtno)ethanesulfonlc acid; "PBS" means phosphate buffered saline; "dPBS" means Dulbecco's phosphate buffered saline; "HEMA" means 2-hydroxy-ethyi methacrylate; “DMEM" means Dulbecco's modified eagle's medium; “FBS" means fetal bovine serum; , “NEAA" means non-essential amino acids; “HEPES"· means N-2-hydroxyethylpiperazine-N’-2-ethanesuifonic acid; and “DMF" means dimethyl formamide. 30 - "

Example 1, ALK-T Immunogen Preparation

The ECD of ALK-1 was cloned from full-length human ALK-1 ORF clone (Invitrogen, Clone ID IOH21048) by PCR using the forward 5’-ACGGCCCAGCCGGCCGACCCTGTGAAGCCGTCT (SEQ ID NO: 96) and 35 reverse 5'-ACTAAGCT7TTAATGATGATGATGATGATGCTGGCCATCTGTTCCCG (SEQ ID NO: 97) primers. The PCR product was purified, treated with the Sfil and Hindlll restriction enzymes, and cloned into the Sfll/HIndlll site of a mammalian expression vector pSecTag2/Hygro (Invitrogen Inc., Catalog No.V910-20). The clone was used to transiently transfect 293T cells with Fugene 6 transfection reagent -78- (Roche Applied Science, Catalog No.1814443) following manufacture's instruction^ Supernatant from the cell culture containing the secreted target protein was harvested 72 hours post-transfection and allowed to bind to Ni-NTA resin (QIAGEN, Catalog No.30430) at 4°C overnight The resin was then washed with buffer containing 20 mM Tris pH 8.0, 25 mM imidazole and 300 mM sodium chloride. The His-Tag protein 5 was eluted off the resin using buffer containing 20 mM Tris pH 8.0, 300 mM imidazole and 300 mM sodium chloride. A CM Sepharose cation exchange resin was used to further purify the protein in 20 mM sodium phosphate (pH 7.0), and the unbound fraction containing the target protein was collected. The protein was buffer exchanged to PBS or 10 mM HEPES, pH 7.4, plus 150 mM sodium chloride by dialysis and concentrated to 0.2-1 mg/mL with a final purity of >90%, judged by SDS PAGE gel stained with 10 Coomassie blue. The ALK-1 ECD His-Tag protein was heavily glycosylated with an apparent MW of 26 KDa, comparing to an 11 KDa theoretical MW for the protein. The ALK-1 ECD His-Tag protein (SEQ ID NO: 98) has been used for generation of hybridomas producing anti-ALK-1 antibody as described in Example 2.

Human ALK-1 ECD His-Tag protein: 15 gene sequence (lowercase partis the secretion signal):

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgcggcccagccggccGACCCTGTGAAGC

CGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGG

GGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCT

GCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGC 20 GACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAG CCGGGAACAGATGGCCAGCATCATCATCATCATCAT(SEQ ID NO: 99) protein sequence: DPVKPSRGPLVTCTCESPHCKGPTCRGAWCTWLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHY CCDSHLCNHNVSLVLEATQPPSEQPGTDGQHHHHHH (SEQ ID NO: 98) 25 ' ...... • Example 2, Generation of Hybridomas Producing Anti-ALK-1 Antibody

Eight to ten week old XENOMOUSE® mice were immunized in their hind footpads with 10 pg/ mouse of either recombinant human ALK-1/Fc chimera (R&amp;amp;D Systems, Inc., Catalog Number 370-AL) or with the ALK-1 ECD His-Tag protein described in Example 1. This dose was repeated five to seven times 30 over a three to five week period. Three or four days before fusion, the mice were given a. final injection of the immunogen in PBS. The lymph node lymphocytes from immunized mice were fused with the non- secretory myeloma P3-X63-Ag8.653 cell line via electro cell fusion (ATCC Cat No. CRL 1580), and these fused cells were subjected to HA-DMEM selection as previously described (DMEM/15%FBS/1% 200 mM L-glutamlne/1% 100X Non-essential amino acid/1% 100X Pen/Strep/10 U/ml 1L-6/1 viai/liter OPI media 35 supplement plus 0,5x HA (Azaserine-Hypoxanthine; Sigma, Cat # A9666)). A panel of hybridomas was recovered that all secrete ALK-1 specific human lgG2 antibodies. ELISA assay was used to detect antibody binding. Immunogen was coated to the 96-well immulon microtiter plate (NUNC-lmmuno™ plate MaxISorp™ surface, Nalge Nunc International, Cat - 79 · 10 15 20 25 30 35 Νο.439454) at 4pg/mL in 50 mM sodium bicarbonate buffer for overnight at 4°C. Plates were washed, and then blocked with PBS with the addition of 0.1% Tween-20 and 0.5% bovine serum albumin. Antibodies were' added to the blocked ELISA plates, incubated for 1 hour, and washed with PBS with Tween-20. The binding was detected by anti-human IgG-horseradish peroxidase (Pierce, Catalog No. 31420) followed by the addition of ABTS (Pierce, Catalog No. 37615). Colorimetric measurements were performed at 405 nm in amicro-plate reader (SpectraMax Plus 384, Molecular Devices).

Twenty five hybridomas were selected for further study. These were single-cell cloned by limiting dilution and were designated 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A);'1.13.1; 1.14.1; 1.T5T.1; 1.162.1; 1.-183:4; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1.

Mouse Hybridoma Cell line LN 15916 (the hybridoma 1.12.1) was deposited under terms In accordance with the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 on June 21, 2005. The hybridoma 1.12.1 has been assigned the following accession number PTA-6808.

Example 3, Sequences of Antt-ALK-1 Antibodies

To analyze the structure of antibodies produced In accordance with the invention, nucleic acids were cloned that encode heavy and light chain fragments from hybridomas producing anti-ALK-1 monoclonal antibodies. Cloning and sequencing was accomplished by standard means.

Poly(A)+ mRNA was isolated using a Fast-Track™ kit (Invitrogen) from approximately 2 X 106 hybridoma cells for each of the ALK-1 ‘ antibodies. cDNA was synthesized from the mRNA by using random primers. The random primed cDNA was amplified by· PCR using human VH or human Vk family specific variable domain primers In conjunction with primers specific for the human Cy2 constant region, or a Ck constant region to amplify the· antibody variable region, including all the framework regions (FRs) and complementarity determining regions (CDRs). Nucleic acid sequences were obtained that encode human heavy and kappa light chain transcripts from the anti- ALK-1 producing hybridomas by direct sequencing of- both strands of PCR products. Sequences were analyzed using Abgenixta proprietary software and publicly available sequence information for human VH and Vk genes, the *V BASE sequence directory", Tomlinson et al., MRC Centre·for Protein Engineering, Cambridge, UK). Identical results could be obtained using publicly available sequence alignment software by someone of ordinary skill! using MacVector and Geneworks software programs.

Specifically, full-length ALK-1 antibody 1.12.1 was cloned into expression vectors as follows: Poly(A)+ mRNA was isolated using an RNeasy Mini Kit (Qiagen) and cDNA synthesized from the mRNA with the Advantage RT-fbr-PCR kit (BD Biosciences) using ollgo(dT) priming. The oligo(dT) primed cDNA for clone 1.12.1 was amplified using primers listed in Table 2. Amplification was achieved using the Pfu Ultra polymerase (Stratagene) and a PTC-200 DNA Engine (MJ Research) with cycling as follows: 3‘@95°C; 25x (20"@95°C, 30"@52°C, 1'20"@72°C); 1O’@72°C. Clones were sequence verified using Grills 16th BDTv3.1/dGTP chemistry (Applied Biosystems Inc) and a 3730x1 DNA Analyzer (Applied -80-

Biosystems Inc). In the process of cloning 1.12.1 Vh, a silent mutation was introduced in the 8"’ codon, converting “GGC" to a “GGT." All sequences were analysed by alignments to the V BASE sequence directory' (Tomlinson, et al, J. Mot. Blot., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-860 (1994); EMBO J., 14,4628-4638 (1995).)

Table 2

Heavy and Light Chain Amplification Primers Used for Cloning full-length 1.12.1

Primer Name Primer Sequence SEQ ID NO 4-61 5' tcttcaagcttgatatctctagaagccgccaccATGAAACACCTGTGGTTCTTCCTCC 3’ 105 G1/2 FL R 5' ttctctgatcagaattcctaCTATTTACCCGGAGACAGGGAGAGGC 3' 106 A11 5' tcttcaagcttcccgggagccgccaccATGGAAACCCCAGCGCAGCTT 3' 107 K FL R 5’ ttctttgatcagaattctcaCTAACACTCTCCCCTGTTGAAGCTCTTTG 3’ 108 Non-hybridiz ng bases in lower case

Example 4, Gene Utilization Analysis and CDR Analysis

From the nucleic acid sequence and predicted amino acid sequence of the antibodies, the gene usage was identified for each antibody chain. Table 3 sets forth the gene utilization of selected hybridoma clones of antibodies in accordance with the invention. . .

Table 3

Heavy and Light Chain Gene Utilization

Clone Heavy Chain Germiine Kappa Light Chain Germiine , SEQ ID NO: VH Dh Jh' SEQID NO: Vk Jk 1.11.1 9 3-33 6-19 ,. JH3B 11 L1 JK4 1.12.1 103 4-31 6-19 JH4B 126 A27 JK5 1.13.1 13. 4-61 6-19 . JH4B . 1.5 . A27 JK5 1.14.1 ...17 . 4-61 , 6-19 - JH4B 19 A27 JK5 1.151.1 21 4-31 3-3 JH3B 23 B3 JK1 14624 25 4-31 JH3B 27 A27 JK5 14834 29 4-59 6-19 JH4B 31 L2 JK3 1.314 4-31 6-19 JH4B A27 JK5 1.84 33 4-31 3-3 JH3B 35 B3 JK1 1.9.1 37 3-11 3-22 JH6B 39 A2 JK1 440.1 41 3-15 3-22 JH4B 43 A3 JK4 4.244 45 4-31 5-12 JH6B 47 A27 JK5 - 81 -

Clone ’ Heavy Chain Germline Kappa Light Chain Germline SEQ ID NO: . VH Dh Jh SEQ ID NO: Vk Jk 4.38.1 . 49 4-31 4-23 JH4B 51 B3 JK1 4.58.1 53 4-31 4-23 JH4B 55 A27 JK5 4.62.1 57 4-31 5-12 JH6B 59 A27 JK5 4.68.1 61 4-31 2-2 JH5B 63 A27 JK5 4.72.1 65 4-31 5-12 JH6B • 67 A27 JK5 5.13.1 69 4-31 JH3B 71 A27 JK4 5.34.1 73 4-31 JH6B 75 A1 JK1 5.53.1 77 3-15 1-1 JH4B 79 B2 JK4 5.56.1 81 3-11 6-19 JH6B 83 A2 JK1 5.57.1 85 3-11 3-10 JH6B 87 A2 JK1 5.59.1 89 3-11 6-6 JH6B 91 A2 JK1

Mutagenesis, in the VH (M29I) and VK (D19A) regions of clone 1.12.1, was conducted with the primers listed in Table 4 and the QuickChange kit (Stratagene) according to the manufacturer's instructions. The mutated variants were sequence verified and cloned into expression vectors by 5 standard procedures.

Table 4

Mutagenic Oligonucleotides (sequences 5'to 3*):

Primer.. Sense. Antisense 1.12.1(D19A) ctccaggggaaagagCcaccctctcctgtagg (SEQ ID NO: 109) cctacaggagagggtgGctctttcccctggag (SEQ ID NO: 110) 1.12.1(M29I) ggtggctccatCagcagtggtgaatactac (SEQ ID NO: 111) gtagtAttcaccactgctGatggagccacc (SEQ ID NO: 112) 10 Mutations are indicated in bold and underlined.

Nucleic acid, molecules encoding the variable domain of heavy chain (SEQ ID NO: 5) and the variable domain light chain (SEQ ID: 7) chain ofthe 1.12.1 (M29I/D19A) antibody were deposited under terms in accordance with the Budapest Treaty with the American Type Culture Collection (ATCC), 10801

15 University Blvd., Manassas, VA 20110-2209 on July 14, 2005. The deposits have been assigned the following accession numbers: ATCC No. PTA-6864 for E. coli DH5a containing plasmid pCR2.1 TOPO

1.12,1 VH (M29I):UC 25502; and ATCC No. PTA-6865 for E. coli DH5a containing plasmid pCR2.1 TOPO /, 1.12.1 VK(D19A):UC 25503. A number of anti-ALK-1 specific human antibodies exhibited a common pattern in CDR1 of the 20 heavy chain variable domain. These lead molecules utilize the 4-31 or 4-61 heavy chain V-gene segments. The FR1 and CDR1 sequences corresponding to these antibody heavy chains are shown in -82-

Table 4A, aligned against the germiine sequences. A dash (-) in the alignment indicates a residue identical to germiine. In ail cases, the GYYWS (SEQ ID NO: 136) pattern at the end of CDR1 has undergone somatic mutations to yield a new sequence pattern, whereby the G residue is changed to an acidic residue (D or E), and the final S residue is changed to an N in 9 out of the 12 examples. Sequence 5 diversity in other regions of VH indicates that these are likely to be independent somatic mutation events leading to the same sequence pattern at the end of CDR1 of VH.

Table 4A ALK-1 Antibody Heavy Chain Sequence Patterns

Clone V-gene D-gene J-gene PRI CDRl Germiine QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 122) GGSISSGGYYWS (SEQ ID NO: 123) 5.34.1 VH4-31 - NA - JH6B -------0---N 4.5B.1· . VH4-31 D4-23 JH4B -------D---ν 4.3B.1 VH4-31 04-23 JH4B -------O---N 5.13.1 VH4-31 - NA - JH3B 1.162.1 VH4-31 - NA - . JH3B 4.72.1 VH4-31 D5-12 JH6B ------KD---N 4.24.1 VH4-31 D5-12 JH6B 4.62.1 ' VH4-31 D5-12 JH6B -------D---n 1.31.1 VH4-31 D6-19 JH4B -------Ο---n 1.12.1 VH4-31 D6-19 JH4B “R-- Germiine QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 124) GGSVSSGGYYWS (SEQ ID NO: 125) 1.13.1 VH4-61 D6-19 JH4B -------D---K 1.14.1 VH4-61 06-19 JH4B Example 5. Preparation of 1.12.1 Fab Molecules

Fab fragment of 1.12.1(M29I/D19A) was prepared by digesting 1.12.1(M29I/D19A) lgG1 using 15 papain. Protein A purified full length 1.12.1 (M29I/D19A) lgG1 was incubated with papain (VWR) at 1:50 ratio (papain: protein) in buffer containing 30 mM sodium phosphate (pH 7.0), 2 mM EDTA and 2 mM cysteine at 37 °C for 2-3 hours. The digestion mixture was then applied to a protein A mini-column to remove undigested full length protein and Fc fragment. Unbound Fab was collected in the flow-through. A size exclusion column (Superdex 200, Amersham Pharmacia Biotech) was then used to further purify 20 the Fab protein and to exchange the buffer into PBS. Endotoxin was removed by applying the protein solution through Detoxi gel (PIERCE) and Vivapure Mini Q ion exchange column (VIvaScience) subsequently. The protein was filtered with 0.2 pm syringe filter and endotoxin level was tested with a LAL pyrogent kit (Cambrex). The final purified protein was at concentration of 2-3 mg/mL, with endotoxin level of <0.1 EU/mg and purity of >95%. 1.12.1(M29I/D19A) Fab fragment has a molecular weight of 25 47,347 under non-reduced condition as shown by electron-spray mass spectrometry. Edman N-termlnal sequencing analysis confirmed the light chain N-termini sequence of EIVLTQSPG (SEQ ID NO: 113) and heavy chain sequence of QVQLQESG (SEQ ID NO: 114), respectively. -83-

Example 6, . Determination of Avidity Values of Fully Human Anti-ALK-1 Monoclonal Antibodies bv

Surface Plasmon Resonance (SPR) using BIACORE™

Avidity measures of purified antl-ALK-1 antibodies by surface plasmon resonance using the BIACQRE™ 3000 instrument were performed as follows using the manufacturer’s protocols. 5 To perform kinetic analyses, recombinant human ALK-1/Fc fusion protein (hALK-1/Fc) and cynomologus ALK-1/Fc fusion protein (cALK-1/Fc) were immobilized on separate flow cells of a CMS BlAcore sensor chip using routine amine coupling. Surfaces were prepared using 10 mM acetate buffer, pH 5.0 as the immobilization buffer and protein densities of 300 and 150 RU were achieved for the hALK-1/Fc and cALK-1/Fc fusion proteins, respectively. Deactivation of unreacted N-hydroxysuccinimlde esters 10 was performed using 1 M ethanolamine hydrochloride, pH 8.5. Antibody samples in running buffer were prepared at concentrations ranging from 0,125 to 2 nM (a 0 nM solution comprising running buffer alone was included as a zero.reference). Samples were randomized and injected in duplicate for 10 minutes each across all 4 flow cells using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as running buffer. On-rates were observed to be independent of flow rates from 1 to 100 15 pL/min indicating no mass transport limitation. A flow rate of 25 pL/min was used to determine avidity values. The dissociation of the antibody was monitored for 10 minutes, the surface regenerated by a 12 second injection of 100 mM H3PO4 (25 pL/min). The raw data were processed using the Scrubber (©BioLogic Software) software package and analyzed using the CLAMP (©BioLogic Software) software package. Multiple data sets from a single surface, six data sets at a time, were simultaneously fit globally 20 to a simple 1:1 Langmuir binding model utilizing a common variable Rmax value. Table 5 lists avidity values for representative anti-ALK-1 antibodies of the present invention. The represented data indicate that the antibodies prepared in accordance with the invention possess high affinities and strong binding constants for human ALK-1. -84-

Table 5

Determination of Avidity Value bv Surface Plasmon Resonance (BlAcore)

Clone hALK-1/Fc Avidity(pM) hALK-1/Fc koff (1/s) cALK-1/Fc Avidity (pM) 1.12.1(M29I/D19A) &lt;6.8 &lt;5.0 χ 104 27 1.14,2 76 sTexio*5 ’ 280 1.27,3 2.9 1.9 x10'6 60 1.31.1 &lt;13 &lt;5.0x10‘6 150 1.162.1 18 1.1 x10'&amp;amp; 62 1.183.2 220 Tix 1O'6 .,.1800 4.24.2 70 4.4x10'6 430 4.38.1 100 . 4.0 x.t.O* 1.50 4.58.2 40 1.6 x10'6 ......130 . 4.62.1 9.6 . “Γβχ 19 . 4.68.2 86 3.8x1 O’5 320 4.72.2 73 3.4x10* 280 5.13.3 91 6.3 x 10‘6 • 190 1,12.1 (M291/D19A) refers to the mAb 1.12,1 variant that was expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 In the heavy chain replaced with Isoleucine and aspartic acid at position 19 In the light chain replaced with alanine). 10

Example 7, Determination of Affinity Constants (Kq) of Varjants of Fully Human Antl-ALK-1 Monoclonal

Antibody 1.12,1 bv Surface Plasmon Resonance fSPR) using BIACORE™

Affinity measures of purified anti-ALK-1 antibodies by surface plasmon resonance using the BIACORE™ 3000 instrument were performed as follows using the manufacturer's protocols. 15 To perform kinetic analyses, variants of fully human anti-ALK-1 monoclonal antibody 1.12.1 were immobilized onto the dextran layer of a CMS biosensor chip using amine coupling. Surfaces were prepared using 10 mM acetate buffer pH 5.0 as the immobilization buffer and protein densities of 3500- . .. 4800 RU were achieved. Deactivation of unreacted N-hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride, pH 8.5. Samples of monomeric ALK-ECD in running buffer were prepared 20 at concentrations ranging from 2.63 to 640 nM (a 0 nM solution comprising running buffer alone was included as a zero reference), Samples were randomized and injected for 2 minutes each across ail 4 flow cells using HBS-EP (10 mM HEPES pH 7.4,150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as running buffer. A flow rate of 25 pL/min was used to determine affinity constants. Dissociation of -85- monomeric ALK-ECD was monitored for 10 minutes, the surface regenerated by a 12 second injection of 100 mM H3PO4 (25 pL/mln). The raw data were processed using the Scrubber (©BioLogfc Software) software package and analyzed using the CLAMP (©BioLogic Software) software package. The data were fit globally to a simple 1:1 Langmuir binding model. Table 6 fists affinity measurements for variants 5 of human anti-ALK-1 monoclonal antibody 1.12.1 ofthe present invention.

Table 6

Determination of mAb 1.12.1 variant affinity constant, Kn, bv surface plasmon resonance (BlAcore)

Antibody on-rate (M'1 s"1) Off-rate (s'1) KD (nM) 1.12.1 1.9X103 7.4x10*° 39 1.12.1(rWT) 2.2 X 10·· 5.8x10"° · 26 1.12.1(D19A) ' 2.6x10a 4.4x10'° 17 1.12,1(M29l) 2.4x10° 9.1 x 10* 38 1.12.1(M291/D19A) (1)* 2.2x10° 9.5x10'° 43 1.12.1(M29l/D19A) (2)* 2.3 x 103 8.4ΧΪ0'5 37 10 *The two affinity constants for 1.12.1 (M29I/D19A) (1) and (2) were obtained using two separate surfaces. 1.12.1 refers to the mAb 1.12.1 variant that was isolated from the hybridoma. 1.12.1 (rWT) refers to the mAb 1.12.1 variant that was expressed recombinant mAb. " 1.12.1(M29I) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the methionine at position 29 in the heavy chain was replaced 15 with isoleuctne. 1.12.1 (D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the aspartic acid at position 19 in the light chain was replaced with alanine. 1.12.1(M29I/D19A) refers to the mAb 1,12.1 variant that was expressed recombinant mAb 20 containing two specific amino acid mutations (methionine at position 29 In the heavy chain replaced with

Isoleucine and aspartic acid at position 19 in the light chain replaced with alanine).

Example 8, Determination of Affinity Constants (Kn) of Representative Fully Human Anti-ALK-1

Monoclonal Antibodies bv Surface Plasmon Resonance (SPR) using BIACORE™ 25 Affinity measures (Ko and koff) of purified anti-ALK-1 antibodies by surface plasmon resonance using the BIACORE™ 3000 instrument were performed as follows using the manufacturer's protocols.

To perform kinetic analyses, affinity-purified mAbs were immobilized onto the dextran layer of a CM5 biosensor chip using amine coupling. Surfaces were prepared using 10 mM acetate buffer pH 5.0 as the immobilization buffer and protein densities of 200-400 RU were achieved. Deactivation of 30 unreacted N-hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride, pH 8.5. Samples of monomeric ALK-ECD In running buffer were prepared at concentrations ranging from 3.125- -86- 400 nM (a 0 ηΜ solution comprising running buffer alone was included as a zero reference). Samples were randomized and injected in duplicate for 2 minutes each across all 4 flow cells using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20).as running buffer. On-rates were observed to be independent of flow rates from 1 to 100 pL/min indicating no mass transport 5 limitation. A flow rate of 25 pL/min was used to determine affinity constants. Dissociation of monomeric ALK-ECD was monitored for 10 minutes, the surface regenerated by a 12 second injection of 100 mM H3PO4 (25 pL/min). The raw data were processed using the Scrubber (©BioLogtc Software) software package and analyzed using the CLAMP (©BioLogic Software) software package. The data were fit globally to a simple 1:1 Langmuir binding modeL Table 7 lists affinity measurements for representative 10 anti-ALK-1 antibodies of the present invention:

Table 7

Determination of Affinity Constant, Kn, for Representative Monoclonal Antibodies bv Surface Plasmon -

Resonance (BIAcore) 15 ...... mAb ori-rate (M'1 s'1) off-rate (s'1) Kg(nM) 1,12.1 (M29I/D19A) 3.3 x 10* 8,2x10·* 25 1.31.1 3.2 x 10* 1.9Χ10*4 .6.0 4.72.1 3.2 x 104 2.5 xW5 0.8 Fab 1,12.1(M29I/D19A) 3.8x10’ 8.2x10” 22

The monomeric ALK-ECD used to generate the data in Example 8 was a different preparation than that used to generate the data in Example 7. 1.12.1 (M29I/D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb 20 containing two specific amino acid mutations mentioned above (aspartic acid at position 19 in the light chain replaced with alanine and methionine at position 29 In the heavy chain replaced with isoleucine).

Fab 1.12.1(M291/D19A) refers to the Fab fragment of mAh 1.12.1(M29I/D19A) prepared by digesting 1.12.1(M29I/D19A) lgG1 using papain. 25 Example 9, Identification of Epitope Selectivity of Anti-ALK-1 Antibodies ·

Cross-competition experiments were performed using the BIACORE™ 3000 instrument* (Biacore

International AB, Uppsala, Sweden and Piscataway, N.J), following the manufacturer's protocols.

Recombinant human ALK-1/FC chimera was immobilized onto the dextran layer of a CMS biosensor chip using amine coupling. Chips were prepared using 10 mM acetate buffer pH 5.0 as the 30 immobilization buffer and a protein density of 940 RU was achieved. Deactivation of unreacted N- hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride, pH 8.5.

Purified mAbs were diluted to a concentration of 50 nM in HBS-EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Polysorbate 20). A primary antibody was chosen and then 87 injected across the flow cell for 600 seconds at a rate of 10 pL/min. After the injection was complete, a secondary antibody was chosen and injected across the same flow cell for 600 seconds at a rate of 10 pL/min. The sensor surface was regenerated by a 12 second injection of 100 mM H3PO4 (25 pL/min).

After regeneration, the primary antibody was again injected across the flow cell for 600 seconds 5 at a rate of 10 pL/min. After the injection was complete, a different secondary antibody was chosen and injected across the same flow cell for 600 seconds at a rate of 10 pL/min. Once the entire panel of 14 antibodies had been used as the secondary antibody a new primary antibody was chosen and the procedure repeated with the new primary antibody. These procedures were carried out until all possible combinations of primary and secondary antibodies had been injected across the flow ceil. Binding of the 10 secondary antibody was considered to have occurred if the total response observed after injecting both antibodies exceeded that observed for both possible threshold values. Threshold values were determined by using the same antibody as both the primary antibody and secondary antibody. Shown in Table 8, a response matrix was created based on whether binding was observed: - indicates no binding of the secondary antibody, x indicates binding was observed (response was greater than the threshold values 15 for the individual antibodies). Grouping the clones that show the same reactivity pattern gives rise to two different epitope bins with 1.11.1 in one bin and all the other antibodies in the other bin.

Table 8. BlAcore epitope binning response matrix

Primary mAb Secondary mAb 1.9.1 1.11.1 1.12.1 (M29I/D19A) 1.27.3 1.31.1 1.162.1 1.183.2 4.24.2 4.38.1 4.58.2 4.62.1 4.68.2 4.72 5.13 1.9.1 x - - - - - - - - - - 1.11.1 x y.i.jT.i’i'hf'ji X X X X X X X X X X X X 1.12.1(M29I/D19A) X :f'" \ , '.i-i 11 f * ...............a.JL/. - - - - - - - - 1.27.3 - X - - - - - - - - - - 1.31.1 X - - j H u»·’ - I 1 F - - - - - - - 1.162.1 - X - - - - - - - - - - 1.183.2 X - - - - - - - - - - 4.24.2 - X - - - - -.(‘'Iii! ! !.Ί - - - - - - 4.38.1 - X - - - - - - - - - 4.58.2 - X - - - - - - - - ί - - - - 4.62.1 - X - - - - - - - - - - - 4.68.2 - X - - - - - - - - - - - 4.72 - X - - - - - - - - - 5.13 X •j μ -88-

ExamplelO. Isolation of Cynomolgus Monkey ALK-1 Gene

Cynomolgus monkey ("Cyno’’) ALK-1 gene was extracted from Cyno lung tissue. Based on the published gene sequence for human ALK-1 (Genebank record L17075), primers were designed to PCR amplify· the full-length Cynomolgus ALK-1. mRNA was prepared from frozen excised cynomolgus lung 5. tissue (ca. 1 g) using the mRNA purification kit (Ambion, Catalog No. 1915) according to the manufacturer’s instructions. 200 ng of the mRNA was reverse transcribed and PCR amplified using the OneStep RT-PCR kit (Qiagen, Catalog No. 210210) utilizing gene-specific oligos: 5-AGCGGGCCCAGAGGGACCA7G (Seq ID NO: 115) (forward) and 5’- CAGAAAGGAATCAGGTGCTCCTGGGCTA (Seq ID NO: 116) (reverse) at an annealing .temperature of. 10 61°C. An RT-PCR product of the appropriate size (~1.5 Kb) was excised and purified from a 0.9 % agarose gei after electrophoresis, then TOPO-TA cloned into the pCR4-TOPO vector (Invitrogen, Catalog No. K4575-01). The insert was sequenced to obtain the ORF nucleotide sequence of Cynomolgus ALK-1. The nucleotide and predicted translated amino acid sequences are shown in SEQ ID NOs: 93 and 94, respectively. While the cytoplasmic portion of the gene encodes identical protein sequences between 15 Cyno and human, there are 5 amino acid differences in the extracellular domain (ECD, which includes positions 22-118) and 1 amino acid difference in the transmembrane domain of the protein . ECD sequence identity between human and Cyno is 94.8%. An alignment of the human and primate ECD is shown In Figure 2. A pair of primers (forward: 5'- G ATTA TGGCCTTGGGCTCCCCC AGG AAA (Seq ID NO: 117) and 20 reverse: 5'- GGGCLATTGAATCACTTTAGGCTTCTCTGGACTGTTG) (Seq ID NO: 118) were used to PCR amplify the full-length Cynomolgus ALK-1 gene.

Example 11. Determination of Cell Surface Binding Characteristics and Primate Crqsshvbridisation bv

Flow Cytometry (FACS) 25 To generate ALK-1-overexpressing cell lines, which can be used to test anti- ALK-1 binding affinity using flow cytometry (FACS), full-iength human, Cyno, and rat ALK-1 genes were cloned .into Invitrogen’s (Catalog No, K6510-20) pcDNA5/FRT/To TOPO vector and transfected into 293 Flp-ln T-Rex host cell (Invitrogen, Catalog No. R780-07), respectively. Selections were carried out using hygromycin to obtain the final stable cell lines. Overexpressing of the respective full-length ALK-1 proteins were 30 achieved by tetracycline (2 pg/mL)induction at 37°C/5% CO2 for 24 hours.

Anti-ALK-1 mAbs were tested for their binding affinities to cell surface ALK-1 using FACS assay, employing 293 stable cells overexpressing ALK-1 proteins. The cells were detached using trypsin-EDTA and washed with cold PBS-SA. After being aliquoted into 96-well plates, the ceils were blocked by serum and incubated with different concentrations of specific mAb for 1 hour at 4°C. Subsequently, the cells 35 were washed and incubated with an anti-human κ secondary antibody conjugated with the R-PE fluorophore before analyzed using a FACSCalibur flow cytometer (BD Blosclences). 10,000 events were collected for each sample without applying any gating. Shown in Table 9, the geometric mean of each sample histogram was plotted as a function of the mAb concentration and Ko was calculated for each -89-

mAb after fitting to a two-state equilibrium model Examples of equivalent human and primate FACS experiments are shown in Figure 3.

Table 9 5 Mean Binding Affinity (Kn) Results of Anti- ALK-1 Monoclonal Antibodies to Cell Surface Human or Cyno

ALK-1 Measured bv FACS

Antibody Kd (nM) Human Cyno 1.12.1 6.7 2.0 1.27.1 3.7 2.2 1.162.1 5.6 3,0 4.38.1 9.3 3.4 4.58.1 14.0 ' 6.7 4.72.1 6.4 3.8 5.13,1 3.2 1.6 1.31.1 3.2 1.7 4.24.1 7.6 3.1 4.62.1 2.3 0.78 4.68.1 8.4 9.0

In addition, 1.12.1 was shown by the FACS assay to have very limited cross-over to rat (Kd>100 10 nM) and is predicted to have very low cross-over to mouse in view of 74% and 68% ECD sequence identity between rat/human and mouse/human ALK-1, respectively). FACS assay was also used to determihe Kd of the recombinant 1.12,1 mAh variants. Shown in

Table 10, the results Indicate similar binding affinity from the recombinant antibody. -90-

Table 10

Mean Binding Affinity (Kn) Results of 1.12.1(Μ291/Ρ19Α) Variants to Cell Surface Human or Cyno ALK-1

Measured by FACS

Antibody KD (nM) Human Cyno 1.12.1 6.7 2.0 1.12.1(rWT) 5.9 6.0 1.12.1(M29I) 6.0 3.3 1.12.1(D19A) 5.7 3.8 i.12.1(M29l/D19A) 7.2 3.4 Fab 1.12.1 (M29I/D19A). 0.77 ND 1.12.1 refers to the mAb 1.12.1 variant that was isolated from the hybridoma, 1.12.1(tWT) refers to the mAb 1.12.1 variant that was expressed recombinant mAb. 1.12.1(M29I) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing a 10 specific single amino acid mutation where the methionine at position 29 in the heavy chain was replaced with isoleucine. 1.12.1(D19A) refers to the mAb 1.12,1 variant that was expressed recombinant mAb containing a specific single amino acid mutation where the aspartic acid at position 19 in the light chain was replaced with alanine. 15 1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that was expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced with isoleucine and aspartic acid at position 19 In the light chain replaced with alanine).

Fab 1.12.1(M29I/D19A) refers to the Fab fragment of mAb 1.12.1(M29I/D19A) prepared by digesting 1.12.1 (M29I/D19A) lgG1 using papain. 20 " ' * '

Example 12. Taqman Assay for Id 1 HUVECs (BioWhittaker, Cat. # CC-2519) were seeded on 24-well plates, 12000 cells/well In 600 pL of complete HUVEC medium (EGM-2 Bullet kit, Biowhlttaker, Cat # CC-3162), and allowed to grow overnight The following day the cells weretypically 50% confluent Complete Medium was removed and 25 200 pL of Starvation Medium (EBM-2 with 0.2% FBS only) was added. Cells were incubated for 2 hrs.

Then the cells were treated with 40 pLof antibody solution ih PBS. Lyophilized Ab was reconstituted with sterile PBS. 'Finally, the cells were treated with 1 %FBS/Basal medium (final concentrations) for 30 minutes, the medium was removed and the cells were lysed in 400 pL of RTL Buffer (Rneasy 96 kit Qiagen, Cat # 74182), according to the manufacturer's protocols. Subsequently, RNA was prepared 30 using RNeasy kit (according to manufacturer's instructions). The RNA was eluted and quantitated with -91 -

RiboGreen® RNA Quantitation Kit (Molecular probes, Cat # R-11490). Equal amount of total RNA was used for real time PCR analysis to detect Idi RNA expression (ABI 7900 instrument). PCR was conducted using the Taqman One Step PCR Master Mix Kit (ABI, Cat. # 4309169) and the ID1 primer/ Probe sequences listed below. PCR was conducted using 40 cycles of the following annealing and 5 amplification conditions: 95 °C, 15 seconds ; 60 °C, 1 min.

TaqMan probe: CPG-conjugated 5’-6-FAM, and 3’-TAMRA.

Name: ID1-Probe

Sequence: 5' CCAGCACGTCATCGACTACATCAGGGA 3’ (Seq ID NO: 119)

Taqman PCR Primers:

10 Name: ID1-F

Sequence: 5' AAGGTGAGCAAGGTGGAGATTC 3' (Seq ID NO: 120)

Name: ID1-R . .

Sequence: 5’ TTCCGAGTTCAGCTCCAACTG 3' (Seq ID NO: 121)

Examples of Id1 titrations for the 1.12.1(M291/D19A) lead molecule (including 1.12.1 sequence 15 variants) and the Fab derivative are shown in Figures 4 and 5. A summary of mean IC50 values for this assay is shown in Table 11. Ail ICSo determinations were run in triplicate.

Example 13, Smadl Phosphorylation Detected by Odyssey Infrared Imaging System from Ll-COR 20 Biosciences f24-well plate) HUVECs (Biowhittaker, Cat # CC-2519) were seeded on 24-well plates, 18000 cells/well in 600 pL of complete HUVEC medium (EGM-2 Bullet kit, Biowhittaker, Cat # CC-3162), and allowed to grow overnight The following day the cells were typically 50% confluent. Complete Medium was removed and 200 pL of Starvation Medium was added (Starvation Medium: EBM-2 with 0.2% FBS only). Cells were 25 Incubated for 2 hrs. Then the cells were treated with 40 pL of antibody solution in PBS for 3 hours. Finally, the cells were treated with 0.3X Complete Medium (final concentration) for 35 minutes. The medium was removed, and the cells were lysed in 80 pL of 1.1X Sample Buffer (Invitrogen, Cat No. NP0007). Phosphorylated Smadl was determined by Western Blotting using X Cell Surelock Mini-Cell &amp;amp; Blot Module (Invitrogen, Cat. # EI0002). Phosphorylated Smadl was detected using rabbit anti-phosphor- 30 Smadl antibody (Cell Signaling, Cat. No. 9511), which is then detected by IRDye™ 800 Conjugated Anti-RABBIT IgG (Rockland Immunochemicals, Cat No.611-732-127). Amount of phosphorylated Smadl was quantified using Odyssey Infrared Imager (Li-Cor). Actin (Santa Cruz, # sc-8432) was used for normalization (anti-mouse Alex 680, Molecular Probe, Cat No. A-21058). A summary of mean ICeo values for this assay is shown in Table 11, All IC50 determinations were run in triplicate. -92-

Table 11

Clone ID1 Taqman ICso nM pSmadl Western ICeo nM 1.11.1 nd nd __ 1.12.1(M29I/D19A) 16 18 1.13.1 - 100 Γ87 1.27.1 82 70 1.29.1 94 82 1.31.1 24 21 1.162.1 75 15 1.183.1 58 17 4.24.1 100 82 4.38.1 87 52 4.58.1 14 15 4.62.1 24 34 4.68.1 141, 110 4.72.1 21 35 5.13.1 30 68 5 .

Example 14, Internalization Characteristics of Anti- ALK-1 Monoclonal Antibodies FACS was used to monitor the time course of the remaining cell-surface receptor ALK-1 as well

as the neutralizing antibody. Remaining cell-surface ALK-1 is monitored by a marker antibody which is capable of binding ceil surface ALK-1 yet recognizing a different epitope from the neutralizing antibody. A 10 mouse anti-human ALK-1 ECD mAb (R&amp;amp;D sytems, Cat No # AF310) was identified and used in the study as the marker antibody.

Time course of internalization was studied using endothelial cell lines HUVEC and HUAEC. The cells were grown at 37°C with 5% CO2 in 24-well plates containing 200 pL of complete culture medium per well. At each of 11 time points over the course of 48 hours, 2 pL of 1 mg/mL antibody solution was added 15 to one well and mixed (final concentration of the neutralizing antibody is 10 pg/mL)?The plate was then, put back into the 37°C incubator until the 0 hour time point, when the plate was placed on ice to stop the internalization. process. Marker antibody was added .to the wells at this, point (10. pg/mL final concentration) and incubated on ice for T hr. The cells were then washed with PBS detached by trypsination and transferred into a 96-well plate. Ceils were then washed, blocked, and treated with 20 secondary antibodies bearing different flurophores in order to monitor both neutralizing antibody and receptor ALK-1 remaining on the cell surface. The samples were assayed on a FACSCalibur flow -93« cytometry instrument, counting 3,000-5,000 events/sample. The Geometric Mean of each sample in the specific fluorescence channel was calculated and plotted as a function of time. The data were fitted to a modified radio-decay equation to obtain the half-time (t1/2) of internalization as well as the percentage of neutralizing antibody or receptor ALK-1 remaining on the cell surface when the internalization reached 5 steady-state. As shown in Figure 6, mAb 1.12.1(M29l/D19A) internalizes at the same rate and to the same extend as the cell surface receptor ALK-1. Half-life of the 1.12.1(M29I/D19A) internalization is ~2 hr. An equilibrium was reached when 50% of the antibody was internalized. A polyclonal antibody purchased from R&amp;amp;D systems (Cat. No # AF370) internalizes at a t1/zof 1 hr and reaches the steady-state with "*70% of the receptor being internalized (Figure 6). Similar internalization characteristics were 10 observed with other human anti-ALK-1 mAbs ofthe invention (not shown).

Example 15, Establishment of Human Foreskin - SCID Chimera Mice

Significant modification of the surgery procedure was made to a procedure published previously by H-C Yan, et al “Human/Severe Combined Immunodeficient Mouse Chimeras, An Experimental In Vivo 15 Model System to Study the Regulation of Human Endothelial Cell-Leukocyte Adhesion Molecules", J. Clin. Invest. 91:986, 1993; J. Varner “Regulation of Angiogenesis in Vivo by Ligation of Integrin a5b1 with the Central Cell-Binding Domain of Fibronectin" Amer. J. Path. 156 (4):1345, 2000; K. Tahtis, et al “Expression and Targeting of Human Fibroblast Activation Protein In a Human Skin/Severe Combined Immunodeficient Mouse Breast Cancer Xenograft Model” Mol. Cancer. Ther. 2(8):729, 2003. Upon arrival 20 from National Disease Research Institute and Cooperative ..Human Tissue Network, human foreskin pieces were trimmed of unhealthy regions and transferred to RPMI media (Cellgro/Mediatech, Cat# MT-15-040-CV supplemented with Penicillin and Streptamycin (Gibco/Life Tech, Cat# 15070-063) (add 5 mLs of the pen/strep stock solution into 500mLs of RPMI). Using a scalpel and cutting In sterile petri dish, the skins were trimmed to ari oval shape of approximately 8 x 13 mm cleaning any ragged ends and 25 connective tissues, and stored on wet Ice prior to surgery, the appropriate volume (4 pL/gram of animal) Cf 100 mg/mL Ketamine (Ketaset™, Fort Dodge Animal Health)/'! mg/mL medetomidine (Pfizer Animal Health - Dormltor) solution was intraperitoneally injected into scid mouse abdomen (i.e., at a 45° angle, under skin but not too deep internally). Once anesthetized, the mice were applied with eye lubricant, a subcutaneuosly injection of Ketoprofen (10 mg/kg, Fort Dodge Animal Health) and hair was shaved over 30 the site of the surgery. The surgical region was surgically scrubbed three times using the Clorahexiderm (Butler, Chclo-Scrub 40, cat# WAB20109) and then the alcohol in a circular motion that started from the center of the surgical site outward and avoided going from a dirty area back into a clean area. The mice were transferred to the prepared surgical hood and placed on the heated water pad (Gaymar Industries, cat# TP500 T/Pump) that was maintained at 37°C. The mice were then placed under isofluorin 35 anesthesia for the duration of the surgery. The dorsal side of a mouse was covered with a surgical drape cut to expose the surgery site. The mouse skin was picked up with forceps and an oval shaped skin tissue was cut with curved scissors with one motion. An appropriate sized human foreskin was placed on the mouse. The human and mouse skin were sutured together using the Ethilon suture (Ethicon cat# -94- 697H.), starting at top of oval, then the bottom, then the farthest right and then farthest left side. More stitches were made in between to further secure the tissues together. Approximately 8 stitches were made equal-distantly around the skin. During the surgery, used a syringe with sterile saline to irrigate the . skin/mouse surgical wound when it became dry. A Bandaid was placed over the wound. A transparent 5 dressing (3MTegaderm™) was then used to loosely wrap around the bandage. The dressing was cut to size to cover an area slightly wider than the Bandaid. The mouse was then given Atipamezole (50-100 pL, Pfizer Animal Health - Antisedan) and the mouse recovered in a heated cage in 5 - 10 min. The dressing and the bandages were removed in 7 - 10 days and by 15th day most of the skins appeared as scabs. Complete healing occurred between 21-28 days after which time skins were ready to be 10 inoculated with tumor ceils. Shown in Figure 8 Is an example of the histological (H&amp;amp;E Staining) analysis of a section of the engrafted skin post surgery. The histology of the engrafted skin closely mimics the characteristics of human skin Implanted in mice described by Tahtis, et al "Expression and Targeting of Human Fibroblast Activation Protein in a Human Skin/Severe Combined Immunodeficient Mouse Breast Cancer Xenograft Model" Moi Cancer. Ther. 2(8):729, 2003. h.e.: human epidermal layer; h.d.: human 15 dermal layer. . '

Example 16, Collagen Model In Human Foreskin - SCID Chimera Mice

Collagen I stock solution (cat# 354236, Becten-Dlcklnson) was diluted to 4 mg/mL with 0.02 N acetic acid and was kept on ice before implantation. The acidic collagen solution (8 parts) was mixed 20. with 10X M199 (Sigma, Cat# M9163) (1 part) and human plasma fibronectin (Fn) (cal# 354008, Becten-Dlcklnson) to reach a final Fn concentration of 90 pL/mL; NaOH (1.0 N) was added to adjust pH to ~ 7.2. The Collagen/Fn mixture was kept on ice until use. The. Implant mix was prepared using the above Collagen/Fn mixture plus angiogenic inhibitor of interest with or without human macrovescular endothelial cells (HMVEC), (Cascade Biologies, Cat# C-010-5C). The HMVECs were prepared as 6 x lOecells/mL In 25 PBS. 50- 100 pL of the Implant mixture Was Injected intradermally Into the foreskin in the sold chimera mouse, 7-14 days later, the collagen plugs were harvested, embedded In the OCT compound (cat# .4583, Sajura Flnetek, CA) and snap frozen for Immunohistochemistry analysis. The collagen plug in the foreskin was Identified with the Trichrome Kit (cat# KC1641, Mater Tech, CA) as blue staining as shown in Figure 9 (A). Human vessels were identified by staining for human P-CAM using the anti-human CD-31 30 antibody (Clone 13.3, Vector Laboratories) (Figure 9 (B)). Table 12 summarizes the human vessel staining and quantification In the collagen model in the foreskin - SCID chimera mice. ... -95-

Table 12. Summary of the Collagen model results

Matrix HMVEC in Matrix Treatment (Rx) Days of Rx Study End Point Human vessel scoring (1x10s) %of Control (human vessels) 1.6 mg/ml Collagen None no treatment 4 human CD-31 Staining 0.036 + 0.001 40 1.6 mg/ml Collagen , . 7x103 no treatment 4 0.071 + 0.022 78 1.6 mg/ml Collagen 1.4 x104 no treatment 4 0.063 + 0.016 69 2.4 mg/ml Collagen None no treatment 4 ' 0.091 +0.056 100 2.4 mg/ml Collagen 7 x 10’ no treatment 4 0.067 + 0.049 . 74 2.4 mg/ml Collagen 1.4 x 104 no treatment 4 0.062 + 0.047 68 3.0 mg/ml Collagen 8.8 x 103 Non- treatment 4 human CD-31 Staining 54 + 9 100 3.0 mg/ml Collagen 8.8 x10s (sotype control antibody 100 pg/ml mixed In gel 4 52 + 13 96 3.0'mg/ml Collagen 8.8 χ 103 1.12.1(M29I/ D19A) antibody 100 pg/ml mixed in gel 4 15 + 3. 28 3.0 mg/ml Collagen none no treatment 4 human CD-31. Staining 0.112 + 0.026 100 5.0 mg/ml Collagen none no treatment 4 0.031+0.012 28 3.0 mg/ml Collagen none Isotype control antibody 100 pg/ml, id. injection 4 human CD-31 Staining ' 75 + 15 " ioo 3.0 mg/mi Collagen none 1.12.1(M29I/ D19A) antibody 100 pg/ml, id. injection 4 39 + 11 52 3.0 mg/ml Collagen none 1.14.1 antibody 100 pg/ml, id injection 4 44 + 28 59 5

Example 17. M24met Tumor Model In Human Foreskin - SCID Chimera Mice -96-

Typically graft age of between 5-10 weeks post surgery were used in these studies. The M24met cell line was described by Mueller and coworkers in "Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells", Cancer Research, 55(8):1629-32, 1995. M24met cell suspension was prepared as following: 80% confluent M24met cells were washed, 5 typsonized using Trpsin/EDTA (Gibco, Cat# 25200-056) and collected in the PRMI (Ceiigro/Mediatech, . cat# MT-15-040-CV) media supplemented with 10% FBS (Cellgro/Mediatech, Cat# AKD-11775) and 2 mM L-glutamine (Cellgro/Mediatech, Cat# 25-005-CI). The cells were centrifuged at 600 rpm for 5 min, resuspended in sterile PBS.. Cell counts were estimated using Coulter Counter (Beckman Coulter, Model . Z2). The Cells were, centrifuged at.600 rpm for. 5 min and were re-suspended in Collagen/ and Fn (3 10 mg/mt) mixture to obtain a 4x107 cells /ml cell suspension for implantation.

To inoculate, 2x10® above cells were injected (50 μΙ of 4x107 cells/ml) intradermally into the engrafted human skin In the mouse. At day 5-7 post Implant, the tumors would be palpable and the mice were randomized into the Control and the Treatment groups before the dosing would start. The Control group is defined as. such that the animals would receive either no dose; dose of the Vehicle in which the 15 anti-ALK-1 antibody was constituted, or dose of the isotype matched lgG2 human monoclonal antibody anti-KLH (Pfizer Inc). The Treatment group is defined as such that the animals would receive a dose of the anti-ALK-1 antibody 1.12.1(M29I/D19A).

Example 18, Human and Mouse CD-31 Immunofluorescence (IF) Dual Staining . ' 20 The frozen tissue sections were air-dried and fix at-20°C in acetone (Fisher, Cat#A16S-4), or 10 ’ min. The samples were air dried again and washed in PBS three times at 5 min each. The sampleswere blocked in 5% rabbit serum (Vector Laboratories, Cat# S-5000) in PBS fro 30 min at room temperature. Primary antibody mixture was prepared in 5% rabbit serum with the anti-human CD-31 antibody (Santa Cruz, Cat# SC1505) and the anti-mouse CD-31 (Pharmingen, Clone Mec1 3.3, Cat# 01951 A) at 1:100 25 and 1:150 dilutions, respectively. The above antibody mixture was added to the tissue samples for 1 hour at RT. The blocks were washed in PBS for three times at 5 min each before incubated with the secondary antibody mixture for 1 hour at RT. The secondary antibody mixture was prepared in PBS/0.05% Tween-20 (Sigma, Cat# P1379), Texas Red rabbit anti-goat antibody (Jackson Labs, Cat# 305-075-003) and FITC rabbit anti-Rat antibody (Jackson Labs, Cat# 312-095-003). The antibodies were 30 diluted at 1:50 If frozen antibodies were used or at 1:100 ff fresh antibodies were used. The slides were washed again in PBS for three times at 5 min each before mounted In Vectashield (Hard Set, Mounting medium with DAPI, Vector Lab, CA, Cat# H-1500). The slides were kept In dark and 4°C until image analysis. The image analysis was performed using an Olympus BX60 fluorescent microscope and photographs were taken using an Olympus microfire digital color camera. Pictures from 3-5 hot 35 spots/sllde, one sllde/antmal, 4-7 anlmals/group were taken and the vessel areas as indicated by positive staining of anti-human CD-31 were quantified by three individuals using Image Pro Plus v4.5 (MedlaCybernetics), The pharmacodynamic end point (group mean) was expressed as either the percent of human CD-31 Inhibition compared to the Control group or as total human vessel area. Statistical 97 significance was determined by ANOVA. Shown in Figure 10 is an immunofluorescent image of human (red) and mouse (green) vessels of the M24met tumor in the human foreskin SCID chimera mouse.

Example 19. Human CD-31 Immunohistochemistry (IHC) Staining

The frozen tissue sections were air-dried and fix at -20°C in acetone for 10 min. The samples were air dried again and washed in PBS twice at 5 min each. The samples were incubated in 0.075% H202/ methanol (Fisher Cat# A433-4) for 15min and wash in PBS three times at 5 min each. The samples were blocked in 5% rabbit serum/PBS for 30 min and applied with antihuman CD-31 antibody 1:100 (Santa Cruz, Cat# SC1505) in 5% rabbit serum for 1 hour at RT. The samples were washed in PBS twice at 5 min each and applied with rabbit anti-goat at 1:200 (Vector Labs, Cat# BA-5000) in 5% rabbit serum for 35 min at RT. The slides were then washed in PBS twice at 5 min each and freshly made streptavidin (Vector Labs, ABC Elite kit, Cat# PK-6100) was added. The slides were washed again in PBS twice at 5 min each and then developed in diaminobenzidine (DAB) (Vector Labs, Cat#SK-4100). The slides were wash in PBS twice at 5 min each followed by Mayers haematoxylin (Sigma, Cat# HHS-32) for 5 seconds. The samples were rinsed well in diH20 and dipped twice briefly in the diluted (5ml stock in 1L of diH20) ammonium hydroxide solution (Sigma, Cat# A-6899) and rinsed in diH20 again. The samples were then dehydrated in the 70%, 90% and then 100% alcohol (Harleco, Cat# 65347/85) 1 minute each and finally in xylene (JT Baker, Cat# 516,09). The slides were mounted with Cytoseal 60 (Stephens Scientific, Cat #8310-4,) and covered with cover slips for image analysis. Shown in Figure 11 is the IHC image of human vessels (brown) of the M24met tumor in the human foreskin-SCID chimera mouse.

Example 20, Therapeutic Treatment with the Anti-ALK-1 Antibody 1.12.KM29I/D19A)

For treatment, the dosing was performed either subcutaneously (sc) or intravenously (iv). Typically one dose ofthe 1.12.1 (M29I/D19A) antibody was given for each study. The second dose ofthe ALK-1 antibody, if necessary, was administered on day 9 or 10. Some times multiple dose levels, i.e., 1, 5, 10, 50 mg/kg, were administered to investigate dose-dependent inhibition of human vessel growth. Animals were monitored daily and tumors were measured three times/week by calipers. By day 14 -17 the tumors were between 250-350 mm3 and were removed from the mice, embedded in OCT and frozen down for IF or IHC analysis. Shown in Figure 12 are representative immunofluorescent images of human (red) and mouse (green) vessels of the Control and 1.12.1 (M29I/D19A) Treated (10 mg/kg) M24met tumors in the human foreskin scid chimera mouse. Dose-dependent inhibition of human tumor vessels by 1.12.1 (M29I/D19A) in the human foreskin SCID chimera mouse model is shown in Figure 13 and a summary of related studies is presented in Table 13.

Table 13. -98-

Summarv of in vivo model characterization and the inhibition of human vessel growth of the M24met tumors in the SClD-chimera model

Protocol Parameters Endpoints General notes Tumor Drug Dose Route Schedule CD31 (% Inhibition Compared to Control) Day of Study MCF-7 none na . na na not quantified 19 Tumors implanted Intradermially. Tested with and with out estradiol and collagen implant. Tumors grew slowly and expressed little human CD31 M24met none na na na not quantified 19 Tumors implanted · intradermially, Tested with and with out colfagen/FN matrix With matrix found superior tumor growth, all future studies will contain matrix supllments. Tumors showed good human CD31 staining M24met (small) none na na na not quantified 9 Tumor size &lt;100 mm3. Little human CD31 M24met (medium) none na na na not quantified 12 Tumor size &lt; 100*200 mm3. Some human CD31 M24met (large) none na . na . . na not quantified 12 Tumor size &lt; 200 mm3. Large M24met tumors have superior numbers of human vessel staining, future studies will be conducted with larger tumors M24met Non- Specific human IgG 10 mg/kg IV 2 doses (day 5 &amp;amp; 9) 0 15 First screening study. .1.12.1 (M29I/D19A) showed significant reduction of human CD31 staining. No tumor growth inhibition observed 1.12.1 (M2 9I/D19A) 10 mg/kg IV 42 M24met Non- Specific human IgG 10 mg/kg IV 2 doses (day 5 &amp;amp; 10) θ 14 Second screening study. . . Confirmed results of GVV-366. No tumor growth inhibition observed 1.12.1(M2 9I/D19A) 10 mg/kg IV 40 M24met Non- Specific human IgG 10 mg/kg SC 2 doses (day 5 &amp;amp; 10) 0 14 First test of dose dependent activity of 1.12.1 (M29I/D19A) against human CD31. Some dose dependent effect observed. PK results that single dose wilt be sufficient 1.12.1(M2 91/019 A) 10 mg/kg QC 43 -99-

Protocol Parameters Endpoints Tumor Drug Dose Route Schedule CD31 (% inhibition Compared to Control) Day of Study General notes for significant reduction In CD31. No tumor growth inhibitor! observed 1.12.1 (M2 9I/D19A) 1 . mg/kg SC 50 1.12.1 (M2 9I/D19A) 0.1 mg/kg SC 20 M24met No Dose 0 mg/kg na na ND '· 16 Second test of dose dependent activity of 1.12.1 (M29I/D19A) against human CD31. Clear dose dependent anti-CD31 effect observed. No tumor growth inhibition observed Isotype matched IgG 10 mg/kg SC one dose (day 5) 0 Non- Specific human IgG 10 mg/kg SC ND - 1.12.1 (M2 9I/D19A) 1 mg/kg • SC 24 1.12.1(M2 9I/D19A) 5 mg/kg sc 59 1.12.1 (M2 91/D19A) 10 mg/kg sc 72 M24met Isotype matched IgG 10 mg/kg sc one dose (day 5) 0 14 Final broad dose range test for 1.12.1(M29I/D19A). Study showed good dose dependent effects. Fitting data to a sigmoidal dose response curve yields an IC$o of 93 nM. No tumor growth Inhlbiton observed. 1,12.1(M2 9I/D19A) 1 mg/kg sc 33 1.12.1(M2 9I/D19A) 3 mg/kg sc 41 1.12.1(M2 9I/D19A) 5 mg/kg sc 60 1.12.1(M2 9I/D19A) 7.5 mg/kg sc 60 1.12.1 (M2 9I/D19A) 10 mg/kg sc 73 1,12.1 (M2 9I/D19A). 50 mg/kg sc 70

Exampie 21, In vivo ECgn Determination

Human foreskin SCID chimera mice were intradermally implanted with M24met cells and were . 5 treated (sc) with anti-ALK-1 antibody1.12.1 (M29I/DT9A) at 1, 3, 5, 7.5, 10 and 50 mg/kg or with isotype match anti-human KLH antibody (10 mg/kg). Upon the conclusion of the experiment, human vessel area In each tumor were quantified as described above. Mouse plasma concentrations of anti-ALK-1 antibody 1.12.1(M29I/D19A) were measured using the method described as following: serum samples from mice were analyzed for anti-ALK-1 antibody 1.12.1 (M29I/D19A) concentration by an ELISA (enzyme-linked 10 immunosorbent Assay). ELISA plates were coated with 10 ug/mi goat anti-human IgG Fc specific antibody (pierce, cat# 31123) in PBS, incubated overnight at 4°C, and then blocked with StartBlock blocking buffer 100 (Pierce, cat# 37542) at room temperature for 1 hr. Serum samples were diluted prior to the analysis 100 and 1000-fold in StartBlock blocking buffer. Two sets of standards were prepared in the blank serum diluted 100 and 100-fold. Standards and diluted serum samples were incubated on the plate for 1hr. Bound anti-ALK-1 antibody 1.12.1(M29I/D19A) was detected using horseradish peroxidase (HRP)-labeled goat anti-human IgG (Fab-specific) antibody (Sigma, cat# A0293). The substrate used was 3, 3’, 5, S'-tetramethyl benzidine (Sigma, cat# T8665). Absorbance was read at 450nm on a Vmax plate reader (Molecular Devices, Menlo Park, CA). A standard curve was fit using nonlinear regression. The detection limit of this assay was 10 ng/ml of anti-ALK-1 antibody 1.12.1 (M29I/D19A). SCID mouse plasma concentration of anti-ALK-1 antibody 1.12.1 (M29I/D19A) is shown in Figure 15.

Figure 15 represents the estimated EC50 for 1.12.1 (M29I/D19A) in the M24met Foreskin SCID-chimera Model. Human vessel area was plotted against the average plasma PK across the study period (14 days) for each treatment group. A fitted curve was produced by the Sigmoidal Dose Dependent program in the Graphpad (Prtzm). EC5o of 93 ng/ml (EC50 is defined as the plasma concentration required for a 50% reduction of human vessel area in the Control group) was derived from the curve fit.

All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it wilt be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. -. 101 189,642/3

SEQUENCE LISTING <11ϋ> Pfizer Inc

Amgen Fremont Inc. <120> HUMAN MONOCLONAL ANTIBODIES TO ACTIVIN RECEPTOR-LIKE KINASE 1

<130> ABX-PF9 PROV <140> 60/715,292 <141> 2005-09-07 <160> 136 <170> Patentln version 3.3 <210> 1 <211> 1332

<212> DNA <213> Human <400> 1 caggtgcagc tgcaggagtc gggtccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgaat actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggalt gggtacatct attacagtgg gagtacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtaltactg t gegagagag 300 tcagtggctg gglttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcclec 360 accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgctc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtagt gaccgtgccc tccagcaact tcggcaccca gacctacacc 600 tgeaaegtag atcacaagcc cagcaacacc aaggtggaca agacagttga gcgcaaatgt 660 tgtgtcgagt gcccaccgtg cccagcacca cctgtggcag gaccglcagt ctlectcttc 720 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 780 102 189,642/3 gtggacgtga gccacgaaga ccccgaggtc cagttcaact ggtacgtgga cggcgtggag 840 gtgcataatg ccaagacaaa gccacgggag gagcagttca acagcacgtt ccgtgtggtc 900 agcgtcctca ccgtcgtgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960 tccaacaaag gcctcccagc ccccatcgag aaaaccatct ccaaaaccaa agggcagccc 1020 cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1080 agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140 aatgggcagc cggagaacaa ctacaagacc acacctccca tgctggactc cgacggctcc 1200 ttcttcctct acagcaagct caccglggac aagagcaggt ggcagcaggg gaacgtcttc 1260 tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1320

Iclccgggla aa 1332 <210> 2 <211> 444

<212> PRT <213> Human <400> 2

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Scr Gly Gly Ser lie Ser Ser Gly 20 25 30

Glu Tyr Tyr Trp Asn Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 103 189,642/3

Cys Ala Arg Glu Ser Val Ala Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 85 90 95

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin 165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190

Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys 210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe 225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val 245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gin Phe 260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 104 189,642/3

Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300

Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Thr 325 330 335

Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350

Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365

Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser 385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <21U> 3 <211> 645

<212> DNA <213> Human <400> 3 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 105 189,642/3 ctctcclgta gggccagtca gagtgtcagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtacatcca gcagggccac tggcatccca 180 gacaggltea gtggcagtgg gtctgggaca gacttcaccc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcgccgat caccttcggc 300 caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccclccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645 <210> 4 <211> 215

<212> PRT <213> Human <400> 4

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Thr Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 65 70 75 80 106 189,642/3

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu lie Lys Arg Thr Val Ala 100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140

Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser 145 150 155 160

Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190

Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys 195 200 205

Scr Phe Asn Arg Gly Glu Cys 210 215 <210> 5 <211> 355

<212> DNA <213> Human <400> 5 caggtgcagc tgcaggagtc gggtccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgaat actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 107 189,642/3 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 tcagtggctg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 <210> 6 <21 i> 118

<212> PRT <213> Human <400> 6

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80

Scr Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Glu Ser Val Ala Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 <210> 7 <211> 325 <212> DNA <213> Human 108 189,642/3 <400> Ί gaaallgtgl tgacgcaglc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgta gggccagtca gagtgtcagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtacalcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcaccc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcgccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 8 <211> 108

<212> PRT <213> Human <400> 8

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Thr Scr Scr Arg Ala Thr Gly He Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95

He Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys 100 105 109 189,642/3 <210> 9 <211> 361

<212> DNA <213> Human <400> 9 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt agtcatggca tgtactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagct atatggtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga atagcctgag agccgaggac acggctgtgt attactgtgc gagagatcag 300 gagcagtggc ccgatgtttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 360 £ 361 <210> 10 <211> 120 <212> PRT <213> Human <400> 10

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30

Gly Met Tyr Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Ala Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr I^eu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 110 189,642/3

Ala Arg Asp Gin Glu Gin Trp Pro Asp Val Phe Asp He Trp Gly Gin 100 105 110 85 90 95

Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 11 <211> 322

<212> DNA <213> Human <400> 11 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggcattaga aattatttag cctggtttca gcagaaacca 120 gggaaagccc ctaagtccct gatctatggt gcatccagtt tgcaaagtgg ggtcccatca 180 aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccaacag tataatagtt acccgctcac tttcggcgga 300 gggaccaagg tggagatcaa ac 322 <210> 12 <211> 107

<212> PRT <213> Human <400> 12

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

Asp Arg Val Thr He Thr Cys Arg Ala Ser Gin Gly lie Arg Asn Tyr 20 25 30

Leu Ala Trp Phe Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu lie 35 40 45

Ill 189,642/3

Tyr Gly Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Leu 85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu He Lys 100 105 <210> 13 <211> 355

<212> DNA <213> Human <400> 13 caggtgcacc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccgtcagc agtggtgatt actactggaa ctggatccgg 120 cagccccccg ggaagggact ggagtggatt gggtatatct attacagtgg gagcaccaac 180 tacaacccct ccctcaagag tcgaatcacc atatcaatag acacgtccaa gaaccagttc 240 tccctgaagc tgaactctgt gaccgctgcg gacacggcct tgtattactg tgcgagagaa 300 tcagtggccg cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 <210> 14 <211> 118

<212> PRT <213> Human <400> 14

Gin Val His Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30 112 189,642/3

Asp Tyr Tyr Trp Asn Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg He Thr lie Ser He Asp Thr Ser Lys Asn Gin Phe 65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Leu Tyr Tyr 85 90 95

Cys Ala Arg Glu Ser Val Ala Ala Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 <210> 15 <211> 325

<212> DNA <213> Human <400> 15 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtattagc agtaggtact tagcctggta ccagcaggaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcaa cactatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 16 <211> 108 <212> PRT <213> Human 113 189,642/3 <400> 16

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Ile Ser Ser Arg 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Glu Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin His Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 17 <211> 355

<212> DNA <213> Human <400> 17 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccgtcagc agtggtgatt actactggaa ctggatccgg 120 cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gagcaccaac 180 tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240 tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagagaa 300 gcagtgtccg cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 114 189,642/1

<212> PRT <213> Human <400> 18

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Ala Val Ser Ala Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 <210> 19 <211> 325

<21.2> DNA <213> Human <400> 19 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agtacctact tagcctggca ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat gglgtatcca gcagggccag tggcgtccca 180 114 189,642/1

<212> PRT <213> Human <400> 18

Gin Val Gin Leu Gin Glu Set Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Scr Gly Gly Scr Val Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Ala Val Ser Ala Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Scr Scr 115 <210> 19 <211> 325

<212> DNA <213> Human <400> 19 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agtacctact tagcctggca ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgtatcca gcagggccag tggcglccca 180 115 189,642/1 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gttcaccgat caccttcggc 300 caagggacac gactggagat laaac 325 <210> 20 <211> 108

<212> PRT <213> Human <400> 20

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Thr 20 25 30

Tyr Leu Ala Trp His Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

Ile Tyr Gly Val Ser Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Scr Gly Thr Asp Phe Thr Leu Thr lie Scr Arg I.xu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95

Ile Thr Phe Gly Gin Gly Thr Arg Leu Glu lie Lys 100 105 <210> 21 <211> 370

<212> DNA <213> Human <400> 21 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 115 189,642/1 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gttcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 20 <211> 108

<212> PRT <213> Human <400> 20

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Thr 20 25 30

Tyr Leu Ala Trp His Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Val Ser Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr he Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys 100 105 <210> 21 <211> 370

<212> DNA <213> Human <400> 21 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 116 189,642/1 acclgcaetg lelclgglgg clccalcagc agtgglgglc aclaclggag clggalccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 lacaacccgl ccclcaagag icgagltacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagttctgt gactgccgcg gacacggccg tatattactg tgcgagagcg 300 gggcgatttt tggagtggtc tgatgttttt gatatctggg gccaagggac aatggtcacc 360 gtctcctcag 370 <210> 22 <211> 123

<212> PRT <213> Human <400> 22

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Gly His Tyr Trp Ser Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Scr Thr Tyr Tyr Asn Pro Scr 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Ala Gly Arg Phe Leu Glu Trp Ser Asp Val Phe Asp He 100 105 110 116 189,642/1 acctgcactg tctctggtgg ctccatcagc aglgglgglc actaclggag ctggatccgc 1 20 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagttctgt gactgccgcg gacacggccg tatattactg tgcgagagcg 300 gggcgatttt tggagtggtc tgatgttttt gatatctggg gccaagggac aatggtcacc 360 gtctcctcag 370 <210> 22 <211> 123

<212> PRT <213> Human <400> 22

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30

Gly His Tyr Trp Ser Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr lie Tyr Tyr 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 Gin Phe 65 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 Ala Gly Arg Phe Leu Glu Trp Ser Asp Val Phe Asp lie 100 105 Π0 117 189,642/1

Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 23 <211> 340

<212> DNA <213> Human <400> 23 gacatcgtga tgacccagtc tccagactcc ctggclgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagclcca acaataagaa clacttagcl 120 tggtaccagc agaaaccagg gcagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatgatact 300 cctccgacgt tcggccaagg gaccaaggtg gaaatcaaac 340 <210> 24 <211> 113

<212> PRT <213> Human <400> 24

Asp He Val Mel Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 117 189,642/1

Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 23 <211> 340

<212> DNA <213> Human <400> 23 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccagg gcagcclcct aagctgclca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatgatact 300 cctccgacgt tcggccaagg gaccaaggtg gaaatcaaac 340 <210> 24 <211> 113

<212> PRT <213> Human <400> 24

Asp lie Val Met Thr Gin Scr Pro Asp Scr Leu Ala Val Ser Leu Gly 1 5 10 15

Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Scr 20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 118 189,642/1

He Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

Tyr Tyr Asp Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu He 100 105 110

Lys <210> 25 <211> 355

<212> DNA <213> Human <400> 25 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cctcacagac cctgtccctc 60 atctgtactg tttctggtgg ctccatcagc agtggtgaat actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgacttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 gggatcggtg ciltlgalal clggggccaa gggacaalgg tcaccgtcic ttcag 355 <210> 26 <211> 118

<212> PRT <213> Human <400> 26

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu He Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Glu Tyr Tyr Trp Ser Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45 118 189,642/1 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

Tyr Tyr Asp Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu lie 100 105 110

Lys <210> 25 <211> 355

<212> DNA <213> Human <400> 25 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cctcacagac cctgtccctc 60 atctgtactg tttctggtgg ctccatcagc agtggtgaat actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgacttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 gggatcgglg cttttgatat ctggggccaa gggacaalgg tcaccgtctc ttcag 355 <210> 26 <211> 118

<212> PRT <213> Human <400> 26

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu He Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 . 30

Glu Tyr Tyr Trp Ser Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45 119 189,642/1

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Leu Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Gly Ile Gly Ala Phe Asp Ile Trp Gly Gin Gly Thr 100 105 110

Met Val Thr Val Ser Ser 115 <210> 27 <211> 325

<212> DNA <213> Human <400> 27 gaaattgtgt tgacgcagtc gccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgltagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcctaggct cctcatctat ggagcalcca gcagggccac tggcatccca 180 gacaggttca glggcagtgg gtctgggaca gacttcactc tcaccalcat cagaclggac 240 cctgaagatt ttgcagtgta ttactgtcag cggtatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 28 <211> 108

<212> PRT <213> Human <400> 28

Glu Ile Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 119 189,642/1

Trp He Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Leu Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Gly He Gly Ala Phe Asp He Trp Gly Gin Gly Thr 100 105 110

Met Val Thr Val Ser Ser 115 <210> 27 <211> 325

<212> DNA <213> Human <400> 27 gaaattgtgt tgacgcagtc gccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagclacl tagcctggta ccagcagaaa 120 cctggccagg ctcctaggct cctcatctat ggagcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcal cagactggac 240 cctgaagatt ttgcagtgta ttactgtcag cggtatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 28 <211> 108

<212> PRT <213> Human <400> 28

Glu He Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 120 189,642/1 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He lie Arg Leu Asp 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Arg Tyr Gly Ser Ser Pro 85 90 95

He Thr Phe Gly Gin Gly Thr Arg Leu Glu lie Lys 100 105 <210> 29 <211> 361

<212> DNA <213> Human <400> 29 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg clccatcagt agttactact ggagclggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgagl caccatalca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agaggacgat 300 agcagtggct gcccctactt tgactactgg ggccagggaa ccctggtcac cgcttcctca 360 s 361 <210> 30 120 189,642/1

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30 10 15

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ile Arg Leu Asp 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Arg Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 29 <211> 361

<212> DNA <213> Human <400> 29 caggtgcagc tgcaggagtc gggcccagga ct ggtgaagc ct tcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccclca agagtcgagt caccatalca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agaggacgat 300 agcagtggct gcccctactt tgactactgg ggccagggaa ccctggtcac cgcttcctca 360 e 361 <210> 30 121 189,642/1 <211> 120

<212> PRT <213> Human <400> 30

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser He Ser Ser Tyr 20 25 30

Tyr Trp Ser Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie 35 40 45

Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60

Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Glu Asp Asp Ser Ser Gly Cys Pro Tyr Phe Asp Tyr Trp Gly Gin 100 105 no

Gly Thr Leu Val Thr Ala Ser Ser 115 120 <210> 31 <211> 322

<212> DNA <213> Human <400> 31 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccagggtcct catctatggt gcatccacca gggccactgg tatcccagtc 180 121 189,642/1 <211> 120

<212> PRT <213> Human <400> 30

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser He Ser Ser Tyr 20 25 30

Tyr Trp Ser Trp He Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie 35 40 45

Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60

Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Glu Asp Asp Scr Scr Gly Cys Pro Tyr Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Ala Ser Ser 115 120 <210> 31 <211> 322

<212> DNA <213> Human <400> 31 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccagggtcct catctatggt gcatccacca gggccactgg tatcccagtc 180 122 189,642/1 aggllcaglg gcagtgggtc Igggacagag llcaetclca ccatcagcag cctgcaglcl 240 gaagattttg cagttlatta ctgtcagcag tataataact ggccattcac tttcggccct 300 gggaccaaag tggatatcaa ac 322 <210> 32 <211> 107

<212> PRT <213> Human <400> 32

Glu lie Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Asn 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Val Leu lie 35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly lie Pro Val Arg Phe Ser Gly 50 55 60

Ser Gly Scr Gly Thr Glu Phe Thr Leu Thr lie Ser Ser Leu Gin Ser 65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Asn Asn Trp Pro Phe 85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp He Lys 100 105 <210> 33 <211> 370 <212> DNA <213> Human <400> 33 122 189,642/1 aggtlcagig gcagtggglc tgggacagag Ueacletca ecalcagcag cctgcaglcl 240 gaagattttg cagtttatta ctgtcagcag tataataact ggccattcac tttcggccct 300 gggaccaaag tggatalcaa ac 322 <210> 32 <211> 107

<212> PRT <213> Human <400> 32

Glu lie Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Asn 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Val Leu lie 35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly lie Pro Val Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Scr Leu Gin Ser 65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Asn Asn Trp Pro Phe 85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp lie Lys too <210> <211> <212> <213> 33 370 DNA Human <400> 33 123 189,642/1 cagatgcagc tgcaggagtc gggcccagga ctggtgaagc cttcgcagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtggtc actactggag ctggatccgc .120 cagcaccccg ggaagggcct ggagtggatt gggtacatct attacagtgg gagcgcctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300 gggcgatttt tggagtggtc tgatgttttt gatatctggg gccaagggac aatggtcacc 360 gtctctttag 370 <210> 34 <211> 123

<212> PRT <213> Human <400> 34

Gin Met Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Gly His Tyr Trp Ser Tip lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Ala Gly Arg Phe Leu Glu Trp Ser Asp Val Phe Asp He 100 105 110 123 189,642/1 cagatgcagc tgcaggagtc gggcccagga ctggtgaagc cttcgcagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtggtc actactggag ctggatccgc 120 cagcaccccg ggaagggcct ggagtggatt gggtacatct attacagtgg gagcgcctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300 gggcgatttt tggagtggtc tgatgttttt gatatctggg gccaagggac aatggtcacc 360 gtctctttag 370 <210> 34 <211> 1.23

<212> PRT <23 3> Human <400> 34

Gin Met Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Gly His Tyr Trp Ser Tip lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Ala Gly Arg Phe Leu Glu Trp Ser Asp Val Phe Asp lie 100 105 110 124 189,642/1

Trp Gly Gin Gly Thr Mel Val Thr Val Ser Leu 115 120 <210> 35 <211> 340

<212> DNA <213> Human <400> 35 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttaact 120 tggtaccagc agaaaccagg acggcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcaacaata ttataatact 300 cctccgacgt tcggccaagg gaccaaggtg gaaatcaagc 340 <210> 36 <211> 113

<212> PRT <213> Human <400> 36

Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30

Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Arg 35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 124 189,642/1

Trp Gly Gin Gly Thr Met Val Thr Val Ser Leu 115 120 <210> 35 <211> 340

<212> DNA <213> Human <400> 35 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttaact 120 tggtaccagc agaaaccagg acggcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcaacaata ttataatact 300 cctccgacgt tcggccaagg gaccaaggtg gaaatcaagc 340 <210> 36 <211> 113 <212> PRT <213> Human <400> 36

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15

Glu Arg Ala Thr He Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30

Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Arg 35 40 45

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 125 189,642/1 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

Tyr Tyr Asn Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu He 100 105 110

Lys <210> 37 <211> 379

<212> DNA <2.13> Human <400> 37 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtaatac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaggaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagggaagcc 300 talgalagta gtggttacla ctactactac tacggtatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctcag 379 <210> 38 <211> 126

<212> PRT <213> Human <400> 38

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Scr Cys Ala Ala Scr Gly Phe Thr Phe Ser Asp Tyr 20 25 30 125 189,642/1

Ile Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

Tyr Tyr Asn Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu lie 100 105 110

Lys <210> 37 <211> 379

<212> DNA <213> Human <400> 37 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtaatac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaggaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagggaagcc 300 latgalagla gtggttacta claclactac tacggtalgg acglctgggg ccaagggace 360 acggtcaccg tctcctcag 379 <210> 38 <211> 126

<212> PRT <213> Human <400> 38

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 126 189,642/1

Tyr Mel Ser Trp He Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Tyr lie Ser Ser Ser Gly Asn Thr He Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Arg Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Ala Tyr Asp Ser Ser Gly Tyr Tyr Tyr Tyr Tyr Tyr Gly 100 105 110

Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 39 <211> 337

<212> DNA <213> Human <400> 39 gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60 atctcctgca agtetagtea gagcctcctg catagtgatg gaaagaccta cttgtattgg 120 tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180 tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgacaatc 240 agccgggtgg aggetgaega tgttggggtt tattactgca tgcaaagtac acaccttcct 300 tggacgttcg gccaagggac caaggtggaa atcaaac 337 <210> 40 <211> 112 <212> PRT <213> Human 126 189,642/1

Tyr Mei Ser Tip lie Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Tyr Ile Ser Ser Ser Gly Asn Thr lie Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Ala Tyr Asp Ser Ser Gly Tyr Tyr Tyr Tyr Tyr Tyr Gly 100 105 110

Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 39 <211> 337

<212> DNA <213> Human <400> 39 gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60 atctcctgca agtctagtca gagcctcctg catagtgatg gaaagaccta cttgtattgg 120 tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180 tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgacaatc 240 agccgggtgg aggctgacga tgttggggtt tattactgca tgcaaagtac acaccttcct 300 tggacgttcg gccaagggac caaggtggaa atcaaac 337 <210> 40 <211> 112 <212> PRT <213> Human 127 189,642/1 <400> 40

Asp He Val Met Thr Gin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15

Gin Pro Ala Ser He Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Pro 35 40 45

Pro Gin Leu Leu lie Tyr Glu Val Scr Asn Arg Phe Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 65 70 75 80

Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gin Ser 85 90 95

Thr His Leu Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys 100 105 110 <210> 41 <211> 376

<212> DNA <213> Human <400> 41 gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60 tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggttggccgt attaaaagca aaagtgatgg tgggacaaca 180 gactacgctg cacccgtgaa aggcagattc accatctcaa gagatgaltc aaaaaacacg 240 ctgtatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300 gggaattact atgatggtag tggttattac tcttttgact actggggcca gggaaccctg 360 gtcaccgtct cctcag 376 127 189,642/1 <400> 40

Asp lie Val Met Thr Gin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Pro 35 40 45

Pro Gin Leu Leu He Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He 65 70 75 80

Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gin Ser 85 90 95

Thr His Leu Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys loo 105 no <210> 41 <211> 376

<212> DNA <213> Human <400> 41 gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60 tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggttggccgt attaaaagca aaagtgatgg tgggacaaca 180 gactacgctg cacccgtgaa aggcagattc accatctcaa gagatgattc aaaaaacacg 240 ctgtatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300 gggaattact atgatggtag tggttattac tcttttgact actggggcca gggaaccctg 360 gtcaccgtct cctcag 376 128 189,642/1 <210> 42 <211> 125

<212> PRT <213> Human <400> 42

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30

Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg lie Lys Ser Lys Ser Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80

Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Thr Gly Asn Tyr Tyr Asp Gly Ser Gly Tyr Tyr Ser Phe 100 105 110

Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 337

<212> DNA <213> Human <400> 43 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 128 189,642/1 <210> 42 <211> 125

<212> PRT <213> Human <400> 42

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30

Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg He Lys Ser Lys Ser Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Lys Gly Arg Phe Thr He Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80

Leu Tyr Leu Gin Mel Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Thr Gly Asn Tyr Tyr Asp Gly Ser Gly Tyr Tyr Ser Phe 100 105 110

Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 337

<212> DNA <213> Human <400> 43 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 129 189,642/1 atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc clgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactcct 300 cccactttcg gcggagggac caaggtggag atcaaac 337 <210> 44 <211> 112

<212> PRT <213> Human <400> 44

Asp Ile Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 15 10 15

Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser 20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45

Pro Gin Leu Leu Ile Tyr Leu Gly Scr Asn Arg Ala Scr Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala 85 90 95

Leu Gin Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110

<210> 45 <211> 355 <212> DNA 129 189,642/1 atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagallllae actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactcct 300 cccactttcg gcggagggac caaggtggag atcaaac 337 <210> 44 <211> 112

<212> PRT <213> Human <400> 44

Asp lie Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 15 10 15

Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser 20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45

Pro Gin Leu Leu He Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala 85 90 95

Leu Gin Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu He Lys 100 105 110

<210> 45 <211> 355 <212> DNA 130 189,642/1 <213> Human <400> 45 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtaalgatl actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 tccacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtcic ctcag 355 <210> 46 <211> 118

<212> PRT <213> Human <400> 46

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser He Ser Ser Asn 20 25 30

Asp Tyr Tyr Trp Asn Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Ser Thr Asp Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110 130 189,642/1 <213> Human <400> 45 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtaatgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 tccacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctcag 355 <210> 46 <211> 118

<212> PRT <213> Human <400> 46

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Asn 20 25 30

Asp Tyr Tyr Trp Asn Trp Ile Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr Ile Tyr Tyr 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 Gin Phe 65 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 Glu Ser Thr Asp Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110 131 189,642/1

Thr Val Thr Val Ser Ser 115 <210> 47 <211> 325

<212> DNA <213> Human <400> 47 gaaaatgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaactact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcttcca gcggggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cattatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 48 <211> 108

<212> PRT <213> Human <400> 48

Glu Asn Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Asn 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Ala Ser Ser Gly Ala Thr Gly He Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 131 189,642/1

Thr Val Thr Val Ser Ser 115 <210> 47 <211> 325

<212> DNA <213> Human <400> 47 gaaaatgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaactact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcttcca gcggggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cattatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 48 <211> 108

<212> PRT <213> Human <400> 48

Glu Asn Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Asn 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Ala Ser Ser Gly Ala Thr Gly He Pro Asp Arg Phe Ser 50 ' 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 132 189,642/1 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin His Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys 100 105 <210> 49 <211> 361

<212> DNA <213> Human <400> 49 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcaatag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 cgtgactacg gtggtggctt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 g 361 <210> 50 <211> 120

<212> PRT <213> Human <400> 50

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Ser Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45 132 189,642/1 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin His Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 49 <211> 361

<212> DNA <213> Human <400> 49 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcaatag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 cgtgactacg gtggtggctt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 g 361 <210> 50 <211> 120

<212> PRT <213> Human <400> 50

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Scr Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45 133 189,642/1

Trp lie Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser He Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Arg Asp Tyr Gly Gly Gly Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 340

<212> DNA <213> Human <400> 51 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca tcaataagat ctacttagcl 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagaltl cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcaccaata ttatagtact 300 ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaac 340 <210> 52 <211> 113

<212> PRT <213> Human <400> 52

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 133 189,642/1

Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser He Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Arg Asp Tyr Gly Gly Gly Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 340

<212> DNA <213> Human <400> 51 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca tcaataagat ctacttagct 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcaccaata ttatagtact 300 ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaac 340 <210> 52 <211> 113

<212> PRT <213> Human <400> 52

Asp He Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 134 189,642/1

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30 10 15

Ser lie Asn Lys Ile Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

Ile Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gin 85 90 95

Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Ile 100 105 110

Lys <2.1.0> 53 <211> 355

<212> DNA <213> Human <400> 53 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atttcagtag ccacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 gctacggagg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 134 189,642/1

Glu Arg Ala Thr He Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30 10 15

Ser lie Asn Lys He Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gin 85 90 95

Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu lie 100 105 110

Lys <210> 53 <211> 355

<212> DNA <213> Human <400> 53 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atttcagtag ccacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 gctacggagg ggttlgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 135 189,642/1 <210> 54 <211> 118

<212> PRT <213> Human <400> 54

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp Ile Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp Ile Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Ala Thr Ser Lys Asn Gin Phe 65 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 Glu Ala Thr Glu Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 <210> 55 <211> 325

<212> DNA <213> Human <400> 55 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagggccacc 60 ctctcctgca gggccagtca gagtgttagc accacctact tagcctggta ccagcagaaa 120 135 189,642/1 <210> 54 <211> 118

<212> PRT <213> Human <400> 54

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser Val Ala Thr Ser Lys Asn Gin Phe 65 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 Glu Ala Thr Glu Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 <210> 55 <211> 325

<212> DNA <213> Human <400> 55 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagggccacc 60 ctctcctgca gggccagtca gagtgttagc accacctact tagcctggta ccagcagaaa 120 136 189,642/1

I I cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 ' gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 ι cctgaagatt ttgcactgta ttactgtcag cactatggta cctcatcgat caccttcggc 300 ! caagggacac gactggagat taaac 325 <210> 56 <211> 108

<212> PRT <213> Human <400> 56

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Thr Thr 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Ala Ser Ser Arg Ala Thr Gly He Pro Asp Arg Phe Ser 50 55 60

Gly Scr Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gin His Tyr Gly Thr Ser Ser 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys 100 <2W> <211> <212> <213> 57 355 DNA Human 136 189,642/1 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcactgta ttactgtcag cactatggta cclcatcgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 56 <211> 108

<212> PRT <213> Human <400> 56

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Thr Thr 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gin His Tyr Gly Thr Ser Ser 85 90 95

Ile Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 <210> <211 > <212> <213> 57 355 DNA Human 137 189,642/1 <400> 57 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 tccacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctcag 355 <210> 58 <211> 118

<212> PRT <213> Human <400> 58

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Scr 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Ser Thr Asp Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110 137 189,642/1 <400> 57 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 tccacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtcic ctcag 355 <210> 58 <211> 118

<212> PRT <213> Human <400> 58

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Scr 50 55 60

Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Ser Thr Asp Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110 138 189,642/1

Thr Val Thr Val Ser Ser 115 <210> 59 <211> 325

<212> DNA <213> Human <400> 59 gaaagtgtgt tgacgcagtc tcctggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatatat ggtgtttcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 60 <211> 108

<212> PRT <213> Human <400> 60

Glu Ser Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Scr Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

Ile Tyr Gly Val Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 75 80 i 138 189,642/1

Thr Val Thr Val Ser Ser 115 <210> 59 <211> 325

<212> DNA <213> Human <400> 59 gaaagtgtgt tgacgcagtc tcctggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatatat ggtgtttcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 60 <211> 108

<212> PRT <213> Human <400> 60

Glu Ser Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Val Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 65 70 75 80 139 189,642/1

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys 100 105 <210> 61 <211> 355

<212> DNA <213> Human <400> 61 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 gatattgcag gattcgaccc ctggggccag ggaaccctgg tcaccgtcic ctcag 355 <210> 62 <211> 118

<212> PRT <213> Human <400> 62

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser He Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 140 189,642/1

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80 50 55 60

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Glu Asp Ile Ala Gly Phe Asp Pro Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 <210> 63 <211> 325

<212> DNA <213> Human <400> 63 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctac ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gactccactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtata ttactgtcag cagtatggta gctcacctat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 64 <211> 108

<212> PRT <213> Human <400> 64

Glu Ile Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15 141 189,642/1

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr lie Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu lie Lys 100 105 <210> 65 <211> 355

<212> DNA <213> Human <400> 65 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgaat actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtatatct tttacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcactag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagaa 300 tccacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctcag 355 <210> 66 <211> 118 <212> PRT <213> Human <400> 66 142 189,642/1

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Glu Tyr Tyr Trp Ser Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Phe Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Leu Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Ser Thr Asp Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110

Thr Val Thr Val Ser Ser 115 <210> 67 <211> 325

<212> DNA <213> Human <400> 67 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcggaaa 120 cctggccagg ctcccaggct cctcatatat ggtgtatcca gtagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag caatatggta gctcaatgat caccttcggc 300 143 189,642/1 caagggacac gactggagat taaac 325 <210> 68 <211> 108

<212> PRT <213> Human <400> 68

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Arg Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

Ile Tyr Gly Val Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Met 85 90 95

Ile Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 69 <211> 355

<212> DNA <213> Human <400> 69 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 144 189,642/1 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 ggcctcgagg cttttgatat ctggggtcaa gggacaatgg tcaccgactc ttcag 355 <210> 70 <211> 118

<212> PRT <213> Human <400> 70

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser He Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 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 Glu Gly Leu Glu Ala Phe Asp He Trp Gly Gin Gly Thr 100 105 110

Met Val Thr Asp Ser Ser 115 <210> 71 <211> 325 145 189,642/1

<212> DNA <213> Human <400> 71 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat gatgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg ctctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ctactgtcag cattatggta gctcacttct cactttcggc 300 ggagggacca aggtggagat caaac 325 <210> 72 <211> 108

<212> PRT <213> Human <400> 72

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Asp Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin His Tyr Gly Ser Ser Leu 85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu He Lys 146 189,642/1 100 105 <210> 73 <211> 355

<212> DNA <213> Human <400> 73 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaactcgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 ggccagaacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctcag 355 <210> 74 <211> 118

<212> PRT <213> Human <400> 74

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Asp Tyr Tyr Trp Asn Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Ser Ser 50 55 60

Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80 147 189,642/1

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Glu Gly Gin Asn Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110

Thr Val Thr Val Ser Ser 115 <210> 75 <211> 340

<212> DNA <213> Human <400> 75 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 atctcctgca ggtctagtca aagcctcgta tacagtgatg gaaacaccta cttgaattgg 120 tttcagcaga ggccaggcca atctccaagg cgcctaattt ataaggtttc taactgggac 180 tctggggtcc cagacagatt cagcggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt tattactgca tgcaaggtac acactggcct 300 ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaac 340 <210> 76 <211> 113

<212> PRT <213> Human <400> 76

Asp Val Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val Tyr Ser 20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gin Gin Arg Pro Gly Gin Ser 35 40 45 148 189,642/1

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Trp Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Gly 85 90 95

Thr His Trp Pro Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu lie ioo 105 no

Lys <210> 77 <211> 358

<212> DNA <213> Human <400> 77 gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60 tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggttggccgt attaaaagca aaactgatgg tgggacaaca 180 gactacgctg cacccgtgaa aggcagattc accatctcaa gagatgattc aaaaaacacg 240 ctgtatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300 ggggatggaa cgcactttga ctactggggc cagggaaccc tggtcaccgt ctcctcag 358 <210> 78 <211> 119

<212> PRT <213> Human <400> 78

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 149 189,642/1

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30

Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg He Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80

Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Thr Gly Asp Gly Thr His Phe Asp Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ser 115 <210> 79 <211> 322

<212> DNA <213> Human <400> 79 gaaacgacac tcacgcagtc tccagcattc atgtcagcga ctccaggaga caaagtcaac 60 atctcctgca aagccagcca agacattgat gatgatatga actggtacca acagaaacca 120 ggagaagctg ctattttcat tattcaagaa gctactactc tcgttcctgg aatcccacct 180 cgattcagtg gcagcgggta tggaacagat tttaccctca caattaataa catagaatct 240 gaggatgctg catattactt ctgtctacaa catgataatt tcccgctcac tttcggcgga 300 gggaccaagg tggagatcaa ac 322 150 189,642/1 <210> 80 <211> 107

<212> PRT <213> Human <400> 80

Glu Thr Thr Leu Thr Gin Ser Pro Ala Phe Met Ser Ala Thr Pro Gly 15 10 15

Asp Lys Val Asn lie Ser Cys Lys Ala Ser Gin Asp He Asp Asp Asp 20 25 30

Met Asn Trp Tyr Gin Gin Lys Pro Gly Glu Ala Ala He Phe He He 35 40 45

Gin Glu Ala Thr Thr Leu Val Pro Gly He Pro Pro Arg Phe Ser Gly 50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr He Asn Asn He Glu Ser 65 70 75 80

Glu Asp Ala Ala Tyr Tyr Phe Cys Leu Gin His Asp Asn Phe Pro Leu 85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 <210> 81 <211> 379

<212> DNA <213> Human <400> 81 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac cacatactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa gtcactgtat 240 151 189,642/1 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagagggg 300 tatagcagtg gctggtacga ggactactac tacggtatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctcag 379 <210> 82 <211> 126

<212> PRT <213> Human <400> 82

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30

Tyr Met Ser Trp He Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Tyr He Ser Ser Ser Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Gly Tyr Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr Gly 100 105 110

Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125

<210> 83 <211> 337 <212> DNA 152 189,642/1 <213> Human <400> 83 gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60 atctcctgca agtttagtca gagcctcctg catagtgatg gaaagaccta tttgtattgg 120 tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttt 180 tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240 agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat acagcttcct 300 cggacgttcg gccaagggac caaggtggaa atcaaac 337 <210> 84 <211> 112

<212> PRT <213> Human <400> 84

Asp Ile Val Met Thr Gin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 15 10 15

Gin Pro Ala Ser Ile Ser Cys Lys Phe Ser Gin Ser Leu Leu His Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Pro 35 40 45

Pro Gin Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ser 85 90 95

Ile Gin Leu Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys ioo 105 no 153 189,642/1 <210> 85 <211> 388

<212> DNA <213> Human <400> 85 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gacttctaca tgagctggat ccgccaggct 120 ccagggaagg ggctggaatg gatttcatac attagtagta gtggtagtac catttactac 180 gcagactctg tgaagggccg attcaccatg tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attattgtgc gagagaagga 300 tactatgatt cggggagtta ttataaggac tacgactact acggtatgga cgtctggggc 360 caagggacca cggtcaccgt ctcctcag 388 <210> 86 <211> 129

<212> PRT <213> Human <400> 86

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30

Tyr Met Ser Trp lie Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp He 35 40 45

Ser Tyr lie Ser Ser Ser Gly Ser Thr lie Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 154 189,642/1

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Gly Tyr Tyr Asp Ser Gly Ser Tyr Tyr Lys Asp Tyr Asp 100 105 110

Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser 115 120 125

Ser <210> 87 <211> 337

<212> DNA <213> Human <400> 87 gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60 atctcctgca agtctagtca gagcctcctg catagtgatg gaaagaccta tttgtattgg 120 tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180 tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240 agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat acagcttcct 300 cggacgttcg gccaagggac caaggtggaa atcaaac 337 <210> 88 <211> 112

<212> PRT <213> Human <400> 88

Asp He Val Met Thr Gin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser 20 25 30 155 189,642/1

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Pro 35 40 45

Pro Gin Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ser 85 90 95

Ile Gin Leu Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 89 <211> 379

<212> DNA <213> Human <400> 89 caggtgcggc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtatttc catatactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagaagga 300 tatagcagct cgtcacatta ctacgactac tacggtatgg acgtctgggg ccaagggace 360 acggtcaccg tctcctcag 379 <210> 90 <211> 126 <212> PRT <213> Human <400> 90 156 189,642/1

Gin Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30

Tyr Met Ser Trp lie Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Tyr He Ser Ser Ser Gly He Ser He Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Gly Tyr Ser Ser Ser Ser His Tyr Tyr Asp Tyr Tyr Gly loo 105 no

Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 91 <211> 337

<212> DNA <213> Human <400> 91 gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60 atctcctgca agtetagtea gagcctcctg catagtgatg gaaagaccta tttgtattgg 120 tacctgcaga agccaggcca gcctccacag gteettatet atgaagtttc caaccggttc 180 tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240 agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtac acagcttcct 300 157 189,642/1 cggacgttcg gccaagggac caaggtggaa atcaaac 337 <210> 92 <211> 112

<212> PRT <213> Human <400> 92

Asp lie Val Met Thr Gin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 15 10 15

Gin Pro Ala Ser Ile Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Pro 35 40 45

Pro Gin Val Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ser 85 90 95

Thr Gin Leu Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys 100 105 110 <210> 93 <211> 503

<212> PRT <213> Cynomolgus Monkey <400> 93

Met Thr Leu Gly Ser Pro Arg Arg Gly Leu Leu Met Leu Leu Met Ala 15 10 15 158 189,642/1

Leu Val Thr Gin Gly Asp Pro Val Lys Pro Ser Arg Gly Pro Leu Val 20 25 30

Thr Cys Thr Cys Glu Ser Pro His Cys Arg Gly Pro Thr Cys Gin Gly 35 40 45

Ala Trp Cys Thr Val Val Leu Val Arg Glu Glu Gly Arg His Pro Gin 50 55 60

Glu His Arg Gly Cys Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg 65 70 75 80

Pro Thr Glu Phe Val Asn His Tyr Cys Cys Asp Ser His Leu Cys Asn 85 90 95

Arg Asn Val Ser Leu Val Leu Glu Ala Thr Gin Thr Pro Ser Glu Gin 100 105 110

Pro Gly Thr Asp Ser Gin Leu Ala Leu He Leu Gly Pro Val Leu Ala 115 120 125

Leu Leu Ala Leu Val Ala Leu Gly Val Val Gly Leu Trp His Val Arg 130 135 140

Arg Arg Gin Glu Lys Gin Arg Gly Leu His Ser Glu Leu Gly Glu Ser 145 150 155 160

Ser Leu He Leu Lys Ala Ser Glu Gin Gly Asp Ser Met Leu Gly Asp 165 170 175

Leu Leu Asp Ser Asp Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe 180 185 190

Leu Val Gin Arg Thr Val Ala Arg Gin Val Ala Leu Val Glu Cys Val 195 200 205

Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu Trp His Gly Glu 159 189,642/1

Ser Val Ala Val Lys He Phe Ser Ser Arg Asp Glu Gin Ser Trp Phe 225 230 235 240 210 215 220

Arg Glu Thr Glu lie Tyr Asn Thr Val Leu Leu Arg His Asp Asn lie 245 250 255

Leu Gly Phe lie Ala Ser Asp Met Thr Ser Arg Asn Ser Ser Thr Gin 260 265 270

Leu Trp Leu lie Thr His Tyr His Glu His Gly Ser Leu Tyr Asp Phe 275 280 285

Leu Gin Arg Gin Thr Leu Glu Pro His Leu Ala Leu Arg Leu Ala Val 290 295 300

Ser Ala Ala Cys Gly Leu Ala His Leu His Val Glu He Phe Gly Thr 305 310 315 320

Gin Gly Lys Pro Ala He Ala His Arg Asp Phe Lys Ser Arg Asn Val 325 330 335

Leu Val Lys Ser Asn Leu Gin Cys Cys He Ala Asp Leu Gly Leu Ala 340 345 350

Val Met His Ser Gin Gly Ser Asp Tyr Leu Asp lie Gly Asn Asn Pro 355 360 365

Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Gin 370 375 380

He Arg Thr Asp Cys Phe Glu Ser Tyr Lys Trp Thr Asp lie Trp Ala 385 390 395 400

Phe Gly Leu Val Leu Trp Glu He Ala Arg Arg Thr lie Val Asn Gly 405 410 415 160 189,642/1 lie Val Glu Asp Tyr Arg Pro Pro Phe Tyr Asp Val Val Pro Asn Asp 420 425 430

Pro Ser Phe Glu Asp Met Lys Lys Val Val Cys Val Asp Gin Gin Thr 435 440 445

Pro Thr lie Pro Asn Arg Leu Ala Ala Asp Pro Val Leu Ser Gly Leu 450 455 460

Ala Gin Met Met Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala Arg Leu 465 470 475 480

Thr Ala Leu Are lie Lys Lys Thr Leu Gin Lys lie Ser Asn Ser Pro 485 490 495

Glu Lys Pro Lys Val Ile Gin 500 <210> 94 <211> 1512

<212> DNA <213> Cynomolgus Monkey <400> 94 atgaccttgg gctccccgag gagaggcctt ctgatgctgc tgatggcctt ggtgacccag 60 ggtgaccccg tgaagccctc tcggggcccg ctggtgacct gcacatgtga gagcccacat 120 tgcagggggc ctacctgcca gggggcctgg tgcacagtag tgctggtgcg ggaggagggg 180 aggcaccccc aggaacatcg gggctgcggg aacttgcaca gggagctctg cagggggcgc 240 cccaccgagt tcgtcaacca ctactgctgt gacagccacc tctgcaaccg caacgtgtcc 300 ctggtgctgg aggccaccca aactccttcg gagcagccgg gaacagacag ccagctggcc 360 ctgatcctgg gccccgtgct ggccttgctg gccctggtgg ccctgggtgt cgtgggcctg 420 tggcatgtcc gacggaggca ggagaagcag cggggcctgc acagcgagct gggagagtcc 480 agtctcatcc tgaaagcatc tgagcagggc gacagcatgt tgggggacct cctggacagt 540 189,642/1 161 gactgcacca cagggagtgg ctcggggctc cccttcctgg tgcagaggac agtggcacgg 600 caggttgcct tggtggagtg tgtgggaaaa ggccgctatg gcgaagtgtg gcggggcttg 660 tggcacggtg agagtgtggc cgtcaagatc ttctcctcga gggacgaaca gtcctggttc 720 cgggagaetg agatctacaa cacagtgttg ctcagacacg acaacatcct aggcttcatc 780 gcctcagaca tgacctcccg caactcgagc acgcagctgt ggctcatcac gcattaccac 840 gagcacggct ccctctacga ctttctgcag agacagacgc tggagccgca tttggctctg 900 aggctagctg tgtccgcagc ctgtggcctg gcacacctgc acgtggagat cttcggtaca 960 cagggcaaac cggccattgc ccaccgtgac ttcaagagcc gcaacgtgct ggtcaagagc 1020 aacctgcagt gttgcattgc tgacctgggc ctggctgtga tgcactcaca gggcagcgat 1080 tacctggaca tcggcaacaa cccgagagta ggcaccaaga ggtacatggc acccgaggtg 1140 ctggatgagc agatccgcac ggactgcttt gagtcctata agtggactga catctgggcc 1200 tttggcctgg tgctgtggga gatcgcccgc cggaccatcg tgaacggcat cgtggaggac 1260 tatagaccac ccttctatga tgtggtgccc aatgacccca gctttgagga catgaagaag 1320 gtggtgtgtg tggatcagca gacccccacc atccctaacc ggctggctgc agacccggtc 1380 ctctcaggcc tagctcagat gatgcgggag tgctggtacc caaacccctc tgcccgactc 1440 actgcgctgc ggatcaagaa gacactacag aaaattagca acagtccaga gaagcccaaa 1500 gtgattcagt ag 1512 <210> 95 <211> 1332

<212> DNA <213> Human <400> 95 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatgagc agtggtgaat actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 162 189,642/1 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 tcagtggctg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcctcc 360 accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgctc tgaccagcgg cgtgcacacc ttcccagctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcaact tcggcaccca gacctacacc 600 tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agacagttga gcgcaaatgt 660 tgtgtcgagt gcccaccgtg cccagcacca cctgtggcag gaccgtcagt cttcctcttc 720 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 780 gtggacgtga gccacgaaga ccccgaggtc cagttcaact ggtacgtgga cggcgtggag 840 gtgcataatg ccaagacaaa gccacgggag gagcagttca acagcacgtt ccgtgtggtc 900 agcgtcctca ccgttgtgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960 tccaacaaag gcctcccagc ccccatcgag aaaaccatct ccaaaaccaa agggcagccc 1020 cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1080 agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140 aatgggcagc cggagaacaa ctacaagacc acacctccca tgctggactc cgacggctcc 1200 ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1260 tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1320 tctccgggta aa 1332 <210> 96 <211> 33

<212> DNA <213> Artificial <220> <223> The forward primer used for cloning ECD of ALK-1 <400> 96 163 189,642/1 acggcccagc cggccgaccc tgtgaagccg tct 33 <210> 97 <211> 47

<212> DNA <213> Artificial <220> <223> The reverse primer used for cloning ECD of ALK-1 <400> 97 actaagcttt taatgatgat gatgatgatg ctggccatct gttcccg 47 <210> 98 <211> 103

<212> PRT <213> Human <400> 98

Asp Pro Val Lys Pro Ser Arg Gly Pro Leu Val Thr Cys Thr Cys Glu 15 10 15

Ser Pro His Cys Lys Gly Pro Thr Cys Arg Gly Ala Trp Cys Thr Val 20 25 30

Val Leu Val Arg Glu Glu Gly Arg His Pro Gin Glu His Arg Gly Cys 35 40 45

Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg Pro Thr Glu Phe Val 50 55 60

Asn His Tyr Cys Cys Asp Ser His Leu Cys Asn His Asn Val Ser Leu 65 70 75 80

Val Leu Glu Ala Thr Gin Pro Pro Ser Glu Gin Pro Gly Thr Asp Gly 85 90 95

Gin His His His His His His 100 164 189,642/1 <210> 99 <211> 387

<212> DNA <213> Human <400> 99 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgcggccc agccggccga ccctgtgaag ccgtctcggg gcccgctggt gacctgcacg 120 tgtgagagcc cacattgcaa ggggcctacc tgccgggggg cctggtgcac agtagtgctg 180 gtgcgggagg aggggaggca cccccaggaa catcggggct gcgggaactt gcacagggag 240 ctctgcaggg ggcgccccac cgagttcgtc aaccactact gctgcgacag ccacctctgc 300 aaccacaacg tgtccctggt gctggaggcc acccaacctc cttcggagca gccgggaaca 360 gatggccagc atcatcatca tcatcat 387 <210> 100 <211> 444

<212> PRT <213> Human <400> 100

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Met Ser Ser Gly 20 25 30

Glu Tyr Tyr Trp Asn Trp lie Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp Ue Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80 165 189,642/1

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Glu Ser Val Ala Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin 165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190

Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys 210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe 225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val 245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gin Phe 260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 166 189,642/1

Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295 300 275 280 285

Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro He Glu Lys Thr lie Ser Lys Thr 325 330 335

Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350

Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365

Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser 385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 101 <211> 645 <212> DNA <213> Human <400> 101 167 189,642/1 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagacacc 60 ctctcctgta gggccagtca gagtgtcagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtacatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcaccc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcgccgat caccttcggc 300 caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645 <210> 102 <211> 215

<212> PRT <213> Human <400> 102

Glu He Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Asp Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Thr Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 75 80 168 189,642/1

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95

He Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys Arg Thr Val Ala 100 105 110

Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140

Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser 145 150 155 160

Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190

Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys 195 200 205

Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 103 <211> 355

<212> DNA <213> Human <400> 103 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc acctgcactg tctctggtgg ctccatgagc agtggtgaat actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtacctac 180 60 169 189,642/1 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 tcagtggctg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 <210> 104 <211> 118

<212> PRT <213> Human <400> 104

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Met Ser Ser Gly 20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr 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 Gin Phe 65 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 Glu Ser Val Ala Gly Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 105 <211> 58 <212> DNA 170 189,642/1 <213> Artificial <220> <223> Amplification Primer Used for Cloning Full-Length 1.12.1 <400> 105 tcttcaagct tgatatctct agaagccgcc accatgaaac acctgtggtt cttcctcc 58 <210> 106 <211> 46

<212> DNA <213> Artificial <220> <223> Amplification Primer Used for Cloning Full-Length 1.12.1 <400> 106 ttctctgatc agaattccta ctatttaccc ggagacaggg agaggc 46 <210> 107 <211> 48

<212> DNA <213> Artificial <220> <223> Amplification Primer Used for Cloning Full-Length 1.12.1 <400> 107 tcttcaagct tcccgggagc cgccaccatg gaaaccccag cgcagctt 48 <210> 108 <211> 49

<212> DNA <213> Artificial <220> <223> Amplification Primer Used for Cloning Full-Length 1.12.1 <400> 108 ttctttgatc agaattctca ctaacactct cccctgttga agctctttg 49 <210> 109 <211> 32 <212> DNA <213> Artificial 171 189,642/1 <220> <223> Mutagenic Oligonucleotide <400> 109 ctccagggga aagagccacc ctctcctgta gg 32 <210> 110 <211> 32

<212> DNA <213> Artificial <220> <223> Mutagenic Oligonucleotide <400> 110 cctacaggag agggtggctc tttcccctgg ag 32 <210> 111 <211> 30

<212> DNA <213> Artificial <220> <223> Mutagenic Oligonucleotide <400> 111 ggtggctcca tcagcagtgg tgaatactac 30 <210> 112 <211> 30

<212> DNA <213> Artificial <220> <223> Mutagenic Oligonucleotide <400> 112 gtagtattca ccactgctga tggagccacc 30 <210> 113 <211> 9 <212> PRT <213> Human 172 189,642/1 <400> 113

Glu Ile Val Leu Thr Gin Ser Pro Gly 1 5 <210> 114 <211> 8

<212> PRT <213> Human <400> 114

Gin Val Gin Leu Gin Glu Ser Gly 1 5 <210> 115 <211> 21

<212> DNA <213> Artificial <220> <223> Forward Primer <400> 115 agcgggccca gagggaccat g 21 <210> 116 <211> 28

<212> DNA <213> Artificial <220> <223> Reverse Primer <400> 116 cagaaaggaa tcaggtgctc ctgggcta 28 <210> 117 <211> 28 <212> DNA <213> Artificial <220> <223> Forward Primer 173 189,642/1 <400> 117 gattatggcc ttgggctccc ccaggaaa 28 <210> 118 <211> 37

<212> DNA <213> Artificial <220> <223> Reverse Primer <400> 118 gggctattga atcactttag gcttctctgg actgttg 37 <210> 119 <211> 27

<212> DNA <213> Artificial <220> <223> TaqMan Probe (IDl-Probe) <400> 119 ccagcacgtc atcgactaca tcaggga 27 <210> 120 <211> 22

<212> DNA <213> Artificial <220> <223> Taqman PCR Primer (ID1-F) <400> 120 aaggtgagca aggtggagat tc 22 <210> 121 <211> 21

<212> DNA <213> Artificial <220> <223> Taqman PCR Primer (ID1-R) <400> 121 174 189,642/1 ttccgagttc agctccaact g 21 <210> 122 <211> 25

<212> PRT <213> Human <400> 122

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 123 <211> 12

<212> PRT <213> Human <400> 123

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser 1 5 10 <210> 124 <211> 25

<212> PRT <213> Human <400> 124

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15 20 <210> <211> <212> <213> 125 12 PRT Human 25 175 189,642/1 <400> 125

Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr Trp Ser 1 5 10 <210> 126 <211> 325

<212> DNA <213> Human <400> 126 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagacacc 60 ctctcctgta gggccagtca gagtgtcagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtacatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcaccc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcgccgat caccttcggc 300 caagggacac gactggagat taaac 325 <210> 127 <211> 108

<212> PRT <213> Human <400> 127

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Asp Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

He Tyr Gly Thr Ser Ser Arg Ala Thr Gly He Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 176 189,642/1 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95

Ile Thr Phe Gly Gin Gly Thr Arg Leu Glu lie Lys 100 105 <210> 128 <211> 1332

<212> DNA <213> Human <400> 128 caggtgcagc tgcaggagtc gggtccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatgagc agtggtgaat actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 tcagtggctg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcctcc 360 accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgctc tgaccagcgg cgtgcacacc ttcccagctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcaact tcggcaccca gacctacacc 600 tgeaaegtag atcacaagcc cagcaacacc aaggtggaca agacagttga gcgcaaatgt 660 tgtgtcgagt gcccaccgtg cccagcacca cctgtggcag gaccgtcagt cttcctcttc 720 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 780 gtggacgtga gccacgaaga ccccgaggtc cagttcaact ggtacgtgga cggcgtggag 840 gtgcataatg ccaagacaaa gccacgggag gagcagttca acagcacgtt ccgtgtggtc 900 agcgtcctca ccgttgtgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960 177 189,642/1 tccaacaaag gcctcccagc ccccatcgag aaaaccatct ccaaaaccaa agggcagccc 1020 cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1080 agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140 aatgggcagc cggagaacaa ctacaagacc acacctccca tgctggactc cgacggctcc 1200 ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1260 tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1320 tctccgggta aa 1332 <210> 129 <211> 355

<212> DNA <213> Human <400> 129 caggtgcagc tgcaggagtc gggtccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatgagc agtggtgaat actactggaa ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 tcagtggctg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 <210> 130 <211> 503

<212> PRT <213> Human <400> 130

Met Thr Leu Gly Ser Pro Arg Lys Gly Leu Leu Met Leu Leu Met Ala 15 10 15

Leu Val Thr Gin Gly Asp Pro Val Lys Pro Ser Arg Gly Pro Leu Val 20 25 30 178 189,642/1

Thr Cys Thr Cys Glu Ser Pro His Cys Lys Gly Pro Thr Cys Arg Gly 35 40 45

Ala Trp Cys Thr Val Val Leu Val Arg Glu Glu Gly Arg His Pro Gin 50 55 60

Glu His Arg Gly Cys Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg 65 70 75 80

Pro Thr Glu Phe Val Asn His Tyr Cys Cys Asp Ser His Leu Cys Asn 85 90 95

His Asn Val Ser Leu Val Leu Glu Ala Thr Gin Pro Pro Ser Glu Gin 100 105 110

Pro Gly Thr Asp Gly Gin Leu Ala Leu He Leu Gly Pro Val Leu Ala 115 120 125

Leu Leu Ala Leu Val Ala Leu Gly Val Leu Gly Leu Trp His Val Arg 130 135 140

Arg Arg Gin Glu Lys Gin Arg Gly Leu His Ser Glu Leu Gly Glu Ser 145 150 155 160

Ser Leu He Leu Lys Ala Ser Glu Gin Gly Asp Thr Met Leu Gly Asp 165 170 175

Leu Leu Asp Ser Asp Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe 180 185 190

Leu Val Gin Arg Thr Val Ala Arg Gin Val Ala Leu Val Glu Cys Val 195 200 205

Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu Trp His Gly Glu 210 215 220

Ser Val Ala Val Lys lie Phe Ser Ser Arg Asp Glu Gin Ser Trp Phe 179 189,642/1 225 230 235 240

Arg Glu Thr Glu lie Tyr Asn Thr Val Leu Leu Arg His Asp Asn lie 245 250 255

Leu Gly Phe lie Ala Ser Asp Met Thr Ser Arg Asn Ser Ser Thr Gin 260 265 270

Leu Trp Leu lie Thr His Tyr His Glu His Gly Ser Leu Tyr Asp Phe 275 280 285

Leu Gin Arg Gin Thr Leu Glu Pro His Leu Ala Leu Arg Leu Ala Val 290 295 300

Ser Ala Ala Cys Gly Leu Ala His Leu His Val Glu lie Phe Gly Thr 305 310 315 320

Gin Gly Lys Pro Ala He Ala His Arg Asp Phe Lys Ser Arg Asn Val 325 330 335

Leu Val Lys Ser Asn Leu Gin Cys Cys lie Ala Asp Leu Gly Leu Ala 340 345 350

Val Met His Ser Gin Gly Ser Asp Tyr Leu Asp lie Gly Asn Asn Pro 355 360 365

Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Gin 370 375 380

He Arg Thr Asp Cys Phe Glu Ser Tyr Lys Trp Thr Asp He Trp Ala 385 390 395 400

Phe Gly Leu Val Leu Trp Glu lie Ala Arg Arg Thr He Val Asn Gly 405 410 415

He Val Glu Asp Tyr Arg Pro Pro Phe Tyr Asp Val Val Pro Asn Asp 420 425 430 180 189,642/1

Pro Ser Phe Glu Asp Met Lys Lys Val Val Cys Val Asp Gin Gin Thr 435 440 445

Pro Thr He Pro Asn Arg Leu Ala Ala Asp Pro Val Leu Ser Gly Leu 450 455 460

Ala Gin Met Met Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala Arg Leu 465 470 475 480

Thr Ala Leu Arg He Lys Lys Thr Leu Gin Lys He Ser Asn Ser Pro 485 490 495

Glu Lys Pro Lys Val He Gin 500 <210> 131 <211> 120

<212> PRT <213> Human <400> 131

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Gly Tyr Tyr Trp Ser Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80 181 189,642/1

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Gly Ile Ala Val Ala Gly Tyr Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 132 <211> 108

<212> PRT <213> Human <400> 132

Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 133 <211> 96 182 189,642/1

<212> PRT <213> Human <400> 133

Glu He Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser 50 55 60

Giy Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro 85 90 95 <210> 134 <211> 99

<212> PRT <213> Human <400> 134

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Gly 20 25 30

Gly Tyr Tyr Trp Ser Trp He Arg Gin His Pro Gly Lys Gly Leu Glu 35 40 45

Trp lie Gly Tyr lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 183 189,642/1

Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80 50 55 60

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg <210> 135 <211> 108

<212> PRT <213> Human <400> 135

Glu He Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ser 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45 lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly He Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin His Phe Gly Ser Ser Pro 85 90 95 lie Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys 100 105 184 189,642/1 <210> 136 <211> 5 <212> PRT <213> Human <400> 136

Gly Tyr Tyr Trp Ser 1 5 , crnxan rwzn ατα inia^n pnvn irn πτ “|»oa ,ρηη ηχΰπ laoana ru^na na^maa np’-ioz .zruwan tto mpnan 7mi7 oxnm □innn 7v

<img img-format="tif" img-content="drawing" file="IL189642AD000215.tif" id="idf0015" />

.(moia nannn) cras&amp;amp;'an -nwa