Human monoclonal antibodies to activin receptor-like kinase-1 (ALK-1)

[0001] The present disclosure relates to human monoclonal antibodies and antigen binding portions thereof that bind to the extracellular domain (ECD) of activin-1 receptor-like kinase (ALK-1). The disclosure also relates to nucleic acid molecules encoding such antibodies and antigen binding portions, methods for making human anti-ALK-1 antibodies and antigen binding portions, compositions comprising these antibodies and antigen binding portions, and methods of using these antibodies, antigen binding portions and compositions.

Background of the Invention [0002] ALK-1 is a type I surface receptor for transforming growth type beta 1 (TGF-beta-1). Human ALK-1 is a 503 amino acid polypeptide that contains a signal sequence (amino acids: 1-21), the N-terminal extracellular ligand binding domain of TGF-beta-1 or ECD (amino acids: 22-118), the single transmembrane domain (amino acids: 119 -141), the glycine / serine (GS) rich regulatory domain (amino acids 142-202) and the C-terminal serine-threonine kinase domain (202-492). The human ALK-1 amino acid sequence disclosed in Attisano et al. Cell, 1993, volume 75, p. 671-680 contains Ser at position 172 (Genbank register L17075), while US 6316217 claims the amino acid sequence of human ALK-1 with Thr at position 172 (Genbank register NM_000020). The ACVRL1 gene encoding full-length human ALK-1 disclosed in Attisano et al. is commercially available from Invitrogen Inc., Clone ID IOH21048. Although ALK-1 has 60-80% general homology with other type I receptors (ALK-2 to ALK-7), ECD ALK-1 is significantly different from ECD of other ALK family kinases. For example, in humans, only ECD ALK-2 is significantly associated with ECD ALK-1 (exhibits approximately 25% amino acid identity). U.S. Patent No. 6,316,217; this Dijke et al. Oncogene, 1993, volume 8, pp. 28792887; Attisano et al. Cell, 1993, volume 75, pp. 671-680.

[0003] Typically, ligands from the TGF-beta superfamily show their biological activity by binding to heteromeric receptor complexes consisting of two types (I and II) of serine / threonine kinases. Type II receptors are constitutively active kinases that phosphorylate the type I receptor upon ligand binding. In turn, activated type I kinases phosphorylate downstream molecules that signal to the cell nucleus, including various Smad proteins that translocate to the nucleus and lead to a transcriptional response. Heldin et al. Nature, 1997, volume 390, pp. 465-471. In the case of ALK-1, we have shown that Smad1 undergoes specific phosphorylation and translocation to the nucleus, where it directly regulates the expression of Smad1 Id1 and EphB2 responsive genes. [0004] ALK-1 is highly and selectively expressed in endothelial cells and in other highly vascularized tissues such as the placenta or brain. We have demonstrated by real-time Affymetrix and RT-PCR profiling methods that expression of ALK-1 in endothelial cells strongly exceeds the expression of its type II activin and endoglin co-receptors, its ligand TGF-beta-1 or ALK-5. Mutations in ALK-1 are associated with congenital hemorrhagic hemangioma (HHT), which indicates the key role of ALK-1 in controlling the formation or repair of blood vessels. Abdalla et al. J. Med. Genet, 2003, volume 40, pp. 494-502; Sadick et al. Hematologica / The Hematology J., 2005, volume 90, 818828. In addition, two independent studies of ALK-1 knockout mice provide key in vivo evidence for ALK-1 function during angiogenesis. Oh et al. Proc Natl Acad Sci USA, 2000, volume 97, p. 2626-2631; Urness et al. Nature Genetics, 2000, volume 26, p. 328331.

[0005] Angiogenesis is a physiological process involving the formation of new blood vessels from pre-existing vessels and / or circulating endothelial stem cells. This is a normal process during growth and development as well as during wound healing. However, this is also the basic stage of cancer transition from dormancy to malignancy. Hanahan and Folkman, "Patterns and Emerging 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 such as cancer, the body loses its ability to maintain balanced angiogenesis. New blood vessels nourish affected tissues, destroy healthy tissues, and in some cancers, new vessels can allow cancer cells to leak into the circulation and colonize other organs (cancer metastases). Angiogenesis inhibitors, including monoclonal antibodies (mAbs), are a very promising class of drugs directed against this abnormal process to block or slow tumor growth.

[0006] In addition to the role in solid tumor growth and metastasis, other noteworthy conditions with the angiogenic component are for example arthritis, psoriasis, age-related neovascular macular degeneration, and diabetic retinopathy. Bonnet et al. "Osteoarthritis, Angiogenesis and Inflammation," Rheumatology, 2005, volume 44, pp. 7-16; Creamer et al. "Angiogenesis in psoriasis," Angiogenesis, 2002, volume 5, pp. 231-236; Clavel et al. "Recent data on the role for angiogenesis in rheumatoid arthritis," Joint Bone Spine, 2003, volume 70, pp. 321-326; Anandarajah et al. "Pathogenesis of psoriatic arthritis," Curr. Opin. Rheumatol., 2004, volume 16, pp. 338-343; Ng et al. "Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration," Can. J. Ophthalmol., 2005, volume 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 al. "Angiogenesis and ophthalmic disease," Angiogenesis, 1999, volume 3, pp. 9-14.

[0007] Antiangiogenic therapies are expected to be chronic. Accordingly, targets with highly selective endothelial activity, such as ALK-1, are preferred to reduce cachexia resulting from side effects. In addition, given the significant discrepancy between ECD ALK-1 and ECD of other ALK family members, it is expected that mAbs generated against human ALK-1 ECD will be selectively targeted for ALK-1. Based on these considerations, a monoclonal antibody against the extracellular domain of ALK-1 is highly desirable, which can prevent dimerization with the type II receptor and thereby block Smad1 phosphorylation and subsequent transcriptional response in the cell nucleus.

[0008] R&D Systems, Inc. produces and sells anti-human ALK-1 monoclonal antibody (cat. no. MAB370) made from hybridoma obtained by fusing mouse myeloma with B lymphocytes obtained from mice immunized with the purified extracellular domain of recombinant human ALK-1 derived from NS0. We have shown that this antibody does not neutralize the interaction between ALK-1 and TGF-beta-1, nor tolerates Smad1 phosphorylation. Rabbit antisera were generated against a synthetic peptide corresponding to a portion of the ALK-1 intracellular juxtaposition region (amino acid residues 145-166), conjugated with keyhole limpet hemocyanin (KLH) (US Patent No. 6692925) and against the entire extracellular ALK-1 domain outside the leader sequence (Lux et al., J. Biol. Chem., 1999, volume 274, p. 99849992). Abdalla et al. (Human Mol. Gen., 2000, volume 9, pp. 1227-1237) report the production of a polyclonal anti-ALK-1 antibody using a recombinant vaccinia virus construct. R&D Systems, Inc. produces and sells polyclonal anti-human ALK-1 antibody (cat. no. AF370) made in goats immunized with the purified extracellular domain of recombinant human ALK-1 derived from NS0.

[0009] There have been no reports to date of fully human monoclonal anti-ECD ALK-1 antibodies and no one has demonstrated the efficacy of any anti-ECD ALK-1 monoclonal antibody in clearing the ALK-1 / TGFbeta-1 / Smad1 signaling pathway.

[0010] Femandez-L et al., "Blood outgrowth endothelial cells from Hereditary Haemorrhagic Telangiectasia patients reveal abnormalities compatible with vascular lesions", Cardiovascular Research, vol. 68, No. 2 pages 235-248 (July 5, 2005) concerns the isolation and characterization of circulating cells endothelial cells from HHT patients, and refers to MAB370 anti-ALK-1 in the "Materials and Methods" section under the headings "Western Blot Analysis" and "Immunofluorescence Microscopy".

Summary of the invention [0011] The invention is defined in the claims. The aspects / instances of the present disclosure that make up the invention are as defined in the claims and in the following forms:

E1. The neutralizing anti-ALK-1 monoclonal antibody or antigen-binding portion thereof, which antibody or antigen-binding portion competes cross-linking for binding to ALK-1 with an antibody comprising: the heavy chain variable domain of SEQ ID NO: 6 and the light chain variable domain of SEQ ID NO : 8; or the amino acid sequence of the heavy chain variable domain encoded by the nucleotide sequence of the insert present in the clone deposited at ATCC under accession number PTA-6864 and the amino acid sequence of the light chain variable domain encoded by the nucleotide sequence of the insert present in the clone deposited at ATCC under accession number PTA-6865.

E2. The antibody or antigen binding portion according to E1, which antibody comprises: a heavy chain that utilizes a human germline VH 4-31 sequence; and a light chain that uses the germline Vk A27 sequence.

E3. The E2 antibody or antigen-binding portion, wherein the amino acid sequence of this heavy chain is at least 90% identical to the amino acid sequence encoded by the germline VH 4-31 sequence, and wherein the amino acid sequence of this light chain is at least identical 90% with the amino acid sequence encoded by the human germline Vk A27 sequence.

E4. The antibody or antigen binding portion according to any of E1-E3, which antibody comprises a VH domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6.

E5. The antibody or antigen binding portion according to any of E1-E3, which antibody comprises a VL domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8.

E6. The antibody or antigen binding portion according to any of E1-E5, which antibody comprises a VH domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6, and a VL domain that is at least 90% identical to the sequence amino acid SEQ ID NO: 8.

E7. The antibody or antigen binding portion according to any of E1-E5, which antibody comprises a VH domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6, and a VL domain that is at least 95% identical to the sequence amino acid SEQ ID NO: 8.

E8. The antibody or antigen binding portion according to any of E1-E5, which antibody comprises a VH domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 6, and a VL domain that is at least 99% identical to the sequence amino acid SEQ ID NO: 8.

E9. An antibody according to any of E1-E8, wherein said antibody is or is an IgG molecule.

E10. The antibody of E9, wherein said antibody is an IgG1 or IgG2 molecule.

E11. The antibody or antigen binding portion according to any one of E1-E10, wherein said antibody or antigen binding portion is derivatized or linked to another molecule.

E12. The E11 antibody or antigen binding portion wherein said molecule is another antibody, detection agent, label, cytotoxic agent, pharmaceutical agent, peptide or protein.

E13. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12; or an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any one of E1-E12.

E14. A vector comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a vector comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12, each of which vectors contains a control expression sequence linked to the nucleic acid molecule; or a vector comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12, which vector contains an expression control sequence operably linked to the nucleic acid molecule.

E15. A host cell comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12; a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any one of E1-E12; a vector comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a vector comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12, each of which vectors contains a control expression sequence linked to the nucleic acid molecule; or a vector comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12, which vector contains an expression control sequence operably linked to the nucleic acid molecule.

E16. A pharmaceutical composition comprising the antibody or antigen binding portion according to any one of E1-E12 and a physiologically acceptable carrier.

E17. Use of the antibody or antigen binding portion according to any one of E1-E12 or the pharmaceutical composition according to E16 for the manufacture of a medicament.

E18. The antibody or antigen binding portion according to any one of E1-E12 or the pharmaceutical composition according to E16, for use in therapy.

E19. Use of the antibody or antigen-binding portion according to any one of E1-E12 or the pharmaceutical composition according to E16 for the manufacture of a medicament for the treatment of cancer, age-related macular degeneration, diabetes-related blindness, endometriosis, ocular neovascularization, psoriasis or rheumatoid arthritis in a mammal.

E20. The antibody or antigen binding portion according to any one of E1-E12 or the pharmaceutical composition according to E16 for use in the treatment of cancer, age-related macular degeneration, diabetes-related blindness, endometriosis, ocular neovascularization, psoriasis or rheumatoid arthritis in a mammal.

E21. Use: a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12; or a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12; for the manufacture of a medicine.

E22. A nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any one of E1-E12, or a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes the light chain of an antibody or antigen binding portion according to any one of E1-E12; for use in therapy.

E23. Use: a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12; or a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12; for the manufacture of a medicament for treating cancer, age-related macular degeneration, diabetes-related blindness, endometriosis, ocular neovascularization, psoriasis or rheumatoid arthritis in a mammal.

E24. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a light chain, antibody or antigen binding portion according to any of E1-E12, or an isolated nucleic acid molecule comprising a nucleotide sequence that encodes heavy chain and nucleotide sequence, which codes for the light chain of the antibody or antigen-binding portion according to any of E1-E12; for use in the treatment of cancer, age-related macular degeneration, diabetes-related blindness, endometriosis, ocular neovascularization, psoriasis or rheumatoid arthritis in a mammal.

E25. The antibody, antigen binding portion or pharmaceutical composition according to E20 for use in the treatment of cancer, or use according to E 19 for the manufacture of a medicament for the treatment of cancer, wherein the cancer is cutaneous or intraocular melanoma, mesothelioma, renal cell carcinoma, breast cancer, head and neck cancer. primary or secondary brain tumor, cervical cancer, urethral cancer, prostate cancer, pancreatic cancer, testicular cancer, hepatobiliary cancer, ductal liver cancer, bile duct cancer, colorectal cancer, bladder cancer, ovarian cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, colon cancer, rectal cancer, or anal cancer.

E26. The use of a monoclonal antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 4 in the manufacture of a medicament for the treatment of hepatobiliary cancer in a mammal.

E27. A monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 2 heavy chain and the amino acid sequence of SEQ ID NO: 4 for use in the treatment of hepatobiliary cancer in a mammal.

E28. An antibody, antigen binding portion or pharmaceutical composition for use according to any of E20, E25 and E27, an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain, and an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a light chain of an antibody or antigen binding portion according to any of E1-E12, or an isolated nucleic acid molecule comprising a nucleotide sequence, which encodes a heavy chain, and a nucleotide sequence that encodes a light chain of an antibody or antigen-binding portion according to any one of E1-E12, for use according to E24, or use according to any of E19, E23, E25 and E26; wherein the mammal is a human.

[0012] The disclosure relates to isolated neutralizing anti-ALK-1 monoclonal antibodies or antigen-binding portions thereof that bind to primate ALK-1, preferably to primate ALK-1 ECD, more preferably to human ALK-1 ECD. In a preferred case, the neutralizing antibodies are fully human monoclonal antibodies or antigen binding portions thereof.

[0013] In a further aspect, the present disclosure is an anti-ALK-1 antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof abrogates the ALK-1 / TGF-beta-1 / Smad1 signaling pathway. In a preferred case, the antibodies are fully human monoclonal antibodies or antigen binding portions thereof.

[0014] In a further aspect, the present disclosure is an anti-ALK-1 antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof is an antagonist of TGF-beta-1 angiogenesis. In a preferred case, the antibodies are fully human monoclonal antibodies or antigen binding portions thereof.

[0015] In another aspect, the present disclosure is a fully human anti-ALK-1 antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof is an antagonist of TGF-beta-1 stimulated tumor angiogenesis.

[0016] In another aspect, the present disclosure is a well-tolerated, injectable, fully human anti-ALK-1 antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof is an antagonist of TGF-beta-1 stimulated angiogenesis.

[0017] In another aspect, the present disclosure is an anti-ALK-1 antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof inhibits regulation leading to stimulation of a specific downstream ALK-1, Id1 target gene. In a preferred case, the antibodies are fully human monoclonal antibodies or antigen binding portions thereof.

[0018] In a further aspect, the present disclosure is a monoclonal anti-ALK-1 antibody or antigen binding portion thereof, which antibody or antigen binding portion thereof is described in terms of at least one of several functional properties described below.

[0019] For example, in one instance, the antibody or antigen-binding portion thereof binds to the extracellular domain of the ALK-1 primate at an avidity value of 1 μΜ or less as measured by surface plasma resonance. In another case, the antibody or portion binds to the extracellular domain of ALK-1 primates with an avidity value of less than 100 nM, less than 5 nM, less than 1 nM, less than 500 pM, less than 100 pM, less than 50 pM, less than 20 pM, less than 10 pM or less than 1 pM, measured by surface plasmon resonance. In some cases, the avidity value is from 0.1 pM to 1 μΜ. In other cases, the avidity value is from 1 pM to 100 nM. In other cases, the avidity value is from 1 pM to 5 nM. In other cases, the avidity value is from 1 pM to 500 pM. In other cases, the avidity value is from 1 pM to 100 pM. In other cases, the avidity is from 1 pM to 10 pM.

[0020] In another case, the antibody or antigen binding portion thereof binds to the extracellular domain of human ALK-1 at an avidity value of 100 nM or less as measured by surface plasmon resonance. In another case, the antibody or portion binds to the extracellular domain of human ALK1 at an avidity value of less than 10 nM, less than 5 nM, less than 1 nM, less than 500 pM, less than 100 pM, less than 50 pM, less than 20 pM , lower than 10 pM or lower than 1 pM, measured by surface plasmon resonance. In some cases, the avidity value is from 1 pM to 100 nM. In other cases, the avidity value is from 1 pM to 5 nM. In other cases, the avidity value is from 1 pM to 500 pM. In other cases, the avidity value is from 1 pM to 100 pM. In other cases, the avidity is from 1 pM to 10 pM.

[0021] Otherwise, the antibody or portion thereof has a dissociation rate constant (koff) for human ALK-1 equal to 5 x 10^-3 s^-1 or lower, measured by surface plasmon resonance. For example, in some cases, the antibody or portion thereof has a human ALK-1 koff lower than 10^-3 s^-1, less than 5 x 10^-4 s^-1, less than 10^-4 s^-1, less than 5 x 10^-5 s^-1, less than 10^-5 s^-1, or lower than 5 x 10^-6 s^-1. In other cases, koff is from 10^-6 s^-1 up to 10^-4 s^-1. In other cases, koff is from 10^-6s^-1 up to 5 x 10^-5 s^-1.

[0022] In another case, the antibody or portion thereof binds to ALK-1 primates with a KD of 1000 nM or less. In a further case, the antibody or portion binds to human ALK-1 with a KD lower than 500 nM, lower than 100 nM, lower than 50 nM, lower than 20 nM, lower than 10 nM or lower than 1 nM, measured by surface plasmon resonance . In some cases, Kd is from 1 μΜ to 100 nM. In other cases, the KD is from 100 nM to 10 nM. In other cases, the KD ranges from 50 nM to 0.1 nM. Such KD values ​​can be measured by any technique known to those skilled in the art, such as ELISA, RIA, flow cytometry or surface plasmon resonance, such as BIACORE^™.

In another case, the antibody or portion thereof has a higher binding affinity for primate ALK-1 (KD (P)) than for rodent ALK-1 (KD (R)). In one case, the antibodies or antigen binding portions thereof of the present disclosure have a KD (R) / KD (P) ratio that is greater than or equal to 1.5. In a further case, the antibodies or antigen binding portions thereof of the present disclosure have a KD (R) / KD (P) that is greater than or equal to 2, greater than or equal to 3, greater than or equal to 5, greater than or equal to 10, greater than than or equal to 20, greater than or equal to 50, greater than or equal to 100, greater than or equal to 200, greater than or equal to 500 or greater than or equal to 1000. Such KD values ​​for both primate ALK-1 and rodent ALK-1 can be measured by any technique known to those skilled in the art, such as flow cytometry, ELISA, RIA, or surface plasmon resonance, such as BIACORE^™.

[0024] In a further case, the anti-ALK-1 antibody or portion thereof has an IC50 of 500 nM or lower, measured on the basis of their ability to inhibit regulation leading to stimulation of a specific downstream ALK-1, Id1 target gene. In another case, the IC50 is lower than 300 nM, lower than 200 nM, lower than 150 nM lower than 100 nM, lower than 50 nM, lower than 20 nM, lower than 10 nM or lower than 1 nM.

In some cases, the IC50 ranges from 1 nM to 500 nM. In other cases, the IC50 ranges from 5 nM to 250 nM. In other cases, the IC50 is between 10 nM and 100 nM. [0025] In another case, the anti-ALK-1 antibody or portion thereof has an IC 50 equal

250 nM or lower, measured on the basis of their ability to inhibit Smad1 phosphorylation determined by Western Blot using an Odyssey infrared imaging system. In another case, the IC50 is lower than 200 nM, lower than 150 nM, lower than 100 nM, lower than 50 nM, lower than 20 nM, lower than 10 nM or lower than 1 nM. In some cases, the IC50 ranges from 1 nM to 250 nM. In other cases, the IC50 is between 5 nM and 200 nM. In other cases, the IC50 is between 10 nM and 100 nM. [0026] In another case, the anti-ALK-1 antibody or portion thereof inhibits human vascular angiogenesis in SCID mice transplanted with human foreskin tissue into which M24met human melanoma tumor cells are intradermally implanted as determined by IHC analysis using human CD signaling assay -31, by at least 40% compared to the control sample. In a further case, the anti-ALK-1 antibody or portion thereof inhibits human vascular angiogenesis in SCID mice transplanted with human foreskin tissue in which human M24met melanoma tumor cells are intradermally implanted with at least 30%, at least 40%, at least 50 % or at least 60% compared to the control sample.

[0027] In a further case, the anti-ALK-1 antibody or portion thereof has an EC50 of 500 nM or lower, measured on the basis of their ability to inhibit human vascular angiogenesis in SCID mice transplanted with tissue from a human foreskin in which tumor cells are intradermally implanted human melanoma M24met. In another case, the EC50 is lower than 400 nM, lower than 300 nM, lower than 200 nM, lower than 150 nM, lower than 100 nM, lower than 50 nM, lower than 25 nM or lower than 5 nM. In some cases, the EC50 is from 5 nM to 500 nM. In other cases, the IC50 ranges from 25 nM to 300 nM. In other cases, the IC50 ranges from 50 nM to 150 nM.

[0028] In a further case, the anti-ALK-1 antibody or portion thereof inhibits human vascular angiogenesis in SCID mice transplanted with human foreskin tissue into which collagen mix with human vascular endothelial cells is implanted intradermally as determined by IHC analysis using human CD31 signaling assay, by at least 25% compared to the control sample. In a further case, the anti-ALK-1 antibody or portion thereof inhibits human vascular angiogenesis in SCID mice transplanted with human foreskin tissue in which collagen is implanted intradermally by at least 50% compared to the control sample. In a further case, the anti-ALK-1 antibody or portion thereof inhibits by at least 75%, by at least 80%, by at least 85%, by at least 90%, or by at least 95% compared to the control.

[0029] In another case, the anti-ALK-1 antibody or portion thereof competes for binding to ALK-1 with an antibody selected from the group consisting of 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 ​​and 5.59.1.

[0030] In a further case, the anti-ALK-1 antibody or portion thereof competes for binding to ALK-1 with an antibody selected from the group consisting of 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 ​​and 5.59.1.

[0031] In a further case, the anti-ALK-1 antibody or portion thereof binds to the same ALK-1 epitope as the antibody selected from the group consisting of 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 ​​and 5.59.1.

[0032] In a further case, the anti-ALK-1 antibody or portion thereof binds to ALK-1 at substantially the same KD as the antibody selected from the group consisting of 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 ​​and 5.59.1.

[0033] In a further case, the anti-ALK-1 antibody or portion thereof binds to ALK-1 with substantially the same koff as the antibody selected from the group consisting of 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 ​​and 5.59.1.

[0034] A further aspect of the present disclosure is an antibody or antigen binding portion thereof, having at least one of the functional properties previously described which comprises a VH domain which is at least 90% identical in amino acid sequence to any of SEQ ID NO: 6 ; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104. In one case, this VH domain is identical in amino acid sequence at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% with any of the SEQ IDs NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104.

[0035] In a further case, the antibody or portion thereof has at least one of the functional properties previously described and comprises a VH domain which is any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or different from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 63; 66; 70; 74; 78; 82; 86; 90; or 104 having at least one conservative amino acid substitution. For example, the VH domain may differ 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104. In a further case, any of these conservative amino acid substitutions may occur in the CDR1, CDR2 and / or CDR3 regions.

[0036] Another aspect of the present disclosure is an antibody or antigen binding portion thereof, having at least one of the functional properties previously described which comprises a VL domain that is at least 90% identical in amino acid sequence to any of SEQ ID NO: 8 ; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127. In one case, this VL domain is identical in amino acid sequence at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% with any of the SEQ IDs NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127.

[0037] In a further case, the antibody or portion thereof has at least one of the functional properties previously described and comprises a VL domain which is any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or is different from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127 having at least one conservative amino acid substitution. For example, the VL domain may differ from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127. In a further case, any of these conservative amino acid substitutions may occur in the CDR1, CDR2 and / or CDR3 regions.

[0038] Another aspect of the present disclosure is an antibody or antigen binding portion thereof, having at least one of the functional properties previously described, wherein each of the VL and VH domains is identical in amino acid sequence at least 90% with the VL and VH domains, respectively any of the monoclonal 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; 5.57.1; and 5.59.1. For example, each of the VL and VH domains is identical in amino acid sequence at least 91%, 93%, 95%, 97%, 99% or 100%, respectively, with the VL and VH domains of any of 1.11.1 monoclonal antibodies, respectively; 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; and 5.59.1.

[0039] Another aspect of the present disclosure is the monoclonal antibody or antigen-binding portion thereof, which are selected from the group consisting of: a) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 6, and the VL domain shown in SEQ ID NO : 8; b) an antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 10, and the VL domain shown in SEQ ID NO: 12; c) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 14 and the VL domain shown in SEQ ID NO: 16; d) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 18, and the VL domain shown in SEQ ID NO: 20; e) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 22 and the VL domain shown in SEQ ID NO: 24; f) an antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 26 and the VL domain shown in SEQ ID NO: 28; g) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 30 and the VL domain shown in SEQ ID NO: 32; h) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 34 and the VL domain shown in SEQ ID NO: 36; i) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 38 and the VL domain shown in SEQ ID NO: 40; j) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 42 and the VL domain shown in SEQ ID NO: 44; k) an antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 46 and the VL domain shown in SEQ ID NO: 48; I) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 50 and the VL domain shown in SEQ ID NO: 52; m) an antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 54 and the VL domain shown in SEQ ID NO: 56; n) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 58 and the VL domain shown in SEQ ID NO: 60; o) an antibody or portion thereof that contains the VH domain shown in SEQ ID NO; 62 and the VL domain shown in SEQ ID NO: 64; p) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 66 and the VL domain shown in SEQ ID NO: 68; q) the antibody or antigen binding portion thereof that comprises the VH domain shown in SEQ ID NO: 70 and the VL domain shown in SEQ ID NO: 72; r) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 74 and the VL domain shown in SEQ ID NO: 76; s) an antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 78 and the VL domain shown in SEQ ID NO: 80; t) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 82 and the VL domain shown in SEQ ID NO: 84; u) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 86 and the VL domain shown in SEQ ID NO: 88; v) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 90 and the VL domain shown in SEQ ID NO: 92; w) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 104 and the VL domain shown in SEQ ID NO: 127; x) the antibody or portion thereof that comprises the VH domain shown in SEQ ID NO: 6 and the VL domain shown in SEQ ID NO: 127; and y) the antibody or portion thereof that contains the VH domain shown in SEQ ID NO: 104 and the VL domain shown in SEQ ID NO: 8. [0040] In a further case, for each of the above described antibodies or parts thereof in groups a) to v) the VH and / or VL domains may differ from the specific SEQ ID NOs enumerated therein with at least one conservative amino acid substitution. For example, the VH and / or VL domains may differ from the calculated SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions. In a further case, each of these conservative amino acid substitutions may occur in the CDR1, CDR2 and / or CDR3 regions.

[0041] In a further case, the present disclosure provides a monoclonal antibody or antigen binding portion thereof, having at least one of the functional properties previously described, wherein the VH domain is selected independently from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence that differs from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, at least one conservative amino acid substitution, and the VL domain is independently selected from any of SEQ ID NOs. 8; 12; 16; twenty; 24; 28; 32; 36, 40; 44; 48, 52; 56; 60: 64; 68; 72; 76; 80; 84; 88; 92; or 127, or a sequence that differs from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, at least one conservative amino acid substitution. For example, each of the VH and VL domains may differ from SEQ ID NO: 6, respectively; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, and 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.

[0042] In a further case, the present disclosure provides a monoclonal antibody or antigen-binding portion thereof, with at least one of the functional properties previously described, wherein the antibody or portion comprises VR CDR1, CDR2 and CDR3 sequences independently selected from CDR1, CDR2 and Heavy chain CDR3 found in any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence that differs from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, at least one conservative amino acid substitution. For example, CDR1, CDR2 and CDR3 VH may differ from CDR1, CDR2 and CDR3 respectively from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.

[0043] In a further case, the present disclosure provides a monoclonal antibody or antigen-binding portion thereof, with at least one of the functional properties previously described, wherein the antibody or portion comprises VR CDR1, CDR2 and CDR3 sequences independently selected from CDR1, CDR2 and Light chain CDR3 residing in any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or a sequence that differs from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, at least one conservative amino acid substitution. For example, CDR1, CDR2 and CDR3 VL may differ from CDR1, CDR2 and CDR3 respectively from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.

[0044] The present disclosure further provides a monoclonal antibody or antigen binding portion thereof, having at least one of the functional properties previously described, wherein the antibody or antigen binding portion thereof comprises CDR1 VH and VL, CDR2

VH and VL and CDR3 VH and VL, found in any of 1.11.1 monoclonal antibodies; 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; 4.38.1; 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.

[0045] The present disclosure further provides a monoclonal antibody or antigen binding portion thereof, with at least one of the functional properties previously described, wherein the antibody or antigen binding portion thereof comprises a heavy chain that utilizes the human VH 4-31, VH 3- gene 11, VH 3-15, VH 3-33, VH 4-61 or VH 4-59. In some cases, the heavy chain uses the human VH 3-33 gene, the human D 6-19 gene and the human JH 3B gene; human VH 4-31 gene, human D 6-19 gene and human JH 4B gene; human VH 4-61 gene, human D 6-19 gene and human JH 4B gene; human VH 4-31 gene, human D 3-3 gene and human JH 3B gene; human VH 4-31 gene and human JH 3B gene; human VH 4-59 gene, human D 6-19 gene and human JH 4B gene; human VH 3-11 gene, human D 3-22 gene and human JH 6B gene; human VH 3-15 gene, human D 3-22 gene and human JH 4B gene; human VH 4-31 gene, human D 5-12 gene and human JH 6B gene; human VH 4-31 gene, human D 4-23 gene and human JH 4B gene; human VH 4-31 gene, human D 2-2 gene and human JH 5B gene; human VH 4-31 gene and human JH 6B gene; human VH 3-15 gene, human D 1-1 gene and human JH 4B gene; human VH 3-11 gene, human D 619 gene and human JH 6B gene; human VH 3-11 gene, human D 3-10 gene and human JH 6B gene; or the human VH 3-11 gene, the human D 6-6 gene and the human JH 6B gene.

[0046] The present disclosure further provides a monoclonal antibody or antigen binding portion thereof, with at least one of the functional properties previously described, wherein the antibody or antigen binding portion thereof comprises a light chain that utilizes the human Vk A27, Vk A2, Vk A1 gene , Vk A3, Vk B3, Vk B2, Vk L1 or Vk L2. In some cases, the light chain uses the human VK L1 gene and the human JK 4 gene; human VK A27 gene and human JK 5 gene or human JK 4 gene; human VK B3 gene and human JK 1 gene; human VK L2 gene and human JK 3 gene; human VK A2 gene and human JK 1 gene; human VK A3 gene and human JK 4 gene; human VK A1 gene and human JK 1 gene; human VK B2 gene and human JK 4 gene; or the human VK A2 gene and the human JK 1 gene.

[0047] The present disclosure further provides a monoclonal antibody or antigen binding portion thereof, having at least one of the functional properties previously described, wherein said antibody or antigen binding portion comprises one or more heavy chain FR1, FR2, FR3 or FR4 amino acid sequences and / or a light chain that are present in any of 1.11.1 monoclonal antibodies; 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 ​​and 5.59.1.

[0048] The present disclosure further provides a monoclonal antibody comprising the amino acid sequences set forth in: a) SEQ ID NO: 2 and SEQ ID NO: 4; b) SEQ ID NO: 2 and SEQ ID NO: 102; c) SEQ ID NO: 100 and SEQ ID NO: 4; and d) SEQ ID NO: 100 and SEQ ID NO: 102.

[0049] Another instance of the present disclosure is any of the previously described antibodies which is or is derived from an IgG, IgM, IgE, IgA or IgD molecule. For example, the antibody may be IgG1 or IgG2.

[0050] Another case provides any of the above-described antibodies or antigen-binding portions, which is a Fab fragment, an F (ab ') 2 fragment, an FV fragment, a single-chain Fv fragment, a single-chain VH fragment, a single-chain VL fragment, a humanized antibody, or a chimeric antibody or dual specificity antibody.

[0051] Another case is a derivatized antibody or antigen binding portion comprising any of the previously described antibodies or portions thereof and at least one additional molecule. For example, at least one additional molecule may be another antibody (e.g. dual specificity antibody or diabody), detection agent, label, cytotoxic agent, pharmaceutical agent and / or protein or peptide that can mediate the binding of the antibody or antibody portion to another molecule (such as streptavidin core region or polyhistidine tag). For example, useful detection agents with which the antibody or antigen-binding portion of the disclosure can be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. The antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. In a further case, the antibodies or portions thereof of the present disclosure may also be labeled with biotin or a predetermined polypeptide epitope recognized by a secondary reporter (e.g. leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In yet a further instance of the present disclosure, each of the antibodies or parts thereof may be derivatized with a chemical group such as polyethylene glycol (PEG), methyl or ethyl, or a carbohydrate group.

[0052] In some cases, the anti-ALK-1 antibodies disclosed herein, or antigen-binding portions thereof, are bound to a solid support.

[0053] In some cases, the C-terminal heavy chain lysine of any of the anti-ALK-1 antibodies of the disclosure is cleaved. In various instances of this disclosure, the heavy and light chains of anti-ALK-1 antibodies may optionally include a signal sequence.

[0054] The present disclosure also provides a pharmaceutical composition comprising any of the above described antibodies or antigen binding portions thereof and a pharmaceutically acceptable carrier.

[0055] In a further case, the disclosure relates to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes any of the antibodies or antigen binding portions described herein. In one particular case, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 1, which sequence encodes the heavy chain. In another specific case, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 3, which sequence encodes the light chain.

[0056] In another specific case, the isolated nucleic acid molecule comprises a polynucleotide comprising an open reading frame for the cDNA sequence of the clone deposited at ATCC under accession number PTA-6864. In another specific case, the isolated nucleic acid molecule comprises a polynucleotide comprising an open reading frame for the cDNA sequence of the clone deposited at ATCC under accession number PTA-6865.

[0057] In another specific case, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 95 or 128, each of which encodes a heavy chain. In another specific case, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 101, which sequence encodes a light chain.

[0058] The disclosure further relates to a vector comprising any of the nucleic acid molecules described herein, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.

[0059] Another case provides a host cell comprising any of the vectors described herein or containing any of the nucleic acid molecules described herein. The present disclosure also provides an isolated cell line that produces any of the antibodies or antigen binding portions described herein, or which produces the heavy chain or light chain of any of these antibodies or antigen binding portions.

[0060] In a further case, the present disclosure relates to a method of producing an anti-ALK-1 antibody or antigen-binding portion thereof, comprising culturing any of the host cells or cell lines described herein under appropriate conditions and recovering said antibody or antigen-binding portion.

[0061] The present disclosure also relates to a transgenic non-human living being or a transgenic plant that comprises any of the nucleic acids described herein, wherein the transgenic non-human living being or the transgenic plant expresses said nucleic acid.

[0062] The present disclosure further provides a method for isolating an antibody or antigen-binding portion thereof that binds to ALK-1, comprising the step of isolating the antibody from a transgenic non-human animal or from a transgenic plant as described herein. [0063] In a further case, the disclosure relates to a hybridoma deposited with ATCC under accession number PTA-6808.

[0064] The present disclosure also provides a method of determining whether a substance inhibits regulation leading to stimulation of a specific downstream ALK-1 target gene, Id1, which method involves contacting a first sample of cells that express Id1 with that substance and determining whether expression of Id1 has been inhibited, wherein the reduced level of Id1 expression in the first sample of cells contacted with this substance compared to the cell control sample is an indicator of the inhibition of this substance by Id1 expression. The present disclosure further provides a method in which the substance is an antibody that binds to the extracellular domain of ALK-1.

[0065] The present disclosure also provides a method of treating abnormal cell growth in a mammal in need thereof, comprising the step of administering to that mammal any of the antibodies or antigen-binding portions thereof, or any of the pharmaceutical compositions as described herein. The present disclosure further provides a method of treating abnormal cell growth in a mammal in need thereof, using an antibody or antigen-binding portion thereof that binds to ALK-1, comprising the steps of administering to said mammal an effective amount of any of the nucleic acid molecules described herein under appropriate conditions that allow expression of these nucleic acid molecules. In a further case, the method of treating abnormal cell growth further comprises administering an amount of one or more substances selected from anti-cancer agents, anti-angiogenesis agents, signal processing inhibitors and antiproliferative agents, which amounts together are effective in treating this abnormal cell growth. In certain cases, this abnormal cell growth is cancerous.

[0066] The present disclosure also provides the cynomolgus isolated ALK-1 protein with the amino acid sequence of SEQ ID NO: 93. The present disclosure further provides an isolated nucleic acid molecule encoding a protein with the amino acid sequence of SEQ ID NO: 93. The present disclosure further provides an isolated nucleic acid molecule about

SEQ ID NO: 94.

Brief description of the drawings [0067]

Figure 1 shows an example of epitope binding data. Antibody 1.12.1 (M29I / D19A) was injected for 10 minutes, and then for a further 10 minutes, antibody 1.12.1 (M29I / D19A) was injected. The maximum response to a 20 minute injection of this antibody was thus determined. This maximum response to a 20-minute injection was determined in a similar manner for the antibody

1.27.1. Antibody 1.12.1 (M29I / D19A) was injected for 10 minutes, followed by the injection of 1.27.1 antibody for 10 minutes. If the overall response is between the specified maximum responses, then these two antibodies must bind to the same epitope. If the overall response exceeds the highest maximum response, then these antibodies must bind to different epitopes. The experiment was then repeated with the injection order reversed, as described in Example 9.

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

Figure 3 shows the determination of KD for binding of recombinant 1.12.1 antibody to ALK-1 on the cell surface. (a) Human. (b) Tin.

1.12.1 (rWT) refers to the variant mAb 1.12.1 that was the expressed recombinant mAb.

1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine).

1.12.1 (M29I) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which methionine at position 29 in the heavy chain was replaced with isoleucine.

1.12.1 (D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which aspartic acid at position 19 in the light chain was replaced with alanine.

Figure 4 shows examples of ID1 titrations using the Taqman ID1 assay for antibody variants 1.12.1.

1.12.1 refers to the mAb 1.12.1 variant that has been isolated from a hybridoma. 1.12.1 (rWT) refers to the variant mAb 1.12.1 that was the expressed recombinant mAb.

1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine).

1.12.1 (M29I) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which methionine at position 29 in the heavy chain was replaced with isoleucine.

1.12.1 (D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which aspartic acid at position 19 in the light chain was replaced with alanine.

Figure 5 shows examples of ID1 titrations using the Taqman ID1 assay for antibody 1.12.1 sequence variants and a Fab derivative.

1.12.1 refers to the mAb 1.12.1 variant that has been isolated from a hybridoma. 1.12.1 (rWT) refers to the variant mAb 1.12.1 that was the expressed recombinant mAb.

1.12.1 (M29I) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which methionine at position 29 in the heavy chain was replaced with isoleucine.

1.12.1 (D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which aspartic acid at position 19 in the light chain was replaced with alanine.

1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine).

Fab 1.12.1 (M29I / D19A) refers to a Fab mAb 1.12.1 fragment (M29I / D19A) generated by digestion with IgG1 1.12.1 (M29I / D19A) using papain.

Figure 6 shows the internalization of ALK-1. (a) Monitoring of the neutralizing antibody remaining on the cell surface. (b) Monitoring of receptor ALK-1 remaining on the cell surface.

Figure 7A shows sequence alignment of variable domains for anti-ALK-1 antibodies according to the disclosure with germline sequences. Mutations are bold compared to germline. CDR sequences are underlined. Figure 7B shows the alignment of the predicted amino acid sequences of the 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 with the A27 Vk sequence from a human germline. Figures 7C and 7D show the alignment of the predicted amino acid sequences of the heavy and light 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 with a 4-31 VH sequence from the human germ line.

Figure 8 shows an example of histological analysis (H&E staining) of a section of transplanted human skin after surgery.

Figure 9 (A) shows tricolor collagen staining in a chimeric mouse with human skin.

Figure 9 (B) shows detection of human vessels in collagen gel implanted into a chimeric human foreskin mouse. Tex-red: human vessels. FITC: mouse dishes. Yellow: co-staining.

Figure 10 shows an immunofluorescent image of human (red) and mouse (green) vessels in an M24met tumor in a chimeric SCID mouse with human foreskin.

Figure 11 shows IHC image of human vessels (brown) in an M24met tumor in a human Foreskin SCID chimeric mouse.

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

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

Figure 14 shows the concentration of antibody 1.12.1 (M29I / D19A) in the plasma of SCID mice. Figure 15 shows the estimated EC50 for antibody 1.12.1 (M29I / D19A) in a chimeric SCID model with foreskin of M24met. A control value of 100% was given for an artificial plasma concentration of 0.1 nM for imaging purposes. This does not affect the observed EC50.

Detailed description of the invention

Definitions and General Techniques [0068] Unless otherwise defined herein, the scientific and technical terms used in connection with this disclosure will have the meaning that is commonly understood by those of ordinary skill in the art. In addition, unless specified otherwise in a given context, singular terms will include the plural and plural terms will include the singular. In general, the terminology and techniques described herein used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics as well as chemistry and hybridization of proteins and nucleic acids are well known and widely used in the art.

[0069] The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and described in a wide variety of general and more detailed references that are cited and discussed throughout this specification, unless otherwise indicated. See, e.g., Sambrook J. and Russell D.. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Enzyme reactions and purification techniques are carried out according to manufacturers' specifications, as is commonly done in the art, or as described herein. The nomenclature and laboratory procedures and techniques described herein used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry are well known and widely used in the art.

[0070] The following terms, unless otherwise indicated, shall be understood in the following meanings:

The term "ALK-1" as used herein refers to a mammalian activin-1 receptor-like kinase. The term ALK-1 is intended to include recombinant ALK1 and recombinant chimeric forms of ALK-1, which can be made using standard recombinant expression methods.

The abbreviation "mAb" as used herein refers to monoclonal antibody.

The term antibody, which is designated by number, is a monoclonal antibody (mAb) that is obtained from a hybridoma of the same number. For example, monoclonal antibody 1.12.1 is obtained from hybridoma 1.12.1.

1.12.1 refers to the mAb 1.12.1 variant that has been isolated from a hybridoma.

1.12.1 (rWT) refers to the variant mAb 1.12.1 that was the expressed recombinant mAb.

1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine).

1.12.1 (M29I) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which methionine at position 29 in the heavy chain was replaced with isoleucine.

1.12.1 (D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which aspartic acid at position 19 in the light chain was replaced with alanine.

[0071] As used herein, the term "abnormal cell growth", unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition).

[0072] As used herein, "contiguous" refers to nucleotide sequences that are directly linked to each other without any nucleotides between them. For example, the 5'-AAAAA-3 'pentanucleotide is adjacent to the 5'-TTT-3' trinucleotide when they are connected in such a way: 5'-AAAAATTT-3 'or 5'-TTTAAAAA-3', but not when they are connected in this way: 5'-AAAAACTTT-3 '.

[0073] The term "agent" is used herein to mean a chemical compound, a mixture of chemical compounds, a biological macromolecule or an extract made from biological materials.

[0074] The term "alleviating" a disease, disorder or condition as used herein means reducing the severity of the symptoms of the disease, disorder or condition. This includes, but is not limited to, the effect on tumor size, growth and / or mass, extent or progression of metastases, and the like, in a patient compared to the same parameters in a patient before or in the absence of a treatment.

[0075] As used herein, the abbreviation "Id1" refers to a specific downstream ALK-1 target gene, the Id1 gene, which is important for angiogenesis. The Id1 gene has been reported to control the angiogenesis pathway in certain cancers excluding the production of the thrombospondin-1 (TSP-1) protein, a naturally occurring angiogenesis suppressor. For example, it has been reported that the Id1 gene, which is highly expressed in melanoma, breast, head and neck cancer, brain, cervix, prostate, pancreas and testicles, results in reduced TSP1 expression and increased tumor blood vessel formation. Volpert, Olga V. et al., "Id1 regulates angiogenesis through transcriptional repression of thrombospondin-1," Cancer Cell, December 2002, volume 2, pp. 473-483.

[0076] As used herein, the term "Smad" refers to domain Smad proteins present in many species from nematodes to humans. These highly conserved proteins contain an MH1 N-terminal domain that contacts DNA and is separated by a short linker region from the MH2 C-terminal domain that exhibits striking similarity with the forkhead-associated domain (FHA). The FHA and Smad (MH2) domains have a common structure consisting of a two-layer sandwich with eleven beta threads with Greek key topology. Smad proteins mediate signal transduction by TGF-beta / activin / BMP-2/4 cytokines from Ser / Thr receptor protein kinases on the cell surface to the nucleus. Smad proteins are divided into three functional classes: Smad receptors regulated by receptors (R-Smad), including Smad1, -2, -3, -5 and -8, each of which is involved in a ligand-specific signaling pathway; comediator Smad (co-Smad) proteins, including Smad4, which interact with R-Smad proteins by participating in signal transduction; and inhibitory Smad (I-Smad) proteins, including Smad-6 and -7, which block the activation of R-Smad and Co-Smad proteins, negatively regulating signaling pathways.

[0077] As used herein, the term "TGF-beta" refers to transforming growth factor beta, which is a family of multifunctional cytokines (TGF-beta 1-5) that regulate cell growth and differentiation. Transforming growth factor (TGF) is one of many growth factors described that exist in nature. It plays a key role in "SCID" mice with severe combined immunodeficiency. Many cells synthesize TGF-beta, and basically all have receptors specific for this peptide. TGF-beta regulates the effects of many other peptide growth factors and determines the positive or negative direction of their effects. TGF-beta is a tumor suppressor cytokine that inhibits growth in epithelial cells. TGF-β may also act as a tumor promoter causing transformation from epithelium to mesenchyme. TGF-β inactivates several proteins involved in cell cycle management and thus exerts its growth inhibitory effect on epithelial cells, leading to their arrest in the G1 phase of the cell cycle. This protein works as a homodimer connected by disulfide bridges. Its sequence is characterized by the presence of several C-terminal cysteine ​​residues that form disulfide linking bonds arranged in a node-like structure. A similar "cystine node" system has been noticed in the structures of some enzyme and neurotoxin inhibitors that bind to voltage-gated Ca channels²⁺, although their exact structure varies. TGF-beta genes are expressed differently, suggesting that different TGF-beta variants may have distinct physiological functions in vivo.

[0078] The term "TGF-beta 1" as used herein refers to the transforming beta-type receptor growth factor 1, which is a peptide of 112 amino acid residues obtained by proteolytic cleavage from the C-terminus of the precursor protein. Examination of TGF-beta 1 mRNA levels in adult mouse tissues indicates that this expression is highest in the spleen, lung and placenta. TGF-beta 1 is thought to play an important role in pathological processes.

[0079] The term "SCID" as used herein refers to mice with severe combined immunodeficiency.

[0080] The term "HUVEC" as used herein refers to human umbilical vein endothelial cells.

[0081] As used herein, "amino acids" are represented by their full names, their corresponding three-letter codes, or their corresponding one-letter codes, as indicated in the following table:

<td>Full name</td><td>Three letter code</td><td>One letter code</td>

<td>Aspartic acid</td><td>Asp</td><td>D</td>

<td>Glutamic acid</td><td>Glu</td><td>E</td>

<td>lysine</td><td>lys</td><td>K</td>

<td>arginine</td><td>Arg</td><td>R</td>

<td>histidine</td><td>His</td><td>H</td>

<td>tyrosine</td><td>Tyr</td><td>Y</td>

<td>Cysteine</td><td>Cys</td><td>C</td>

<td>asparagine</td><td>own</td><td>N</td>

<td>glutamine</td><td>Gln</td><td>^Q</td>

<td>serine</td><td>Cheese</td><td>S</td>

<td>threonine</td><td>Thr</td><td>T</td>

<td>glycine</td><td>Gly</td><td>G</td>

<td>alanine</td><td>ala</td><td>AND</td>

<td>valine</td><td>val</td><td>V</td>

<td>leucine</td><td>Leu</td><td>L</td>

<td>isoleucine</td><td>How much</td><td>AND</td>

<td>methionine</td><td>Underworld</td><td>M</td>

<td>proline</td><td>Pro</td><td>P</td>

<td>phenylalanine</td><td>phe</td><td>F</td>

<td>tryptophan</td><td>Trp</td><td>IN</td>

[0082] In the present application, the twenty basic amino acids and their abbreviations are consistent with standard use. See Immunology-A Synthesis (Edition 2, ES

Golub and DR Gren, Ed., Sinauer Associates, Sunderland, Mass. (1991)).

[0083] A "conservative amino acid substitution" is one in which the amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more sequences differ from each other by conservative substitutions, the percentage identity or degree of sequence similarity may be increased to correct for the conservative nature of the substitution. Methods for such matching are well known to those skilled in the art. See, e.g., Pearson, Methods Mol. Biol. 243: 307-31 (1994).

[0084] Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) side chains containing amide moieties: 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 groups for conservative amino acid substitutions are: valine-leucineisoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate, aspartate and asparagine-glutamine.

[0085] Alternatively, the conservative replacement is any change having a positive value in the PAM250 log-probability matrix disclosed in Gonnet et al., Science 256: 1443-45 (1992).

[0086] A "moderately conservative" replacement is any change having a non-negative value in the PAM250 log-probability matrix.

[0087] In some cases, amino acid substitutions in the anti-ALK-1 antibody or antigen-binding portion thereof are those that (1) reduce the susceptibility to proteolysis, (2) reduce the susceptibility to oxidation, (3) change the binding affinity to form protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogues, but still retain the specificity of binding to ALK-1. Analogs may contain different substitutions in the normal peptide sequence. For example, in a normally occurring sequence, for example, in a portion of a polypeptide outside of the domain (s) involved in intermolecular interactions, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, can be made. Alternatively, amino acid substitutions can be made in the domain (s) that are involved in intermolecular interactions that can improve the activity of the polypeptide. Conservative amino acid substitution should not substantially alter the structural properties of the parent sequence, e.g. amino acid replacement should not alter the anti-parallel β-sheet structure that creates the immunoglobulin binding domain found in the parent sequence, nor damage other types of secondary structures that the parent sequence is characterized by. In general, glycine and proline will not be used in the anti-parallel β-sheet structure. Examples of known in the art of secondary and tertiary polypeptide structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, ed., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354: 105 (1991).

[0088] Sequence similarity for polypeptides, which is also referred to as sequence identity, is usually measured using sequence analysis software. Protein analysis software aligns similar sequences using measures of similarity attributed to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit" that 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 wild-type protein and his mutein. See, e.g., GCG Version

6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters, the FASTA program in GCG Version 6.1. (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlap regions between the query and wanted sequence (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185- 219 (2000)). Another preferred algorithm when comparing the sequences of the disclosure with a database containing a large number of sequences from different organisms is the BLAST computer program, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997).

[0089] 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, most often 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 that consist of a stretch of at least 25 amino acids that are substantially identical to the deduced portion of the naturally occurring amino acid sequence and which have at least one of the properties of a naturally occurring polypeptide. Typically, polypeptide analogs contain a conservative amino acid substitution (either addition or deletion) for a naturally occurring sequence. Analogs usually have a length of at least 20 amino acids, preferably at least 50 amino acids or more, and can often be as long as a naturally occurring full-length polypeptide.

[0090] Peptide analogs are widely used in the pharmaceutical industry as non-peptide drugs with properties analogous to the reference peptide. These types of non-peptide compounds are called "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).

[0091] Such compounds are often developed with the help of computer molecular modeling.

[0092] Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to a paradigm polypeptide (i.e. a polypeptide that has a biochemical property or pharmacological effect), such as a human antibody, in which optionally one or more peptide bonds have been replaced by a bond selected from the group consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, -CH = CH- (cis and trans), -COCH2-, -CH (OH) CH2- and CH2SO-, by methods well known in the art. Systematic substitution of one or more amino acids in a consensus sequence with a D-amino acid of the same type (e.g. D-lysine instead of L-lysine) can be used to produce more stable peptides. In addition, rigid peptides containing a consensus sequence or a substantially identical consensus sequence variant can be prepared using methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61: 387 (1992); for example by adding internal cysteine ​​residues capable of forming intramolecular disulfide bridges that lead to peptide cyclization. [0093] Intact "antibodies" or "immunoglobulins" (Ig) contain at least two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) connected together by disulfide bonds. There are only two types of light chains: λ and κ. They are similar in humans, but only one type is present in each antibody. Heavy chains are classified as mu, delta, gamma, alpha or epsilon and determine the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd Edition Raven Press, NY (1989)). In a preferred case, the antibody is of the IgG type and the IgG1, IgG2, IgG3 or IgG4 subtype. In a more preferred case, the anti-ALK-1 antibody is of the IgG2 subclass.

[0094] Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, wherein the heavy chain also contains a "D" region of about 3 or more amino acids. The VH and VL regions can be further subdivided into hypervariable regions, called "complementarity determining regions" (CDRs), separated by more conserved regions, called "framework regions" (FRs). Each VH and VL consists of three CDR and four FR regions, arranged from amino to carboxy in the following order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The allocation of amino acids to each domain is in accordance with the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989).

[0095] The variable domains of each heavy / light chain pair (VH and VL) form an antibody binding site that interacts with an antigen. Thus, an intact antibody, for example IgG, for two binding sites. In addition to bifunctional or dual specificity antibodies, the two binding sites are the same. Antibody constant regions can mediate the binding of 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.

[0096] Antibodies must have sufficient antigen binding diversity to recognize every possible pathogen (multiple V regions) while maintaining the biological effectiveness of their C regions (several C regions). Ig genes are randomly assembled from gene segments, which makes it possible to use multiple V regions with several C regions. Gene segments encoding kappa and lambda Ig H chains are located on three different chromosomes. During B-lymphocyte development, recombinase enzymes remove introns and some exons from DNA and assemble segments into functional Ig genes.

[0097] The Ig gene segments in mammals are arranged in groups of "variable" (V), "diversity" (D), "connecting" (J) and "constant" (C) exons. Each of the V kappa (Vk) segments encodes the first two CDR regions and three FRs from the V region of the kappa chain, and several residues from CDR3. Each of the J kappa (Jk) segments encodes the remainder of CDR3 and the fourth FR. C kappa (Ck) encodes the entire C region of the kappa light chain. DNA encoding the human kappa chain includes about 40 functional V kappa (Vk) segments, five J kappa (Jk) segments and one kappa C segment (Ck) gene segment, as well as some gene segments that contain stop codons ("pseudogens"). Human lambda (λ) DNA contains about 30 functional V lambda (VX) segments and four functional sets of lambda J (lambda ^ λ) and C lambda ^ λ) segments. The specified J lambda 0 ~ λ) always forms a pair with the corresponding C lambda (CX), unlike J kappa (Jk), which always forms a pair with the same C kappa (Ck). DNA for human H chain includes about 50 functional VH segments, 30 DH segments and six JH segments. The first two CDR regions and the three FR heavy chain variable domains are encoded by VH. CDR3 is encoded by several nucleotides from VH, whole DH and part of JH, while FR4 is encoded by the remainder of the JH gene segment. There are also individual gene segments in DNA for each heavy chain domain and for the membrane region of each isotype, arranged in the order in which they are expressed in B lymphocytes.

[0098] The term "polypeptide" includes native or artificial proteins, protein fragments, and polypeptide analogues of the protein sequence. The polypeptide may be monomeric or polymeric.

[0099] The term "isolated protein", "isolated polypeptide" or "isolated antibody" means a protein, polypeptide or antibody that, due to its origin or source of origin (1), is not associated with naturally bound components that accompany them in a state native, (2) are deprived of other proteins of the same species, (3) are expressed by cells from other species, or (4) are not found in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. Protein can also be made substantially free of its naturally associated components by isolation using well-known protein purification techniques.

[0100] Examples of isolated antibodies include, but are not limited to, an anti-ALK-1 antibody that has been purified for affinity using ALK-1, and an anti-ALK-1 antibody that has been synthesized by an in vitro cell line.

[0101] Proteins or polypeptides are "substantially pure", "substantially homogeneous" or "substantially purified" when at least about 60 to 75% of the sample is one type of polypeptide. These polypeptides or proteins can be monomeric or multimeric. A substantially pure polypeptide or protein can usually be about 50%, 60%, 70%, 80% or 90% w / w. protein samples, usually around 95%, and preferably can be over 99% pure. Protein purity or homogeneity can be determined in a variety of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band after gel staining with a dye well known in the art. For some purposes, greater resolution may be provided using HPLC or other methods well known in the art of purification.

[0102] As used herein, the term "polypeptide fragment" refers to a polypeptide that contains an amino and / or carboxy terminus deletion, but wherein the remaining amino acid sequence is identical to the positions corresponding to it in the naturally occurring sequence. In some cases, the fragments are at least 5, 6, 8 or 10 amino acids long. In other cases, 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.

[0103] As used herein, the term "analog" or "polypeptide analog" refers to a polypeptide that has a segment that is substantially identical to a portion of the amino acid reference sequence and has substantially the same function or activity as the reference amino acid sequence. Typically, polypeptide analogs contain a conservative amino acid substitution (or insertion or deletion) relative to the reference sequence. Analogs can be at least 20 or 25 amino acids in length, or they can be at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids in length or more, and can often be as long as a full-length polypeptide. Some instances of disclosure include polypeptide fragments or antibody polypeptide analogs containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions with respect to the amino acid sequence from germ. Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art according to the directions in this description.

[0104] As used herein, the term "antigen-binding antibody portion" (or simply "antibody portion") refers to one or more antibody fragments that retain the ability of specific antigen binding (e.g., ALK-1 or ECD 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 by the term "antigen binding portion of the antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) the F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) the Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains from a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546) which consists of the VH domain; and (vi) an isolated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombination methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair with two24 running monovalent molecules (known as a single Fv chain (scFv)); see, e.g., Bird et al. Science 242: 423-426 (1988) and Huston et al. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). Such single chain antibodies are also included in the term "antigen binding portion of the antibody". This also applies to other forms of single chain antibodies, such as diabodies. Diabodies are bivalent dual specificity antibodies in which the VH and VL domains are expressed as a single polypeptide chain, but using a linker that is too short to allow pairs between two domains from the same chain, thereby forcing domains to form pairs with complementary domains from the second chain to form two antigen binding sites (see, e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90: 64446448 (1993); Poljak et al. Structure 2: 1121-1123 (1994)).

[0105] Further, the antibody or antigen-binding portion thereof may be part of larger immunoadhesive molecules formed by the covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immunoadhesive molecules include the use of the streptavidin core region to form the tFameric scFv molecule (Kipriyanov et al. Human Antibodies and Hybridomas 6: 93-101 (1995)) and the use of a cysteine ​​residue, marker peptide and C-terminal polyhistidine tag to form divalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31: 1047-1058 ( 1994)). Other examples include those in which one or more CDR regions from an antibody are introduced into the molecule, covalently or non-covalently, to obtain an immunoadhesin that specifically binds to the antigen of interest, such as ALK-1 or ECD ALK-1. In such cases, the CDR region (s) may be introduced as part of a larger polypeptide chain, covalently linked to another polypeptide chain, or non-covalently linked.

[0106] Parts of antibodies such as Fab and F (ab ') 2 fragments can be made from whole antibodies using traditional techniques such as whole antibody digestion with papain or pepsin, respectively. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein.

[0107] The term "human antibody" as used herein means any antibody in which the variable and constant domain sequences are human sequences. This term includes antibodies with sequences derived from human genes that have been altered, e.g., to lower possible immunogenicity, increase affinity, remove cysteines that could cause unwanted splicing, etc. The term includes equally recombinantly produced antibodies in non-human cells, which may lead to atypical glycosylation of human cells. These antibodies can be produced in various ways as described below.

[0108] The term "neutralizing antibody," inhibitory antibody "or antagonist antibody as used herein means an antibody that inhibits the ALK-1 / TGF-beta-1 / Smad1 signaling pathway. In a preferred case, the antibody inhibits the ALK-1 / TGF-beta-1 / Smad1 signaling pathway by at least 20%, more preferably 40%, more preferably 60%, even more preferably 80% or even more preferably 85%. The neutralizing or inhibitory potential of human anti-ALK-1 antibodies can be determined, for example, on the basis of their ability to inhibit regulation leading to stimulation of a specific downstream ALK-1 specific gene, Id1, as shown in Example 12; for inhibiting Smad1 phosphorylation determined by Western Blot using the Odyssey Infrared Imaging System from the LI-COR Biosciences as shown in Example 13.

[0109] The term "chimeric antibody" as used herein means an antibody that contains regions from two or more different antibodies. For example, one or more CDR regions of the chimeric antibody may be derived from human anti-ALK-1 antibody. In another example, all CDR regions may be derived from human anti-ALK-1 antibodies. In another example, the CDR regions of more than one human anti-ALK-1 antibody can be combined in a chimeric antibody. For example, the chimeric antibody may comprise CDR1 from the light chain of the first human anti-ALK-1 antibody, CDR2 from the light chain of the second human anti-ALK-1 antibody and CDR3 from the light chain of the third human anti-ALK-1 antibody, and the heavy chain CDRs may be derived from one or more other anti-ALK-1 antibodies. Further, the framework regions may be derived from one of the anti-ALK-1 antibodies from which one or more CDR regions are taken, or from one or more other human antibodies. In addition, as discussed herein before, a chimeric antibody includes an antibody comprising a portion derived from germline sequences of more than one species.

[0110] In some cases, the chimeric antibody of the disclosure is a humanized anti-ALK-1 antibody. The humanized anti-ALK-1 antibody of the disclosure includes the amino acid sequence of one or more framework regions and / or an amino acid sequence from at least a portion of the constant region of one or more human anti-ALK-1 antibodies of the disclosure, and further includes sequences derived from non-human antibodies against ALK-1, for example CDR sequences.

[0111] The term "ELISA" as used herein refers to an enzyme immunoassay. This test is well known to those skilled in the art. Examples of this test can be found in Vaughan, TJ et al., Nature Biotech. 14: 309-314 (1996) as well as in Example 2 of this application.

[0112] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows for real-time analysis of biospecific interactions based on the detection of changes in protein concentrations on a biosensor matrix, for example using the BIACORE ™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Further instructions can be found in Jonsson et al., Ann. Biol. Clin. 51: 1926 (1993); Jonsson et al., Biotechniques 11: 620-627 (1991); Jonsson et al., J. Mol. Recognit. 8: 125-131 (1995); and Johnsson et al., Anal. Biochem. 198: 268-277 (1991).

[0113] The term "affinity" refers to a measure of attraction between an antigen and an antibody. The innate attraction of an antibody to an antigen is usually expressed as a binding affinity equilibrium constant (KD) for a particular antibody-antigen interaction. The antibody is said to specifically bind to an antigen when the Kd is &lt;1 mM, preferably &lt;100 nM. The Kd binding affinity constant can be measured by surface plasmon resonance, for example using the BIACORE system^™as discussed in Examples 7 and 8.

[0114] The term "koff" refers to a dissociation rate constant of a particular antibody-antigen interaction. The koff dissociation rate constant can be measured by surface plasmon resonance, for example using a BIAcore system, as discussed in Examples 7 and 8.

[0115] The term "avidity" refers to the strength of the functional binding of an antibody to its antigen, which is based on both the affinity and valence of the antibody. As used herein, the term describes increased affinity that occurs as a result of multiple antigen binding sites on an immunoglobulin.

[0116] The term "molecular selectivity" as used herein refers to the binding affinity of the antibody for a specific antigen that is greater than for other antigens. For example, the antibodies of the present disclosure may be selective for ALK-1 compared to ALK-2 to ALK-7, which means that the binding affinity of this antibody for ALK-1 is at least 2 times higher, e.g. times or 50 or 100 times or greater than for ALK-2 to ALK-7. Such binding affinities can be measured using standard techniques known to those skilled in the art.

[0117] The term "epitope" includes any protein determinant capable of binding specifically to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or carbohydrates or side chain sugar, and usually have specific tertiary structure properties as well as specific charge properties. An epitope can be "linear" or "conformational." In a linear epitope, all sites of interaction between a protein and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, interaction sites occur on amino acid residues of a protein that are separated from each other. Once the desired epitope on the antigen has been determined, it is possible to generate antibodies for that epitope, e.g. using the techniques described in this disclosure. Alternatively, during the discovery process, the production and characterization of antibodies provides information on desired epitopes. Using this information, it is then possible to competitively screen antibodies for binding to the same epitope. The approach to achieve this goal is to perform cross-competition studies to find antibodies that competitively bind to each other, i.e. the antibodies compete for binding to the antigen. A high-throughput process for "shoveling" antibodies based on cross-competition is described in International Patent Application No. WO 03/48731.

[0118] As used herein, the term "labeling" refers to a method of grouping antibodies based on their antigen binding properties. Assignment of drawers is largely arbitrary, depending on how the observed binding patterns differ for all tested antibodies. Therefore, drawers will not always correlate with epitopes defined by other methods and should not be used to define epitopes.

[0119] The term "compete" as used herein to refer to an antibody, means that the first antibody or antigen-binding portion thereof competes for binding to the antigen with the second antibody or antigen-binding portion thereof, wherein the binding of the first antibody to its associated epitope is detectably reduced in the presence of a second antibody, compared to binding of the first antibody in the absence of a second antibody. The reverse case in which binding of the second antibody to its epitope is detectably reduced in the presence of the first antibody may or may not occur. That is, the first antibody can inhibit the binding of the second antibody to its epitope, without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, when each of the antibodies detectably inhibits the binding of the other antibody to its associated epitope or ligand to the same, to a greater or lesser extent, it is said that these antibodies "cross-compete" for binding to the appropriate (- i) epitope (s). Both competing and cross-competing antibodies are included in the present disclosure. Regardless of the mechanism responsible for such competition or cross competition (e.g., spatial hindrance, conformational change or binding to a common epitope or part thereof, and the like), a person skilled in the art will understand from the information provided herein that such competing and / or competing cross-covered antibodies are included and may be useful in the methods disclosed herein.

[0120] The term "polynucleotide" as used herein means a polymeric form of nucleotides of at least 10 bases in length, ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single and double stranded forms.

[0121] The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA or synthetic origin, or some combination thereof, which "isolated polynucleotide" due to its origin (1) is not associated with all or some polynucleotides with which this "isolated polynucleotide" occurs in nature, (2) it is operably linked to a polynucleotide with which it is not connected in nature, or (3) does not occur in nature as part of a larger sequence.

[0122] The term "naturally occurring nucleotides" 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" as used herein includes oligonucleotide linkages such as phosphorothioate, dithiophosphate, selenophosphate, diselenophosphate, thioanilophosphate, anilophosphate, phosphoramidate and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14: 9081 (1986); Stec et al., J. Am. Chem. Soc. 106: 6077 (1984); Stein et al., Nucl. Acids Res. 16: 3209 (1988); Zon et al., Anti-Cancer Drug Design 6: 539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990). If required, the oligonucleotide may contain a detection tag.

[0123] "operably linked" sequences include both expression control sequences that are adjacent to a given gene and expression control sequences that act in trans or from a distance in controlling a given gene.

[0124] The term "expression control sequence" as used herein means polynucleotide sequences that are necessary for the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the appropriate transcription initiation sequences, transcription termination, promoter and enhancer; effective signals for RNA processing, such as signals for slicing and polyadenylation; cytoplasmic mRNA stabilizing sequences; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, protein secretion enhancing sequences. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, ribosome binding site and transcription termination sequence; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" is intended to include at least all components whose presence is necessary for expression and processing, and may also include additional components whose presence is beneficial, for example, leader sequences and fusion partner sequences.

[0125] The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. In some cases, the vector is a plasmid, i.e. a circular double-stranded segment of DNA into which additional DNA segments can be inserted. In some cases, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some cases, the vectors are capable of autonomous replication in the host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other cases, vectors (e.g., mammalian non-episomal vectors) may integrate into the host cell genome after they have been introduced into the host cell, and thus replicate together with the host genome. In addition, some vectors are capable of directing the expression of genes with which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0126] As used herein, the term "recombinant host cell" (or simply "host cell") means a cell into which the recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the cell in question, but also the progeny of such a cell. As modifications due to mutation or environmental influences may occur in subsequent generations, such offspring may not actually be identical to the stem cell, but are still included in the term "host cell" as used herein.

[0127] As used herein, "germline" refers to the nucleotide and amino acid sequences of genes and antibody gene segments that are passed from parents to offspring through germ cells. This germline sequence is distinguished from nucleotide sequences encoding antibodies in mature B cells that have been altered by recombination and hypermutation during B cell maturation. An antibody that "utilizes" a particular germline has the nucleotide or amino acid sequence that is most similar to the nucleotide sequence of the germline or amino acid sequence that the first one specifies. Such antibodies are often mutated compared to the germline sequence.

[0128] The term "percent sequence identity" in the context of a nucleic acid sequence means residues in two sequences that are the same when aligned at maximum matching. Comparison of sequence identity can occur over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more often at least about 24 nucleotides, usually at least about 28 nucleotides, even more often at least about 32 nucleotides, and preferably at least about 36 nucleotides , 48 or more nucleotides. There are several different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs from the Wisconsin version 10.0 package, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, for example, the FASTA2 and FASTA3 programs, provides alignment and percent sequence identity of the best overlap regions between the query and wanted sequence (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132 : 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71-84 (1998)). Unless otherwise specified, the default parameters for the specified program or algorithm are used. For example, the percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (word size 6 and NOPAM factor for counting matrix) or using Gap with default parameters, as provided in GCG version 6.1.

[0129] Reference to a nucleotide sequence includes its complement, unless otherwise stated. Thus, reference to a nucleic acid of a particular sequence should be understood to include its complementary strand of complementary sequence.

[0130] The term "substantial similarity" or "substantial sequence similarity" with respect to a nucleic acid or fragment thereof means that with optimal alignment with another nucleic acid (or its complementary strand) taking into account relevant insertions or deletions of nucleotides, the identity of the nucleotide sequences is at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of nucleotide bases, measured by any well-known algorithm for sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0131] The term "percent sequence identity" in the context of amino acid sequences means residues in two sequences that are the same when aligned at maximum matching. Comparison of sequence identity can occur over a section of at least about five amino acids, usually at least about 20 amino acids, more often at least about 30 amino acids, usually at least about 50 amino acids, more often at least about 100 amino acids, and even more often about 150, 200 or more amino acids. There are several different algorithms known in the art that can be used to measure amino acid sequence identity. For example, amino acid sequences can be compared using FASTA, Gap or Bestfit, which are programs from the Wisconsin version 10.0 package, Genetics Computer Group (GCG), Madison, Wisconsin.

[0132] As used herein for polypeptides, the term "substantial identity" or "essential similarity" means that two amino acid sequences, when optimally matched, such as using GAP or BESTFIT programs at the default gap weights provided in these programs , are at least 70%, 75% or 80% identical, preferably at least 90% or 95%, and more preferably at least 97%, 98% or 99%. In some cases, residue positions that are not identical differ by conservative amino acid substitutions.

[0133] The term "signal sequence", also called a signal peptide, leader peptide, refers to a segment of about 15 to 30 amino acids at the N-terminus of a protein that allows secretion of this protein (passage through the cell membrane). The signal sequence is deleted after protein secretion.

[0134] As used herein, the terms "tag" or "labeled" refer to the introduction of another molecule into an antibody. In one case, the label is a detectable marker, e.g., the introduction of a radiolabeled amino acid or attachment to a polypeptide of biotinyl groups that can be detected using labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Otherwise the label or marker may be therapeutic, e.g., drug conjugate or toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g.³H ¹⁴C ¹⁵N ³⁵S ⁹⁰Y ⁹⁹tc ¹¹¹in, ¹²⁵AND, ¹³¹I), fluorescent markers (e.g. FITC, rhodamine, lanthanide phosphors), enzyme markers (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by e.g. . leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalazine B, gramicidin D, ethidium bromide, emetin, mitomycin , etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogues or homologues. In some cases, markers are attached through spacer arms of varying lengths to reduce potential spatial hindrance.

[0135] The term "primates" refers to non-human primates that includes apes and monkeys, with improved development of hands and feet, a short nose, and a large brain. The order of non-human primates includes humans, apes, monkeys, and lower primates.

[0136] A "therapeutically effective amount" refers to that amount of therapeutic agent administered that to some extent alleviates one or more of the symptoms of the disorder being treated. With respect to the treatment of cancer, a therapeutically effective amount refers to that amount which has at least one of the following effects: tumor size reduction; inhibiting (i.e., slowing to some extent, preferably stopping) tumor metastasis; inhibiting to some extent (i.e., slowing to some extent, preferably stopping) tumor growth, and alleviating to some extent (or preferably eliminating) one or more symptoms associated with cancer.

[0137] "Treating", "treatment" or "therapy" refers to a method of alleviating or abolishing a biological disorder and / or its associated symptoms. In relation to cancer, these terms simply mean that the life expectancy of the subject affected by the cancer will be increased or one or more symptoms of the disease will be reduced.

[0138] "Contacting" refers to contacting the antibody or antigen binding portion thereof of the present disclosure and the target ALK-1, or epitope thereof, in such a way that the antibody can affect the biological activity of ALK-1. Such "contacting" can be achieved "in vitro", ie in a test tube, petri dish or similar. In a test tube, contacting may include only the antibody or antigen binding portion thereof and ALK-1 or epitope thereof, or may include whole cells. Cells may also be maintained or cultured in cell culture vessels and in this environment contacted with the antibodies or antigen-binding portions thereof. In this context, before attempting to use a particular antibody in vivo on more complex living organisms, the ability of the antibody or antigen-binding portion thereof to affect an ALK-1 related disorder, e.g., the IC50 value of that antibody, can be determined. For cells found outside the body, there are many methods for contacting ALK1 with antibodies or antigen-binding portions thereof, and they are well known to those skilled in the art.

[0139] The abbreviation "FACS" refers to fluorescence activated cell sorting. The abbreviation FACS and flow cytometry are used interchangeably. Fluorescent labeling allows you to study the structure and function of cells. Immunofluorescence, the most widely used, involves staining cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin. This method is often used to label molecules on the surface of cells, but antibodies can be directed to target molecules in the cytoplasm. In direct immunofluorescence, the anti-molecule antibody is directly conjugated to a fluorescent dye and the cells are stained in one step. In indirect immunofluorescence, the primary antibody is unlabelled and a second antibody conjugated to a fluorescent dye that is specific for the first antibody is added. Anti-ALK-1 Antibodies [0140] This disclosure relates to isolated neutralizing anti-ALK-1 monoclonal antibodies or antigen-binding portions thereof that bind to primate ALK-1, preferably to primate ALK-1 ECD, more preferably to human ALK-1 ECD. In a preferred case, the disclosure relates to isolated neutralizing antibodies that are fully human monoclonal antibodies, or antigen-binding portions thereof. Preferably, human antibodies are recombinant human anti-ALK-1 antibodies that have greater affinity for ALK-1 than for ALK-2 to ALK-7. In some cases, human anti-ALK-1 antibodies are produced by immunizing a transgenic non-human animal, e.g. a rodent whose genome contains human immunoglobulin genes so that this transgenic living being produces human antibodies. Various aspects of the disclosure relate to such antibodies and antigen binding portions and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for forming such antibodies and antigen binding portions. Methods of using the antibodies and antigen-binding portions of the present disclosure for abolishing the ALK-1 / TGF-beta-1 / Smad1 signaling pathway or for detecting ALK-1, in vitro or in vivo, are also encompassed by this disclosure. [0141] The anti-ALK-1 antibody of the disclosure may comprise a human kappa or lambda light chain or an amino acid sequence derived therefrom. In some cases involving the kappa light chain, the light chain variable domain (VL) uses the human A27, A2, A1, A3, B3, B2, L1 or L2 Vk gene. In some cases, the light chain uses the human Vk L1 gene and the human Jk 4 gene; human Vk A27 gene and human Jk 5 gene or human Jk 4 gene; human Vk B3 gene and human Jk 1 gene; human Vk L2 gene and human Jk 3 gene; human Vk A2 gene and human Jk 1 gene; human Vk A3 gene and human Jk 4 gene; human Vk A1 gene and human Jk 1 gene; human Vk B2 gene and human Jk 4 gene; or the human Vk A2 gene and the human Jk 1 gene.

[0142] In some cases, the VL anti-ALK-1 antibody contains one or more amino acid substitutions, deletions, or insertions relative to the germline Vk amino acid sequence. In some cases, the Vl anti-ALK-1 antibody contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid substitutions relative to the germline Vk amino acid sequence. In some cases, one or more germline substitutions are in the light chain CDRs. In some cases, the amino acid substitutions in the Vk relative to the germline are at one or more of the same positions as the germline substitutions found in any one or more VL antibodies provided herein as shown, for example in Figure 7. In some cases, the amino acid changes are in one or more of the same positions, but include a different substitution than the reference antibody.

[0143] In some cases, germline substitutions occur at one or more of the same positions as germline substitutions at any of the 1.11.1 VL antibodies; 1.12.1; 1.12.1 (RWT); 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, but substitutions may be conservative amino acid substitutions at this (these) position (s) relative to the amino acid in the reference antibody. For example, if a certain position in one of these antibodies is changed relative to the germline and is glutamate, aspartate may be substituted at this position. Similarly, if the amino acid substitution compared to the germline in the exemplary antibody is serine, you can conservatively substitute threonine for serine at this position. Conservative amino acid substitutions have been discussed above.

[0144] In some cases, the anti-ALK-1 antibody comprises the amino acid sequence of the light chain SEQ ID NO: 4. In other cases, the light chain comprises the amino acid sequence of the light chain 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.

[0145] In some cases, the light chain of a 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: 12); 1.13.1 (SEQ ID NO: 16); 1.14.1 (SEQ ID NO: 20); 1.151.1 (SEQ ID NO: 24);

1.162.1 (SEQ ID NO: 28); 1.183.1 (SEQ ID NO: 32); 1.8.1 (SEQ ID NO: 36); 1.9.1 (SEQ ID NO: 40); 4.10.1 (SEQ ID NO: 44); 4.24.1 (SEQ ID NO: 48); 4.38.1 (SEQ ID NO: 52);

4.58.1 (SEQ ID NO: 56); 4.62.1 (SEQ ID NO: 60); 4.68.1 (SEQ ID NO: 64); 4.72.1 (SEQ ID NO: 68); 5.13.1 (SEQ ID NO: 72); 5.34.1 (SEQ ID NO: 76); 5.53.1 (SEQ ID NO: 80);

5.56.1 (SEQ ID NO: 84); 5.57.1 ​​(SEQ ID NO: 88) or 5.59.1 (SEQ ID NO: 92); or a given sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions and / or up to 3 non-conservative amino acid substitutions in total. In other cases, the light chain of human anti-ALK-1 antibody comprises the VL amino acid sequence of 1.27.1; 1.29.1 or 1.31.1. In some cases, the light chain comprises the amino acid sequence from the beginning of CDR1 to the end of CDR3 of any of the above antibodies.

[0146] In some cases, the light chain may comprise the amino acid sequences of the CDR1, CDR2 and CDR3 regions independently selected from 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 (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, or given CDR regions, each containing less than 3 or less than 2 conservative amino acid substitutions and / or a total of three or less than three non-conservative amino acid substitutions.

[0147] In some cases, the anti-ALK-1 antibody light chain comprises the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the antibody light chain 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; 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 given CDR regions, each containing less than 3 or less than 2 conservative amino acid substitutions and / or a total of three or less than three non-conservative amino acid substitutions.

[0148] As for the heavy chain, in some cases the variable domain (VH) uses the human VH 4-31, VH 3-11, VH 3-15, VH 3-33, VH 4-61 or VH 4-59 gene. In some cases, the VH sequence of the anti-ALK-1 antibody contains one or more substitutions, deletions or insertions (additions), together, "mutation," amino acids relative to the germline VH amino acid sequence. In some cases, the heavy chain variable domain contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations relative to the germline VH amino acid sequence. In some cases, these mutations are non-conservative amino acid substitutions compared to the germline amino acid sequence. In some cases, these mutations are located in the heavy chain CDRs. In some cases, the heavy chain uses the human VH 3-33 gene, the human D 6-19 gene and the human JH 3B gene; human VH 4-31 gene, human D 6-19 gene and human JH 4B gene; human VH 4-61 gene, human D 6-19 gene and human JH 4B gene; human VH 4-31 gene, human D 3-3 gene and human JH 3B gene; human VH 4-31 gene and human JH 3B gene; human VH 4-59 gene, human D 6-19 gene and human JH 4B gene; human VH 3-11 gene, human D 322 gene and human JH 6B gene; human VH 3-15 gene, human D 3-22 gene and human JH 4B gene; human VH 4-31 gene, human D 5-12 gene and human JH 6B gene; human VH 4-31 gene, human D 4-23 gene and human JH 4B gene; human VH 4-31 gene, human D 2-2 gene and human JH 5B gene; human VH 4-31 gene and human JH 6B gene; human VH 3-15 gene, human D 1-1 gene and human JH 4B gene; human VH 3-11 gene, human D 6-19 gene and human JH 6B gene; human VH 3-11 gene, human D 3-10 gene and human JH 6B gene; or the human VH 3-11 gene, the human D 6-6 gene and the human JH 6B gene.

[0149] In some cases, amino acid substitutions occur at one or more of the same positions as germline substitutions in one or more VH 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; 5.57.1 ​​or 5.59.1. In other cases, the amino acid changes occur in one or more of the same positions, but include a different substitution than that of the reference antibody.

[0150] In some cases, the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. In other cases, the heavy chain comprises the amino acid sequence of the heavy chain 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 cases, the heavy chain comprises the VH amino acid sequence of antibody 1.12.1 (SEQ ID NO: 6); 1.11.1 (SEQ ID NO: 10); 1.13.1 (SEQ ID NO: 14); 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 a given 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 cases, the heavy chain comprises the VH amino acid sequence of 1.27.1; 1.29.1 or 1.31.1. In some cases, the heavy chain comprises the amino acid sequence from the beginning of CDR1 to the end of CDR3 of any of the above antibodies.

[0151] In some cases, the heavy chain comprises the CDR1, CDR2 and CDR3 regions of the heavy chain 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, or given CDR regions, each of which has less than 8, less than 6, less than 4, or less than 3 conservative amino acid substitutions and / or a total of three or less than three non-conservative amino acid substitutions. [0152] In some cases, the heavy chain CDRs are independently selected from the CDRs of two or more 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 (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 another case, the antibody comprises a light chain as disclosed above and a heavy chain as disclosed above. In another case, the light chain CDRs and heavy chain CDRs are from the same antibody.

[0153] In various cases, anti-ALK-1 antibodies have the full-length heavy chain and light chain amino acid sequence (s), VH and VL amino acid sequences, heavy chain CDR1, CDR2 and CDR3 amino acid sequences, and CDR1, CDR2 and CDR3 light chain or heavy chain amino acid sequence from the beginning of CDR1 to the end of CDR3 and the light chain amino acid sequence from the beginning of CDR1 to the end of CDR3 of the anti-ALK-1 antibody provided herein. [0154] One type of amino acid substitution that can be made is the replacement of one or more cysteines in the antibody, which may be chemically reactive, with another residue, such as alanine or serine, which is not limiting. In one case, substitution of non-canonical cysteine ​​occurs. This substitution can be made in the CDR or framework region of the variable domain or in the constant domain of the antibody. In some cases, cysteine ​​is canonical.

[0155] Another type of amino acid substitution that can be made is removal of potential proteolysis sites in the antibody. Such sites may exist in the CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine ​​residues and removal of proteolysis sites may reduce the risk of heterogeneity of the antibody product and thus increase its homogeneity. Another type of amino acid substitution is the removal of asparagine-glycine pairs that create potential deamination sites by changing one or both of these residues.

[0156] In some cases, the C-terminal lysine of the heavy chain anti-ALK-1 antibody of the disclosure is cleaved. In various instances, the disclosures of the heavy and light chains of anti-ALK-1 antibodies may optionally include a signal sequence.

[0157] In one aspect, the disclosure provides twenty-five inhibitory human monoclonal anti-ALK-1 antibodies and hybridoma cell lines that produce them. In some cases, the antibodies of the present disclosure are IgG designated as: 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 ​​and 5.59.1. In preferred cases, 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.

[0158] Antibodies recognize surface exposed epitopes or antigens based on regions with a linear (primary) sequence or a structural (secondary) sequence. The BIAcore method was used to define the functional epitope system and determine the epitope exclusivity of exemplary anti-ALK-1 antibodies according to this disclosure.

[0159] Table 1 lists the sequence identification numbers (SEQ ID NO) of nucleic acids encoding full length heavy and light chains of the 1.12.1 antibody variants and containing the variable domain of the portion of the anti-ALK-1 antibody from the disclosure and the corresponding deduced amino acid sequences.

Table 1

SEQUENCE IDENTIFICATION NUMBERS (SEQ ID NO)

<td rowspan="3">Antibody</td><td colspan="4">FULL LENGTH</td><td colspan="4">INCLUDING PART DOMAIN V</td>

<td colspan="2">Heavy</td><td colspan="2">Light</td><td colspan="2">Heavy</td><td colspan="2">Light</td>

<td>GOUT</td><td>PROTEIN</td><td>GOUT</td><td>PROTEIN</td><td>GOUT</td><td>PROTEIN</td><td>GOUT</td><td>PROTEIN</td>

<td> 1.11.1</td><td></td><td></td><td></td><td></td><td> 9</td><td> 10</td><td> 11</td><td> 12</td>

<td>1.12.1 (M29I / D19A)</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td>

<td> 1.12.1</td><td> 95</td><td> 100</td><td> 101</td><td> 102</td><td> 103</td><td> 104</td><td> 126</td><td> 127</td>

<td>1.12.1 (RWT)</td><td> 128</td><td> 100</td><td> 101</td><td> 102</td><td> 129</td><td> 104</td><td> 126</td><td> 127</td>

<td> 1.13.1</td><td></td><td></td><td></td><td></td><td> 13</td><td> 14</td><td> 15</td><td> 16</td>

<td> 1.14.1</td><td></td><td></td><td></td><td></td><td> 17</td><td> 18</td><td> 19</td><td> 20</td>

<td> 1.151.1</td><td></td><td></td><td></td><td></td><td> 21</td><td> 22</td><td> 23</td><td> 24</td>

<td> 1.162.1</td><td></td><td></td><td></td><td></td><td> 25</td><td> 26</td><td> 27</td><td> 28</td>

<td> 1.183.1</td><td></td><td></td><td></td><td></td><td> 29</td><td> 30</td><td> 31</td><td> 32</td>

<td> 1.8.1</td><td></td><td></td><td></td><td></td><td> 33</td><td> 34</td><td> 35</td><td> 36</td>

<td> 1.9.1</td><td></td><td></td><td></td><td></td><td> 37</td><td> 38</td><td> 39</td><td> 40</td>

<td> 4.10.1</td><td></td><td></td><td></td><td></td><td> 41</td><td> 42</td><td> 43</td><td> 44</td>

<td> 4.24.1</td><td></td><td></td><td></td><td></td><td> 45</td><td> 46</td><td> 47</td><td> 48</td>

<td> 4.38.1</td><td></td><td></td><td></td><td></td><td> 49</td><td> 50</td><td> 51</td><td> 52</td>

<td> 4.58.1</td><td></td><td></td><td></td><td></td><td> 53</td><td> 54</td><td> 55</td><td> 56</td>

<td> 4.62.1</td><td></td><td></td><td></td><td></td><td> 57</td><td> 58</td><td> 59</td><td> 60</td>

<td> 4.68.1</td><td></td><td></td><td></td><td></td><td> 61</td><td> 62</td><td> 63</td><td> 64</td>

<td colspan="9">SEQUENCE IDENTIFICATION NUMBERS (SEQ ID NO)</td>

<td rowspan="3">Antibody</td><td colspan="4">FULL LENGTH</td><td colspan="4">INCLUDING PART DOMAIN V</td>

<td colspan="2">Heavy</td><td colspan="2">Light</td><td colspan="2">Heavy</td><td colspan="2">Light</td>

<td>GOUT</td><td>PROTEIN</td><td>GOUT</td><td>PROTEIN</td><td>GOUT</td><td>PROTEIN</td><td>GOUT</td><td>PROTEIN</td>

<td> 4.72.1</td><td></td><td></td><td></td><td></td><td> 65</td><td> 66</td><td> 67</td><td> 68</td>

<td> 5.13.1</td><td></td><td></td><td></td><td></td><td> 69</td><td> 70</td><td> 71</td><td> 72</td>

<td> 5.34.1</td><td></td><td></td><td></td><td></td><td> 73</td><td> 74</td><td> 75</td><td> 76</td>

<td> 5.53.1</td><td></td><td></td><td></td><td></td><td> 77</td><td> 78</td><td> 79</td><td> 80</td>

<td> 5.56.1</td><td></td><td></td><td></td><td></td><td> 81</td><td> 82</td><td> 83</td><td> 84</td>

<td> 5.57.1</td><td></td><td></td><td></td><td></td><td> 85</td><td> 86</td><td> 87</td><td> 88</td>

<td> 5.59.1</td><td></td><td></td><td></td><td></td><td> 89</td><td> 90</td><td> 91</td><td> 92</td>

<td colspan="9">1.12.1 (M29I / D19A) refers to an anti-ALK-1 antibody containing a single specific amino acid mutation in the heavy chain where the methionine at position 29 has been replaced with isoleucine and a single specific light chain mutation where aspartic acid at position 19 has been replaced with alanine as described in Example 4. 1.12.1 refers to the mAb 1.12.1 variant that has been isolated from a hybridoma. 1.12.1 (rWT) refers to the variant mAb 1.12.1 that was expressed as the recombinant mAb described in Example 3.</td>

[0160] The disclosure further provides heavy and / or light chain variants of the above-mentioned human anti-ALK-1 antibodies; containing one or more amino acid modifications. In the designations of these variants, the first letter is the single letter amino acid symbol of the naturally occurring antibody chain, the number refers to the amino acid position (where the first position is the N-terminal amino acid of FR1), and the second letter is the single letter amino acid symbol of the variant.

[0161] In yet further cases, the disclosure includes antibodies comprising variable domain amino acid sequences with sequence identity over 80%, over 85%, over 90%, over 95%, over 96%, over 97%, over 98% or over 99% of the amino acid sequence of the variable domain of any of the above human anti-ALK-1 antibodies.

Class and subclass of anti-ALK-1 antibodies [0162] The class and subclass of anti-ALK-1 antibodies can be determined by any method known in the art. In general, the class and subclass of antibodies can be determined using antibodies that are specific for the particular class and subclass of the antibody. Such antibodies are commercially available. Class and subclass can be determined by ELISA, Western Blot as well as other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant domains of the heavy and / or light chains of antibodies by comparing their amino acid sequences with known amino acid sequences of immunoglobulins of different classes and subclasses, and determining the class and subclass of these antibodies.

[0163] The class of anti-ALK-1 antibody obtained as described above can be switched to another. In one aspect of the disclosure, the nucleic acid molecule encoding VL or VH is isolated using methods well known in the art such that it does not include a nucleic acid sequence encoding CL or CH. "Antibody Engineering" (Kontermann and Dubel, Ed., Springer-Verlag, Berlin (2001)). The nucleic acid molecules encoding VL or VH are then operably linked to the nucleic acid sequence encoding respectively CL or CH from another class of immunoglobulin molecules. This can be accomplished using a vector or nucleic acid molecule that contains the CL or CH chain as described above. For example, an anti-ALK-1 antibody that was originally IgM class can be switched to IgG class. Class switching can be further used to convert one IgG subclass to another, e.g. from IgG1 to IgG2. A preferred method of producing the antibody of the disclosure including the desired isotypes includes the steps of isolating the nucleic acid molecule encoding the anti-ALK-1 antibody heavy chain and the nucleic acid molecule encoding the anti-ALK-1 antibody light chain, obtaining the heavy chain variable domain, ligating the heavy chain variable domain to the domain heavy chain constant with the desired isotype, expressing the light chain and ligated heavy chain in the cell, and collecting the anti-ALK-1 antibody with the desired isotype.

[0164] In some cases, the anti-ALK-1 antibody is a monoclonal antibody. The anti-ALK-1 antibody may be an IgG, IgM, IgE, IgA or IgD molecule. In a preferred case, the anti-ALK-1 antibody is an IgG antibody and is a subclass of IgG1, IgG2, IgG3 or IgG4. In another preferred case, the antibody is of the IgG2 subclass.

Identification of ALK-1 epitopes recognized by anti-ALK-1 antibodies [0165] This disclosure provides a human anti-ALK-1 monoclonal antibody that binds to ALK-1 and competes or crosses with and / or binds to the same epitope as: ( a) 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; 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 VH domain amino acid sequence from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104, (c) an antibody that comprises a light chain variable domain with a VL domain amino acid sequence from any of SEQ ID NO: 8; 12; 16; twenty; 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 the heavy chain variable domain as defined in (b) and the light chain variable domain as defined in (c).

[0166] Using methods known in the art, it can be determined whether the antibody binds to the same epitope or cross-competes for binding to an anti-ALK-1 antibody. In one case, the anti-ALK-1 antibody of the disclosure is allowed to bind to ALK-1 under saturation conditions, and then the test antibody's ability to bind to ALK-1 is measured. If the test antibody is capable of binding 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 capable of binding to ALK-1 at the same time, then the test antibody binds to the same epitope, overlapping epitope or epitope that lies close to the epitope bound by the anti-ALK-1 antibody of the disclosure. 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, the two-way competition method described above can be used, i.e., checking that the known antibody blocks the antibody being tested, and vice versa. In a preferred case, this experiment is carried out using BIACORE^™. Binding affinity of anti-ALK-1 antibodies with ALK-1 [0167] The binding affinity (KD) and dissociation constant (koff) of the anti-ALK-1 antibody or antigen-binding portion thereof of ALK-1 can be determined by methods known in the art. Binding affinity can be measured by ELISA, RIA, flow cytometry or surface plasmon resonance, such as BIACORE^™. The dissociation constant can be measured by surface plasmon resonance. Preferably, the binding affinity and dissociation constant are measured by surface plasmon resonance. More preferably, the binding affinity and dissociation constant are measured using BIACORE^™. Using methods known in the art, it can be determined whether the antibody has substantially the same KD as the anti-ALK-1 antibody. Such methods for determining KD and koff can be used during the initial screening step as well as during subsequent optimization steps.

Inhibition of ALK-1 activity by an anti-ALK-1 antibody [0168] Monoclonal anti-ALK-1 antibodies that inhibit the binding of ALK-1 can be identified using several tests. For example, neutralizing anti-ALK-1 antibodies can be identified by their inhibition of regulation leading to stimulation of a specific downstream ALK-1 target gene, Id1, as described in Example 12. Preferred ALK-1 antibodies have an IC 50 not higher than 500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM or 1 nM.

[0169] The ability of the anti-ALK-1 antibody to inhibit Smad1 phosphorylation as measured by Western Blott using the Odyssey infrared imaging system as described in Example 13 can also be determined. In various cases, the anti-ALK-1 antibody has an IC50 value in this assay not higher than 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM or 1 nM.

Inhibition of angiogenesis by an anti-ALK-1 antibody [0170] In another case, the anti-ALK-1 antibody or portion thereof inhibits human vascular angiogenesis as demonstrated in SCID mice transplanted with human foreskin tissue in which M24met human melanoma tumor cells were intradermally implanted, determined by IHC analysis using the human CD-31 signaling assay by a factor of at least 40% compared to the control sample, as described in Example 17 and shown in Table 13.

[0171] In another case, the anti-ALK-1 antibody or portion thereof inhibits human vascular angiogenesis as demonstrated in SCID mice transplanted with human foreskin tissue in which collagen was implanted intradermally as determined by IHC analysis using human CD-31 signaling assay , with a factor of at least 50% compared to the control sample as described in Example 16 and shown in Table 12.

Species and molecular selectivity [0172] In another aspect of the disclosure, anti-ALK-1 antibodies exhibit both species and molecular selectivity. According to the information in this specification, species or molecular selectivity for an anti-ALK-1 antibody can be determined using methods well known in the art. For example, species selectivity can be determined using Western blot, surface plasmon resonance, e.g. BIAcore, ELISA, immunoprecipitation or RIA.

[0173] In some cases, the anti-ALK-1 antibody binds to KK primate ALK-1, which is at least two times lower than the rodent KK ALK-1. In another case, the KD value for ALK-1 primates is at least 3 times, at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times or at least 1000 times lower than KD for rodent ALK-1, measured by flow cytometry.

[0174] In other cases, the anti-ALK-1 antibody exhibits selectivity for ALK-1 compared to ALK-2 to ALK-7. In some cases, the anti-ALK-1 antibody does not show any significant specific binding to any protein other than ALK-1. Preferably, the anti-ALK-1 antibody binds to ECD of human ALK-1.

Methods for producing antibodies and antibody-producing cell lines

Immunogen ALK-1 [0175] In some cases, the ALK-1 immunogen or antigen is isolated and / or purified ALK-1. In some cases, the ALK-1 immunogen is human ALK-1. In preferred cases, the ALK-1 immunogen is ECD human ALK-1. Human ALK-1, or antigenic parts thereof may be prepared according to methods well known in the art, or may be purchased from commercial sellers. The human ALK-1 amino acid and nucleotide sequences are known (see e.g. Registry at Genbank under accession number L17075). The ACVRL1 gene encoding full length ALK-1 is commercially available from Invitrogen Inc., Clone No. IOH21048. For example, R&amp;D Systems, Inc. sells a recombinant human ALK-1 / Fc chimera (Catalog Number 370-AL) produced by expressing a DNA sequence encoding the amino acid residues 1-118 ECD ALK-1, which DNA sequence was fused to the DNA sequence encoding the human IgG Fc region through a DNA sequence encoding a polypeptide linker in a murine melanoma cell line. The mature recombinant human ALK-1 / Fc chimera is a homodimeric disulfide bonded protein having Asp 22 amino terminus. In addition, Example 1 describes the production of the ECD ALK-1 His-Tag protein that was used to produce hybridomas producing the anti-ALK-1 antibody of the present disclosure.

[0176] In other cases, the ALK-1 antigen is a cell in which ALK-1 is expressed or overexpressed. In other cases, the ALK-1 antigen is a recombinant protein expressed in yeast, insect cells, bacteria such as E. coli, or in other sources using recombinant technology.

Immunization [0177] In some cases, human antibodies are produced by immunization with an ALK-1 antigen of a transgenic non-human animal containing in part or all of its heavy or light chain human immunoglobulin loci in its genome. In a preferred case, the non-human living being is an XENOMOUSE® animal. (Abgenix, Inc., Fremont, CA).

[0178] XENOMOUSE® mice are genetic engineered mouse strains that contain large fragments of the heavy and light chain loci of human immuboglobulins and do not produce murine antibodies. See, e.g., Green et al., Nature Genetics 7: 13-21 (1994) and US Patent Nos. 5,916,771, 5,939598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6114598, 6130364, 6162963 and 6150584. See also WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 / 09560 and WO 00/037504.

[0179] In another aspect, the disclosure provides a method of producing anti-ALK-1 antibodies from non-human living beings and mice, by immunization with the ALK-1 antigen of transgenic living beings that contain human immunoglobulin loci. Such living things can be produced using the methods described in the documents cited above. The methods disclosed in these documents can be modified as described in US Patent 5,994619. U.S. Patent 5,994,619 describes methods for the production of new cultured cells and embryonic node cell lines (CICM) from pigs and cows and transgenic CICM cells into which heterologous DNA has been introduced. CICM transgenic cells can be used to produce cloned transgenic embryos, fetuses and offspring. The '619 patent also describes methods of producing transgenic living beings that are able to pass heterologous DNA to their offspring. In preferred cases of the present disclosure, non-human living entities are mammals, preferably rats, sheep, goats, cattle, horses or chickens.

[0180] XENOMOUSE® mice produce a repertoire of fully human antibodies, such as in adult humans, and produce human antigen-specific antibodies. In some cases, the XENOMOUSE® mouse contains approximately 80% of the repertoire of human antibody V genes by introducing human heavy chain and kappa light chain loci fragments with germline configuration and megase base size in the artificial yeast chromosome (YAC). In other cases, XENOMOUSE® mice further contain approximately the entire locus for the human lambda light chain. See Mendez et al., Nature Genetics 15: 146-156 (1997), Green and Jakobovits, J. Exp. Med. 188: 483-495 (1998) and WO 98/24893.

[0181] In some cases, non-human living entities having human immunoglobulin genes are animals carrying a "minilocus" for human immunoglobulin. In the minilocus approach, the exogenous Ig locus is mimicked by the incorporation of single genes from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, mu constant domain and second constant domain (preferably constant gamma domain) are constructing a construct for introduction into animal. This approach is described, inter alia, in US Patent Nos. 5545807, 5545806, 5569825, 5625126, 5633425, 5661016, 5770429, 5789650, 5814318, 5591669, 5612205, 5721367, 5789215 and 5643763.

[0182] In another aspect, the disclosure provides a method of producing humanized anti-ALK-1 antibodies. In some cases, non-human living beings are immunized with the ALK-1 antigen as described below and under conditions that allow for the production of antibodies. Antibody-producing cells are isolated from living beings, and the nucleic acids encoding the heavy and light chain of a given anti-ALK-1 antibody are isolated from isolated antibody-producing cells or from an immortalized cell line made from such cells. These nucleic acids are then engineered using techniques known to those of skill in the art and further described below to reduce the content of non-human sequences, i.e. humanize the antibody to reduce the immune response in humans. [0183] Immunization of living things can be carried out by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human living creatures such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. field. See, e.g., Harlow and Lane, supra, and US Patent 5,994619. In a preferred case, the ALK-1 antigen is administered with an adjuvant to stimulate the immune response. Examples of adjuvants include Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatory complexes). Such adjuvants may protect the polypeptide from being rapidly dispersed by depositing it in a local bed, or may contain substances that stimulate the host to secrete chemotactic agents on macrophages and other components of the immune system. Preferably, if the polypeptide is administered, the immunization schedule will include two or more administrations of the polypeptide carried out within a few weeks. Example 2 shows a method for producing anti-ALK-1 monoclonal antibodies in XENOMOUSE® mice.

Production of Antibodies and Antibody-Producing Cell Lines [0184] After immunizing the animal with the ALK-1 antigen, the antibodies and / or antibody-producing cells can be obtained from the animal. In some cases, serum containing anti-ALK-1 antibodies is obtained from an animal by bleeding or killing the animal. The serum can be used as it is obtained from the animal, the immunoglobulin fraction can be obtained from the serum or the anti-ALK-1 antibodies can be purified from the serum.

[0185] In some cases, antibody-producing cell lines are generated from cells isolated from the immunized animal. After immunization, the animal is sacrificed and B lymphocytes from the lymph nodes and / or spleen are immortalized in any manner 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 growing them under conditions of selection of immortalized cells, exposing them to carcinogenic or mutagenic compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating the suppressor gene. cancer. See, e.g., Harlow and Lane, above. If fusion with myeloma cells is used, these myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line). The immortalized cells are screened using ALK-1 or part thereof. In a preferred case, the pre-screening is carried out using an enzyme immunoassay (ELISA) or radioimmunoassay. An example of ELISA screening is provided in WO 00/37504.

[0186] Cells producing anti-ALK-1 antibodies, e.g., hybridomas, are selected, cloned and further screened for desired properties, including rapid cell growth, high antibody production, and desired antibody properties, as discussed below. Hybridomas can be further propagated in vivo in syngeneic animals, in animals that do not have an immune system, e.g. in nude mice or in vitro cell culture. Methods for selecting, cloning and propagating hybridomas are well known to those skilled in the art.

[0187] In a preferred case, the immunized animal is a non-human animal that expresses human immunoglobulin genes and splenic B lymphocytes are fused to a myeloma cell line of the same species as the non-human animal. In a more preferred case, the immunized animal is XENOMOUSE® and the myeloma cell line is a non-secretory mouse myeloma cell line. In an even more preferred case, the myeloma cell line is P3-X63Ag8.653 (American Cell Culture Collection). See e.g. Example 2.

[0188] Thus, in one instance, the disclosure provides methods for producing a cell line that produces a human monoclonal antibody or fragment thereof directed against ALK-1, including (a) immunizing the non-human transgenic animal described herein, using ALK-1, a portion of ALK -1 or ALK-1 expressing cell or tissue; (b) enabling a transgenic animal to induce an ALK-1 immune response; (c) isolating the antibody-producing cells from a transgenic live animal; (d) immortalizing antibody producing cells; (e) generating single monoclonal populations of immortalized antibody producing cells; and (f) screening the immortalized antibody-producing cells to identify an anti-ALK-1 antibody. [0189] In another aspect, the disclosure provides a cell line that produces human anti-ALK-1 antibody. In some cases, the cell line is a hybridoma cell line. In some cases, hybridomas are mouse hybridomas as described above. In other cases, hybridomas are produced in a species other than humans and mice, such as rats, sheep, pigs, goats, cattle or horses. In another case, hybridomas are human hybridomas.

[0190] In another case, the transgenic animal is immunized using the ALK-1 antigen, primary cells, e.g., B-cells from spleen or peripheral blood, are isolated from the immunized transgenic animal and individual cells producing antibodies specific for the desired antigen are identified. Polyadenylated mRNA is then isolated from each individual cell and subjected to reverse transcription polymerase chain reaction (RT-PCR) using sense primers that attach to variable domain sequences, e.g. degenerate primers that recognize almost all or all of the FR1 regions of human genes heavy and light chain variable domains, and antisense primers that attach to the constant or linker region sequence. The cDNA of the heavy and light chain variable domains is then cloned and expressed in any suitable host cell, e.g., a myeloma cell, as chimeric antibodies with appropriate immunoglobulin constant regions, such as heavy and κ or λ constant domains. See Babcook, JS et al., Proc. Natl. Acad. Sci. USA 93: 7843-48, 1996.

[0191] Antibodies against ALK-1 can then be identified and isolated as described herein.

[0192] Otherwise, phage display techniques can be used to provide libraries containing antibody repertoire with differentiated affinity for ALK-1. To produce such repertoires, it is not necessary to immortalize B lymphocytes from the immunized animal. Rather, B cells can be used directly as a DNA source. A mixture of cDNA obtained from B lymphocyte, e.g. derived from the spleen, it is used to produce an expression library, for example, a phage display library transfected into E. coli. The obtained cells are tested for immunoreactivity with ALK-1. Techniques for identifying high affinity human antibody libraries are described by Griffiths et al., EMBO J., 13: 3245-3260 (1994); Nissim et al., Ibid, pp. 692-698 and by Griffiths et al., Ibid, 12: 725-734.

[0193] Finally, clones from the library are identified that give binding affinities with the desired strength for the antigen, and the DNA encoding the product responsible for such binding is recovered and subjected to standard recombinant expression. Phage display libraries can also be built using previously changed nucleotide sequences and searched in a similar way. Typically, cDNA encoding the heavy and light chain for production in the phage library is independently provided or combined to form Fv analogs.

[0194] The phage library is then screened for the antibodies with the highest affinities for ALK-1 and genetic material is recovered from the appropriate clone. Subsequent screenings may increase the affinity of the original isolated antibody.

Nucleic acids, vectors, host cells and recombinant methods of producing antibodies

Nucleic acids [0195] The present disclosure also includes nucleic acid molecules encoding anti-ALK-1 antibodies or antigen-binding fragments thereof. In some cases, different nucleic acid molecules encode the anti-ALK-1 immunoglobulin heavy chain and light chain. In other cases, the same nucleic acid molecule encodes the anti-ALK-1 immunoglobulin heavy chain and light chain.

[0196] In some cases, the nucleic acid molecule encoding the light chain variable domain (Vl) uses the human Vk A27, A2, A1, A3, B3, B2, L1 or L2 gene and the human Jk5, Jk1, Jk3 or Jk4 gene. In some cases, the nucleic acid molecule uses the human Vk A27 gene and the human Jk5 gene. In other cases, the nucleic acid molecule uses the human A2 gene and the human Jk1 gene. In some cases, the nucleic acid molecule encoding the light chain encodes an amino acid sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions with respect to the amino acid sequence (s) germ line. In some cases, the nucleic acid molecule comprises a nucleotide sequence that encodes a VL amino acid sequence containing 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 germline Vk and Jk sequences . Substitutions may occur in CDR regions, in framework regions or in the constant domain.

[0197] In some cases, the nucleic acid molecule encodes a VL amino acid sequence containing one or more mutations compared to a germline sequence that is identical to the germline mutations found in the VL of any of 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.

[0198] In some cases, the nucleic acid molecule encodes at least three amino acid substitutions compared to the germline sequence that are identical to germline mutations found in the VL of any of 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. [0199] In some cases, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 encode the VL amino acid sequences of monoclonal antibodies 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.

[0200] In some cases, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 127. In some cases, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3 or a portion thereof. In some cases, the nucleic acid encodes the amino acid sequence of one, two or all three CDRs of the given light chain of the antibody. In some cases, this portion encodes the continuous light chain CDR1-CDR3 region of the anti-ALK-1 antibody.

[0201] In some cases, the nucleic acid molecule encodes a VL amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical with the VL amino acid sequence any of 1.11.1 antibodies; 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, or with the amino acid sequence of the VL region of SEQ ID NO: 4. The nucleic acid molecules of the disclosure include nucleic acids that hybridize under highly stringent conditions as described above, or which are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 identical % or 99% with a nucleic acid coding for the amino acid sequence of the VL region of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 126 or with a nucleic acid comprising the nucleotide sequence of the VL region of SEQ ID NO: 4.

[0202] In other preferred cases, the nucleic acid molecule encodes a heavy chain variable domain (VH) that utilizes the sequence of the human gene VH 431, VH 3-11, VH 3-15, VH 3-33, VH 4-61 or VH 4- 59 or a sequence derived therefrom. In some cases, the nucleic acid molecule uses the human VH 4-31 gene, the human DH6-19 gene and the human JH4B gene.

[0203] In some cases, the nucleic acid molecule encodes an amino acid sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations compared to the amino acid sequence from the germline of human V, D or J genes In some cases, these mutations are in the VH region. In some cases, these mutations are located in the CDR regions.

[0204] In some cases, the nucleic acid molecule encodes a VH sequence containing one or more amino acid mutations compared to a germline VH sequence that is identical to the amino acid mutations found in VH any of monoclonal 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; 5.57.1 ​​or 5.59.1. In some cases, the nucleic acid encodes at least three amino acid mutations compared to germline sequences that are identical to at least three amino acid mutations found in one of the above-mentioned monoclonal antibodies.

[0205] In some cases, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 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.

[0206] In some cases, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of SEQ ID NO: 2; 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104. In various preferred cases, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of SEQ ID NO: 1 or 95. In some cases, this portion contains the VH region, the CDR3 region, all three CDR regions, or the contiguous region comprising CDR1-CDR3.

[0207] In some cases, 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 with the VH amino acid sequence from any of SEQ ID NO: 2; 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104. Nucleic acid molecules of the disclosure include nucleic acids that hybridize under highly stringent conditions such as those described above, or which are at least 70%, 75%, 80%, 85%, 90%, 90%, 95%, 97% identical , 98% or 99% with a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2; 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90, 100 or 104, or with their VH region, or with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 5, 9, 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 with the nucleotide sequence that encodes their VH region.

[0208] In a further case, the nucleic acid encodes the 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 (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; 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 NO: 1 or 95.

[0209] The nucleic acid molecule encoding the heavy or light chain of an anti-ALK-1 antibody or portion thereof can be isolated from any source that produces such an antibody. In various cases, nucleic acid molecules are isolated from B lymphocyte, which expresses an anti-ALK-1 antibody, obtained from a living animal immunized with ALK-1 or from an immortalized cell obtained from such a B lifmocyte. Methods for isolating nucleic acids encoding antibodies are well known in the art. See, e.g., Sambrook J. and Russell D. Molecular Cloning: A Laboratory Manual, ed. 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000). MRNA can be isolated and used to produce cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In a preferred case, the nucleic acid molecule is isolated from a hybridoma, which one of the fusion partners is a cell from a transgenic non-human animal, which cell produces human immunoglobulin. In an even more preferred case, the cell producing human immunoglobulin is obtained from an XENOMOUSE® animal. In another case, the human immunoglobulin producing cell is obtained from a transgenic non-human animal and mouse as described above. In another case, the nucleic acid is isolated from a non-transgenic non-human animal. Nucleic acid molecules isolated from a non-transgenic non-human animal can be used, e.g. to humanized antibodies that contain one or more amino acid sequences from a human anti-ALK-1 antibody of the present disclosure.

[0210] In some instances, the nucleic acid encoding the heavy chain of the anti-ALK-1 antibody of the disclosure may comprise the nucleotide sequence encoding the VH domain of the disclosure linked in-frame to the nucleotide sequence encoding the heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding the light chain of an anti-ALK-1 antibody of the disclosure may comprise a nucleotide sequence encoding a VL domain of the disclosure linked in-frame to a nucleotide sequence encoding a light chain constant domain from any source.

[0211] In another aspect of this disclosure, nucleic acid molecules encoding heavy (VH) and / or light (VL) chain variable domains are "transformed" into full-length antibody genes. In one case, the nucleic acid molecules encoding the VH or VL domains are converted into full-length antibody genes by inserting them into an expression vector already encoding the heavy chain constant domain (CH) or light chain constant domain (CL), respectively, such that the VH segment is operably linked to the CH segment (s) on this vector and / or the VL segment is operably linked to the CL segment on this vector. In another case, nucleic acid molecules encoding the VH and / or VL domains are transformed into full-length antibody genes by joining e.g. by ligation, a nucleic acid molecule encoding the VH and / or VL domains with a nucleic acid molecule encoding the CH domain and / or CL using standard molecular biology techniques. The nucleic acid sequences of human immunoglobulin heavy and light chain constant domain genes are known in the art. See e.g. Kabat et al., Sequences of Proteins of Immunological Interest, ed. 5, NIH Publication No. 91-3242, 1991. Nucleic acid molecules encoding full length heavy and / or light chains can then be expressed from the cell into which the anti-ALK-1 antibody has been introduced and isolated.

[0212] Nucleic acid molecules can be used to recombinantly express large amounts of anti-ALK-1 antibodies. Nucleic acid molecules can also be used to produce chimeric antibodies, dual specificity antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives as described herein below. If the nucleic acid molecules are derived from a non-transgenic non-human animal, these nucleic acid molecules can be used to humanize the antibody, also as described below.

[0213] In a further case, the nucleic acid molecule of the disclosure is used as a PCR probe or primer for a specific antibody sequence. For example, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify DNA regions that can be used, inter alia, to isolate additional nucleic acid molecules encoding anti-ALK-1 antibody variable domains. In some cases, the nucleic acid molecules are oligonucleotides. In some cases, the oligonucleotides are derived from the highly variable heavy and light chain domains of a given antibody. In some cases, the oligonucleotides encode all or part of one or more CDR regions 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; 5.57.1; or 5.59.1 or variants thereof as described herein.

Vectors [0214] The disclosure provides vectors containing nucleic acid molecules encoding the anti-ALK-1 heavy chain of the disclosure or an antigen binding portion thereof. The disclosure also provides vectors containing nucleic acid molecules encoding the light chain of such antibodies or an antigen binding portion thereof. The disclosure further provides vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

[0215] In some cases, the anti-ALK-1 antibodies of the disclosure or antigen-binding portions are expressed by inserting DNA encoding part or all of the light and heavy chains obtained as described above in expression vectors, such that these genes are operably linked to necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YAC, EBV-derived episomes, and the like. The antibody gene is ligated into the vector so that the transcription and translation control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression in the host cell used. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors. In a preferred case, both genes are inserted into the same expression vector. Antibody genes are inserted into the expression vector using standard methods (e.g. by ligation of complementary restriction sites on an antibody gene fragment and on a vector or ligation of blunt ends when no restriction sites are present).

[0216] A convenient vector is one that encodes a functionally complete human immunoglobulin CH or CL sequence with appropriate restriction sites designed so that any VH or VL sequence can be easily inserted and expressed as described above. In such vectors, splicing usually occurs between the donor splice site in the inserted J region and the acceptor splice site preceding the human C domain, as well as in the splice regions that occur in human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0217] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. One of ordinary skill in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the choice of transformed host cell, the desired level of protein expression, etc. Preferred regulatory sequences for expression in a mammalian host cell include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTR, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), virus Simian Virus 40 (SV40) (such as SV40 promoter / enhancer), adenovirus, (e.g. 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 their sequences, see, e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,966,815. Methods for expressing antibodies in plants, including promoters and vectors, as well as plant transformations are known. in the field. See e.g. U.S. Patent 6,517,529.

[0218] Methods for expressing polypeptides in bacterial or fungal cells, e.g., yeast cells, are also well known in the art.

[0219] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., US Patent Nos. 4,392,916, 4,634,665 and 5,179,017). For example, a selectable marker gene typically confers host cell into which the vector has been introduced resistance to drugs such as G418, hygromycin or methotrexate. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells in methotrexate selection / multiplication), the neo gene (for selection on G418), and the glutamate synthetase gene.

Non-hybridoma host cells and methods for recombinant protein production [0220] Nucleic acid molecules encoding anti-ALK-1 antibodies and vectors containing these nucleic acid molecules can be used to transfect a suitable mammalian, plant, bacterial or yeast host cell. Transformation can be accomplished using any known method of introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include transfection with dextran, calcium phosphate precipitation, polybrene transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes and direct DNA microinjection into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells using viral vectors. Cell transformation methods are well known in the art. See e.g. Methods for transforming plant cells are well known in the art and include, e.g., transformation using Agrobacterium, transformation using a gene shotgun, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0221] Mammalian cell lines available as expression hosts are well known in the art and include many immortalized cell lines available from the American Cell Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, Freestyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, newborn hamster kidney cells (BHK), jumbo kidney cells green (COS), human hepatocellular carcinoma cells (e.g. Hep G2), A549 cells and several other cell lines. Particularly preferred cell lines are selected by determining which cell lines are highly expressed. Other cell lines that can be used are insect cell lines, 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 sufficient time to allow expression of the antibody in the host cells 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., Nicotiana, Arabidopsis, eyelash, maize, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

[0222] In addition, the expression of the antibodies of the disclosure from the cell lines producing them can be increased using several known techniques. For example, the glutamate synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in full or in part in connection with European patents Nos. 0216846, 0256055, 0323997 and 0338841. [0223] It is possible that antibodies expressed by different cell lines or transgenic living things will differ from each other by glycosylation. However, all antibodies encoded by the nucleic acid molecules provided herein or including the amino acid sequences provided herein are part of the present disclosure regardless of the glycosylation of the antibodies.

Transgenic Living Animals and Plants [0224] The anti-ALK-1 antibodies of the disclosure can also be made transgenically by creating a mammal or plant that is transgenic in terms of the heavy and light chain sequences of a given immunoglobulin and producing the antibody in a recoverable form therefrom. Due to transgenic production in mammals, anti-ALK-1 antibodies can be made and recovered from the milk of goats, cows and other mammals. See e.g. U.S. Patent Nos. 5827690, 6756687, 5750172 and 5741957.

[0225] In some cases, transgenic non-human living creatures that contain human immunoglobulin loci are immunized with ALK-1 or an immunogenic portion thereof, as described above. Methods for producing antibodies in plants are described, e.g., in US Patent Nos. 6046037 and 5,959177.

[0226] In some instances, transgenic non-human living creatures are produced by introducing one or more nucleic acid molecules encoding an anti-ALK-1 antibody of the disclosure into that living creature or plant using standard transgenic techniques. See Hogan and United States Patent 6,414,29, above. The transgenic cells used to create the transgenic animal can be embryonic stem cells or somatic cells or fertilized egg cells. Non-human transgenic organisms may be chimeric, non-chimeric heterozygous and non-chimeric homozygous. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, 2. ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999). In some cases, transgenic non-human living entities have a targeted interruption or replacement of the target construct encoding the given heavy chain and / or light chain. In a preferred case, the transgenic living entities contain and express nucleic acid molecules encoding heavy and light chains that specifically bind to ALK-1, preferably human ALK-1. In some cases, the transgenic live creatures contain nucleic acid molecules encoding the modified antibody, such as a single chain antibody, a chimeric antibody, or a humanized antibody. Antibodies against ALK-1 can be produced in any transgenic animal. In a preferred case, the non-human living creatures are mice, rats, sheep, pigs, goats, cattle or horses. A non-human transgenic living being expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other body fluids.

Phage display libraries [0227] The disclosure provides a method of producing an anti-ALK-1 antibody or antigen-binding portion thereof comprising the steps of synthesizing a human phage antibody library, screening this library using ALK-1 or an antigen-binding portion thereof, isolating the phage that binds ALK-1 and obtain antibodies from this phage. By way of example, one method of producing an antibody library for use in phage display techniques includes the steps of immunizing a non-human animal containing a human immunoglobulin loci using ALK-1 or its antigenic portion to elicit an immune response, extracting antibody-producing cells from immunized animal, RNA isolation encoding heavy and light antibody chains of the disclosure from extracted cells, reverse transcribing RNA to generate cDNA, amplifying cDNA using primers, and inserting cDNA into a phage display vector so that the antibodies are expressed on that phage. In this way, recombinant anti-ALK-1 antibodies of the disclosure can be obtained.

[0228] Recombinant human anti-ALK-1 antibodies of the disclosure can be isolated by screening a recombinant combinatorial antibody library. Preferably, this library is a scFv phage display library generated using human VL and VH cDNA generated from mRNA isolated from B cells. Methods for producing and screening such libraries are known in the art. Sets for creating phage display libraries are commercially available (e.g. Pharmacia Recombinant Phage Antibody System, catalog numbers 27-9400-01; and presentation set on the Stratagene SurfZAP phage^™, catalog number 240612). There are also other methods and reagents that can be used to generate and screen antibody display libraries (see, e.g., US Patent No. 5,223,409; PCT Publication No. 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 et al., Hum. Antibod. Hybridomas 3: 81-85 (1992); Huse et al., Science 246: 1275-1281 (1989); McCafferty et al., Nature 348: 552-554 (1990); Griffiths et al., EMBO J. 12: 725-734 (1993); Hawkins et al., J. Mol. Biol. 226: 889-896 (1992); Clackson et al., Nature 352: 624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA 89: 3576-3580 (1992); Garrad et al., Bio / Technology 9: 1373-1377 (1991); Hoogenboom et al., Nuc. Acid Res. 19: 4133-4137 (1991); and Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991).

[0229] In one case, to isolate and produce human anti-ALK-1 antibodies with the desired properties, the human anti-ALK-1 antibody described herein is first used to select human heavy and light chain sequences with similar ALK-1 binding activity, using epitope imprinting methods described in PCT Publication No. WO 93/06213. The antibody libraries used in this method are preferably scFv libraries generated 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 (1993). The scFv antibody libraries are preferably screened using human ALK-1 as the antigen.

[0230] After pre-selection of human VL and VH domains, "mixing and matching" experiments are performed in which different pairs of preselected VL and VH segments are screened for ALK-1 binding to select preferred combinations of VL / VH pairs. In addition, to further improve the quality of the antibody, the VL and VH segments from the preferred VL / VH pair (s) can be randomly mutated, preferably in the CDR3 region of VH and / or VL, by a process analogous to the in vivo somatic mutation process responsible for maturation affinity of antibodies during a natural immune response. This in vitro affinity maturation can be achieved by propagating the VH and VL domains using PCR primers complementary to CDR3 with VH or CDR3 with VL, respectively, which primers were "labeled" with a random mixture of four nucleotide bases at certain positions so that the resulting PCR products encode segments VH and VL that have been randomly mutated in the VH and / or VL CDR3 regions. These randomly mutated VH and VL segments can be screened again for ALK-1 binding.

[0231] After screening and isolation of the anti-ALK-1 antibody of the disclosure from a recombinant immunoglobulin display library, nucleic acids encoding the selected antibody can be recovered from the display kit (e.g., from the phage genome) and cloned into other expression vectors using standard DNA combination re49 techniques . If desired, the nucleic acid may be further maipulated to create other forms of the antibody of the disclosure as described below. To express a recombinant human antibody isolated by screening a combinatorial library, DNA encoding the antibody is cloned into a recombinant expression vector and introduced into mammalian host cells as described above.

Deimmunized Antibodies [0232] In another aspect of the disclosure, the antibody can be deimmunized to reduce its immunogenicity using techniques described in, e.g., PCT Publication Nos. WO98 / 52976 and WO00 / 34317.

Mutant Antibodies [0233] In another instance, the nucleic acid molecules, vectors and host cells can be used to generate mutated anti-ALK-1 antibodies. Antibodies can be mutated in the heavy and / or light chain variable domains e.g. to change the binding properties of the antibody. For example, the mutation may be introduced in one or more CDR regions to increase or decrease the KD of the ALK-1 antibody, to increase or decrease the koff, or to change the binding specificity of the antibody. Techniques of site-directed mutagenesis are well known in the art. See, e.g., Sambrook et al. and Ausubel et al., supra. Otherwise, one or more mutations are introduced at an amino acid residue that is known to be altered 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 (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. Mutations can be introduced in the CDR region or in the variable region framework region or in the constant domain. In a preferred case, the mutations are introduced in the variable domain. In some cases, one or more mutations are introduced at an amino acid residue that is known to be altered compared to the germline in the CDR or framework region of the variable domain with the amino acid sequence of 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 nucleotide sequence is depicted 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.

[0234] In another case, the framework is mutated such that the obtained framework (s) has (have) an amino acid sequence corresponding to the germline gene sequence. The mutation can be introduced in the 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. A mutation may also be introduced into the framework or constant domain to change the immunogenicity of the antibody, provide a site for covalent or non-covalent binding of another molecule, or change properties such as complement binding, FcR binding, and antibody dependent cellular cytotoxicity (ADCC). According to the disclosure, one antibody may contain mutations in any one or more of the CDRs or framework regions of the variable domain or constant domain.

[0235] In some cases, there are from 1 to 13, including any number in this range, of amino acid mutation in the VH or VL domains of the mutant anti-ALK-1 antibody compared to the anti-ALK-1 antibody before the mutation. In any of the above, mutations may occur in one or more CDR regions. In addition, each of the mutations may be a conservative amino acid substitution. In some cases, no more than 5, 4, 3, 2 or 1 amino acid changes occur in the constant domains.

Modified Antibodies [0236] In a further case, a fusion antibody or immunoadhesin may be prepared that comprises all or part of the anti-ALK-1 antibody of the disclosure, linked to another polypeptide. In a preferred case, only the variable domains of the anti-ALK-1 antibody are linked to the polypeptide. In another preferred case, the VH domain of the anti-ALK-1 antibody is fused to the first polypeptide, while the VL domain of the anti-ALK-1 antibody is fused to the second polypeptide that interacts with the first polypeptide in such a way that these VH and VL domains can combine with each other interact to form an antigen binding site. In another preferred case, the VH domain is separated from the VL domain by a linker, so that the VH and VL domains can interact with each other (see single chain antibodies below). The VH-linker VL antibody is then linked to the given polypeptide. In addition, fusion antibodies can be prepared in which two (or more) single chain antibodies are linked to each other. This is useful when there is a need to produce a divalent or multivalent antibody on a single polypeptide chain or to produce a dual specificity antibody.

[0237] To generate a single chain antibody (scFv), DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser) 3, so that the VH and VL sequences can be expressed as continuous single chain protein, with VL and VH domains connected by a flexible linker. See, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); McCafferty et al., Nature 348: 552-554 (1990). A single chain antibody can be monovalent when only single VH and VL are used, divalent when two VH and VL are used, or multivalent when more than two VH and VL are used. Bispecific or multivalent antibodies can be generated that bind specifically to ALK-1 and to another molecule.

[0238] In other cases, other modified antibodies can be produced using nucleic acid molecules encoding anti-ALK-1 antibody. For example, "standard kappa bodies" (III et al., Protein Eng. 10: 949-57 (1997)), "mini-bodies" (Martin et al., EMBO J. 13: 5303- 9 (1994)), "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)) or "Januzine" (Traunecker et al., EMBO J. 10: 3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7: 51-52 (1992) as described in this specification.

[0239] Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods, including by hybridoma fusion or combination of Fab 'fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In addition, dual specificity antibodies can be formed in the form of "diabodies" or "janazines". In some cases, the dual specificity antibody binds to two different epitopes of ALK-1. In some cases, the dual specificity antibody has a first heavy chain and a first light chain 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 heavy chain and light chain of antibody. In some cases, this additional heavy chain and light chain may also be in the form of one of the abovementioned monoclonal antibodies, but different from the first heavy and light chain.

[0240] In some cases, the above-described modified antibodies are produced using one or more variable domains or CDRs from the human anti-ALK-1 monoclonal antibody provided herein.

Derivatized and labeled antibodies [0241] The anti-ALK-1 antibody or antigen binding portion of the disclosure can be derivatized or combined with another molecule (e.g., another peptide or protein). Generally, antibodies or parts thereof are derivatized so that derivatization or unpacking does not adversely affect ALK-1 binding. Accordingly, the antibodies and antibody portions of the disclosure are intended to include both intact and modified forms of the human anti-ALK-1 antibodies described herein. For example, the antibody or antibody portion of the disclosure can be operably linked (via chemical conjugation, genetic fusion, non-covalent binding or otherwise) to one or more other molecules, such as another antibody (e.g. dual specificity antibody or diabody), detection agent, pharmaceutical agent and / or protein or peptide that can mediate the binding of the antibody or antibody portion to another molecule (such as streptavidin core region or polyhistidine tag).

[0242] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types, e.g., to generate dual specificity antibodies). Suitable crosslinking agents include those that are heterodifunctional, containing two separate reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homodifunctional (e.g., disuccinimidyl subperate). Such linkers are available from Pierce Chemical Company, Rockford, II.

[0243] Another type of derivatized antibody is labeled antibody. Useful detection agents that can be derivatized with the antibody or antigen binding portion thereof of the disclosure include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. The antibody can also be labeled with enzymes 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 this enzyme uses to produce a reaction product that can be recognized. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product that is detectable. The antibody can also be labeled with biotin and detected in indirect measurement of avidin or streptavidin binding. The antibody can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g. leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, marker epitopes). In some cases, markers are attached through spacer arms of varying lengths to reduce potential spatial hindrance.

[0244] The anti-ALK-1 antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), methyl or ethyl or carbohydrate. These groups are useful for improving the biological properties of an antibody, e.g., for extending serum half-life.

Pharmaceutical compositions and administration [0245] The disclosure also relates to a pharmaceutical composition for the treatment of conditions associated with undesirably increased angiogenesis in a mammal, including a human, comprising such an amount of an anti-ALK-1 antibody or antigen-binding portion thereof as described herein that is effective in the treatment of such conditions, and a pharmaceutically acceptable carrier.

[0246] The antibodies and antigen binding portions of the present disclosure can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibody or antigen binding portion thereof of the disclosure and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Additional examples of pharmaceutically acceptable carriers are wetting agents or small amounts of excipients such as wetting or emulsifying agents, preservatives or buffers that increase the shelf life or effectiveness of the antibody.

[0247] The compositions of this disclosure may be in a variety of forms, for example liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., solutions for injection and infusion), 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 solutions for injection or infusion, such as compositions similar to those used for passive immunization of humans. Parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular) administration is the preferred method of administration. In a preferred case, the antibody is administered by intravenous infusion or injection. In another preferred case, the antibody is administered by intramuscular or subcutaneous injection. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or multi-dose containers, with or without the addition of preservatives. The compositions may also be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active substance may be in the form of a powder to be reconstituted in a suitable carrier, e.g. in sterile pyrogen-free water before use.

[0248] 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 other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by introducing the anti-ALK-1 antibody in the required amount in a suitable solvent with one of or a combination of the ingredients listed above as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active substance into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods for their preparation are vacuum drying and freeze drying, which provide the powder with the active substance and any additional desired ingredient from its solution previously sterilized by filtration. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable compositions can be achieved by including in the composition an absorption delaying agent, e.g., monostearate and gelatin salts.

[0249] The antibodies or portions of the antibodies of the present disclosure can be administered in a variety of ways known in the art, however, for many therapeutic applications, the preferred route / method of administration is subcutaneous, intramuscular or intravenous infusion. As will be appreciated by one of skill in the art, the route and / or method of administration will vary depending on the results desired.

[0250] In some cases, the antibody compositions of the present disclosure can 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 biocompatible polymers may be used, such as vinyl acetate / ethylene, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and poly lactic acid. Many methods for preparing such preparations are well known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0251] Additional active compounds can also be incorporated into the composition. In some cases, the inhibitory anti-ALK-1 antibody of the disclosure is co-formulated and / or co-administered with one or more additional therapeutic agents. These agents include, but are not limited to, antibodies that bind to other target molecules, anti-cancer agents, anti-angiogenic agents, signal transduction inhibitors, antiproliferative agents, chemotherapeutic agents, or peptide analogs that inhibit anti-ALK-1. Such combination therapies may require lower doses of inhibitory anti-ALK-1 antibody as well as co-administered agents, avoiding possible toxicity or complications associated with various monotherapies.

[0252] As noted above, the compositions of the present disclosure may optionally additionally contain a pharmaceutically acceptable antioxidant in addition to the chelating agent. Suitable antioxidants include, 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, especially about 27 mM. For example, the aqueous preparation may contain: 10 mg / ml anti-ALK1 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-methionine.

[0253] The compositions of the disclosure may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen binding portion of the disclosure. "Therapeutically effective amount" refers to the effective amount at the doses and periods of time necessary to achieve the desired therapeutic effect. The therapeutically effective amount of the antibody or antigen-binding portion may vary depending on factors such as disease stage, age, sex and weight of the subject, and the ability of the antibody or antibody portion to elicit the desired response in the subject. A therapeutically effective amount is also the amount at which any toxic or harmful effects of the antibody or antigen-binding portion are overcome by the therapeutically beneficial effects. "Prophylactically effective amount" refers to the effective amount at the doses and periods of time necessary to achieve the desired preventive effect. Typically, because the prophylactic dose is used in patients before or at the onset of the disease, the prophylactically effective amount may be lower than the therapeutically effective amount.

[0254] Dosage regimens can be adjusted to provide the optimal desired response (e.g., therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses over a period of time, or the dose may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It is particularly preferred to formulate parenteral compositions in dosage unit form to facilitate administration and to harmonize the dose. Dosage unit forms as used herein refer to physically discrete units suitable as unitary dosages for the treated mammalian subjects; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for unit dosage forms of the disclosure is dictated by and directly dependent on (a) the unique characteristics of the anti-ALK-1 antibody or portion thereof, and the specific therapeutic or prophylactic effect to be achieved, and (b) associated limitations in the art regarding the production of such antibodies for treating sensitivity in individuals. [0255] An exemplary, non-limiting therapeutic or prophylactic effective amount of the antibody or antibody portion of the disclosure 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 cases, the preparation contains 5 mg / ml of antibody in a buffer based on 20 mM sodium citrate, pH 5.5, 140 mM NaCl and 0.2 mg / ml polysorbate 80. It should be noted that dose values ​​may vary with the type and severity of the condition to be alleviated. It should further be understood that for any particular individual, specific modes of administration should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the composition, and that the dose ranges provided herein are exemplary only and do not limit the scope or practical use of the claimed composition .

[0256] A further aspect of the present disclosure provides kits comprising an anti-ALK-1 antibody or antigen binding portion of the disclosure or a composition comprising such an antibody or portion. The kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents. The kit may also contain instructions for use in a diagnostic or therapeutic method. In a preferred case, the kit comprises the antibody or composition containing it and a diagnostic agent that can be used in the method described below. In another preferred case, the kit comprises the antibody or a composition containing it and one or more therapeutic agents that can be used in the method described below.

Diagnostic methods of use [0257] Anti-ALK-1 antibodies or antigen-binding portions thereof can be used in diagnostic methods for detecting ALK-1 in a biological sample in vitro or in vivo. For example, anti-ALK-1 antibodies can be used in traditional immunoassays including, but not limited to, ELISA, RIA, flow cytometry, tissue immunohistochemistry, Western blot or immunoprecipitation. The anti-ALK-1 antibodies of the disclosure can be used to detect ALK-1 in humans. Antibodies against ALK-1 can also be used to detect ALK-1 in other primates, e.g. cynomolgus macaque.

[0258] The disclosure further provides a method of detecting ALK-1 in a biological sample comprising contacting said biological sample with an anti-ALK-1 antibody of the disclosure and detecting bound antibody. In one case, the anti-ALK-1 antibody is directly labeled with a detectable label. In another case, the anti-ALK-1 antibody (first antibody) is not labeled, and the second antibody or other molecule that can bind to the anti-ALK-1 antibody is labeled. As is well known to those skilled in the art, the second antibody is selected to be able to specifically bind to a particular type and class of the first antibody. For example, when the anti-ALK-1 antibody is human IgG, then the secondary antibody may be an anti-human IgG antibody. Other molecules that can bind to antibodies include, but are not limited to, Protein A and Protein G, both of which are commercially available, e.g. from Pierce Chemical Co. [0259] Suitable labels for the antibody or secondary antibody have previously been discussed and 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 / biotin and avidin / biotin; examples of suitable fluorescent materials include umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansyl chloride or phycoerythrin; an example of luminescent material includes luminol; and examples of suitable radioactive materials include ¹²⁵AND, ¹³¹AND, ³⁵Wedding ³H.

[0260] In other instances, ALK-1 can be tested in a biological sample in a competition immunoassay using ALK-1 standards labeled with a detectable substance and unlabeled anti-ALK-1 antibody. In this test, the biological sample, labeled ALK-1 standards and anti-ALK-1 antibody are combined and the amount of labeled ALK-1 pattern associated with 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.

[0261] The immunoassays disclosed above can be used for many different purposes. For example, anti-ALK-1 antibodies can be used to detect ALK-1 in cultured cells. In a preferred case, anti-ALK-1 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 the test compound for some time, while the other sample is left untreated. If total ALK-1 levels are to be measured, cells are lysed and total ALK-1 levels are measured using one of the immunoassays described above. The total ALK-1 level in the treated cells is compared to untreated cells to determine the effect of the test compound.

[0262] A preferred immunoassay for measuring total ALK1 levels is flow cytometry or immunohistochemistry. Methods such as ELISA, RIA, flow cytometry, Western blot, immunohistochemistry, labeling of membrane-integrated proteins on the surface of cells and immunoprecipitation are well known in the art. See, e.g., Harlow and Lane, above. In addition, immunoassays can be adapted for high throughput screening to test a large number of compounds for activation or inhibition of ALK-1 expression.

[0263] Anti-ALK-1 antibodies of the disclosure can also be used to determine ALK-1 levels in tissue or cells derived from that tissue. In some cases, the tissue is diseased tissue. In some cases of this method, tissue or biopsy material is collected from a patient. The biopsy tissue or material is then used in an immunoassay to determine, e.g., total ALK-1 levels or ALK-1 localization by the methods discussed above.

[0264] Antibodies of the present disclosure can also be used in vivo to identify tissues and organs that express ALK-1. One of the benefits of using human anti-ALK-1 antibodies of the present disclosure is that they can be safely used in vivo without eliciting a substantial immune response to this antibody after administration, as opposed to non-human or humanized or chimeric antibodies.

[0265] The method includes the steps of administering detectably labeled anti-ALK-1 antibodies or compositions containing them to a patient in need of such diagnostic testing and subjecting said patient to imaging analysis to determine the location of ALK-1 expressing tissues. Imaging analysis is well known in the medical field and includes, but is not limited to, 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 gadolinium chelate, which can be used for MRI or CT. Other labeling agents include, but are not limited to, radioisotopes such as⁹⁹Tc. Otherwise, the anti-ALK-1 antibody will not be labeled and will be imaged by the administration of a second antibody or other molecule that is detectable and can bind to the anti-ALK-1 antibody. In one case, biopsy material is taken from the patient to determine if the tissue of interest expresses ALK-1.

Therapeutic methods of use [0266] In a further instance, the disclosure provides a method of inhibiting ALK1 activity by administering an anti-ALK-1 antibody to a patient in need thereof. Any of the antibodies or antigen binding portions described herein can be used therapeutically. In a preferred case, the anti-ALK-1 antibody is human, chimeric or humanized. In another preferred case, the anti-ALK-1 antibody is a human antibody and the patient is a human patient. Alternatively, the patient may be a mammal that expresses ALK-1 with which the anti-ALK-1 antibody cross-reacts. The antibody can be administered to a non-human mammal expressing ALK-1 with which the antibody cross-reacts (e.g., Cynomolgus macaque) for veterinary purposes or as an animal model of human disease. Such animal models may be useful for assessing the therapeutic efficacy of the antibodies of this disclosure.

[0267] Otherwise, the anti-ALK-1 antibody or binding member thereof can be administered to a patient who expresses abnormally high levels of ALK-1. The antibody can be administered once, but multiple administration is more preferable. The antibody can be administered from three times a day to once every six months or more. Administration can be carried out three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once every three months and once for six months. The antibody can also be administered continuously using a minipump. The antibody can be administered by the mucosal, transbuccal, intranasal, inhalational, intravenous, subcutaneous, intramuscular, parenteral or tumor routes. The antibody may be administered once, at least twice, or for at least the period during which the condition is cured, relieved or cured. The antibody will usually be administered as long as the condition persists. The antibody will typically be administered as part of the pharmaceutical composition described above. Dosage of the 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. Serum antibody concentration can be measured by any method known in the art. [0268] In one case, the antibody is administered as a sterile aqueous solution with a pH in the range of from about 5.0 to about 6.5, containing from about 1 mg / mL to about 200 mg / mL of antibody, from about 1 millimole to about 100 millimoles of histidine buffer, from about 0.01 mg / ml to about 10 mg / ml polysorbate 80, from about 100 millimoles to about 400 millimoles of trehalose, and from about 0.01 millimoles to about 1.0 millimoles of EDTA disodium salt dihydrate .

[0269] The present disclosure further contemplates administering any of the compositions described herein to a patient susceptible to or suffering from a condition associated with increased angiogenesis ("angiogenic condition").

[0270] Examples of angiogenic conditions that can be treated or prevented using the compositions / methods of the present disclosure include, but are not limited to, cancer (both solid and hematological), age-related macular degeneration (AMD), developmental abnormalities (organogenesis), diabetes-related blindness, endometriosis, ocular neovascularization, psoriasis, rheumatoid arthritis (RA) and skin discoloration (e.g. hemangioma, vascular flat or simple nevus).

[0271] For example, the present disclosure relates to methods of treating or preventing conditions associated with ocular neovascularization using any of the compositions / methods described herein. Conditions associated with ocular neovascularization include, but are not limited to, diabetic retinopathy, age-related macular degeneration ("ARMD"), rubeosis glaucoma, interstitial keratitis, premature retinopathy, ischemic retinopathy (e.g. sickle cell), pathological myopia, ocular histoplasmosis, pterygium, punctiform internal choroidopathy and the like.

Treatment of abnormal cell growth [0272] This disclosure also relates to a method of treating 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 for the treatment of abnormal cell growth.

[0273] In one case of this method, the abnormal cell growth is a cancer including, but not limited to, mesothelioma, hepatobiliary carcinoma (liver and bile duct cancer), primary or secondary CNS tumor, primary or secondary brain tumor, lung cancer ( NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal (stomach) cancer. large intestine and duodenum), breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, cancer adrenal gland sarcoma, urethral cancer, penis cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myelogenous leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) cancers, primary CNS lymphoma, non-Hodgkin's lymphoma, spinal tumors, brain stem glioma, pituitary adenoma, adrenal cortex carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroma a combination of one or more of the above cancers.

[0274] In a preferred case of the present disclosure, the cancer is selected from lung cancer (NSCLC and SCLC), head or neck cancer, ovarian cancer, colon cancer, rectal cancer, rectal cancer, stomach cancer, breast cancer, kidney or ureter cancer, cancer renal cell carcinoma, pelvic carcinoma, central nervous system (CNS) cancers, primary CNS lymphoma, non-Hodgkin's lymphoma, spinal tumors, or a combination of one or more of the above tumors.

[0275] In another preferred case of the present disclosure, the cancer is selected from lung cancer (NSCLC and SCLC), ovarian cancer, colon cancer, rectal cancer, anal cancer or a combination of one or more of the above cancers.

[0276] In another case of said method, said abnormal cell growth is benign proliferative disease, including but not limited to psoriasis, benign prostatic hyperplasia or restenosis.

[0277] This disclosure also relates to a method of treating 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 mitosis inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifying compounds, antibodies, cytotoxics, antihormones and antiandrogens.

[0278] The disclosure also relates to a pharmaceutical composition for the treatment of abnormal cell growth in a mammal, including a human, that comprises such 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 in combination with a pharmaceutically acceptable carrier and an anti-cancer agent selected from the group consisting of mitosis inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifying compounds, antihormones and antiandrogens.

[0279] The disclosure also relates to a method of treating 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-cancer agent selected from the group consisting of antiproliferative agents. kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-I inhibitors, cox-II inhibitors, mitosis inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, irradiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifying compounds, antibodies, cytotoxics, antihormones, statins and antiandrogens.

[0280] In one instance of the present disclosure, the anti-cancer agent used in combination with the anti-ALK-1 antibody or antigen-binding portion thereof and in the pharmaceutical compositions described herein is an anti-angiogenic agent, a pan-kinase inhibitor or a growth factor inhibitor. Preferred pan kinase inhibitors include Sutent (Pfizer Inc., SU-11248), described in US Patent No. 6,572,393 (Pfizer, Inc, NY, USA).

[0281] Anti-angiogenic agents include, but are not limited to, the following agents such as EGF inhibitor, EGFR inhibitors, VEGF inhibitors, VEGFR inhibitors, TIE2 inhibitors, IGF1R inhibitors, COX-II inhibitors (cyclooxygenase II), MMP-2 inhibitors (matrix metalloproteinases) 2) and MMP-9 (extracellular matrix metalloproteinase 9) inhibitors. Preferred VEGF inhibitors include, for example, Avastin (bevacizumab), a monoclonal anti-VEGF antibody from Genentech, Inc. from South San Francisco, California.

[0282] Additional VEGF inhibitors include CP-547,632 (Pfizer Inc., NY, USA), Axitinib (Pfizer Inc .; AG-013736), ZD-6474 (AstraZeneca), AEE788 (Novartis), AZD-2171), VEGF Trap (Regeneron, / Aventis), Vatalanib (also known as PTK-787, ZK-222584: Novartis &amp; Schering AG), Macugen (octasodium pegaptanib, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862 ( Cytran Inc. from Kirkland, Washington, USA); and angiozyme, synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California) and combinations thereof. VEGF inhibitors useful in the practical application of the present disclosure are disclosed in US Patent Nos. 6,534,524 and 6,235,764.

[0283] Particularly preferred VEGF inhibitors include CP-547,632, AG13736, Vatalanib, Macugen and combinations thereof.

[0284] Additional VEGF inhibitors are described, for example, in WO 99/24440 (published May 20, 1999), WO 1999/062890 (published December 9, 1999), in WO 95/21613 (published August 17, 1995) , WO 99/61422 (published December 2, 1999), United States Patent No. 6,535,324 (discloses AG13736), United States Patent No. 5,834,504 (filed November 10, 1998), WO 98/50356 (published November 12, 1998) . U.S. Patent No. 5,883,113 (filed March 16, 1999), U.S. Patent No. 5,886,020 (filed March 23, 1999), U.S. Patent No. 5,797,83 (filed August 11, 1998), U.S. Patent No. 6,653,308 (filed 25 November 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 on December 3, 1998), WO 98/02438 (published on January 22

1998 r.), WO 99/16755 (published April 8, 1999) and WO 98/02437 (published January 22, 1998).

[0285] Other antiproliferative agents that can be used with the antibodies or antigen binding portions thereof of the present disclosure include farnesyl protein transferase enzyme inhibitors and PDGFr receptor tyrosine kinase inhibitors, including compounds disclosed and claimed in United States Patent Nos. 6080769; 6194438; 6258824; 6586447; 6071935; 6495564 and 6150377.

[0286] For additional PDGRr inhibitors see WO01 / 40217, published July 7, 2001 and

WO2004 / 020431, published March 11, 2004. Preferred PDGFr inhibitors include

CPizer and its pharmaceutically acceptable salts CP-868,596.

[0287] Preferred GARF inhibitors include Pfizer's AG-2037 (pelitrexol and its pharmaceutically acceptable salts). GARF inhibitors useful in the practical application of the present disclosure are disclosed in US Patent No. 5,608,082.

[0288] Examples of useful COX-II inhibitors that can be used in combination with the anti-ALK-1 antibody or antigen-binding portion thereof as described herein and in the 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-sulfamoylphenyl) -1H-pyrrole, 2- (4-Ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H- pyrrole, T-614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125 and Arcoxia (etoricoxib). For additional COX-II inhibitors, see US Patent Application Publication Nos. 2005-0148627 and 2005-0148777.

[0289] In one preferred case, the anti-cancer agent is celecoxib, see U.S. Patent No. 5,466,823. The structure of celecoxib is shown below:

<img file="PL2447283T3_D0001.tif" />

[0290] In one preferred case, the anti-cancer agent is valecoxib, see US Patent No. 5633272. The structure of valdecoxib is shown below:

<img file="PL2447283T3_D0002.tif" />

[0291] In one preferred case, the anti-cancer agent is parecoxib, see U.S. Patent No. 5,932,598. The structure of parecoxib is shown below:

<img file="PL2447283T3_D0003.tif" />

[0292] In one preferred case, the anti-cancer agent is deracoxib, see US Patent No. 5,521,207. The structure of deracoxib is shown below:

<img file="PL2447283T3_D0004.tif" />

[0293] In one preferred case, the anti-cancer agent is SD-8381, see US Patent No. 6,034,256. The structure of SD-8381 is shown below:

<img file="PL2447283T3_D0005.tif" />

[0294] In one preferred case, the anti-cancer agent is ABT-963, see International Publication Number WO 2002/24719.

[0295] The structure of ABT-963 is shown below:

<img file="PL2447283T3_D0006.tif" />

[0296] In one preferred case, the anti-cancer agent is rofecoxib, as shown below:

<img file="PL2447283T3_D0007.tif" />

[0297] In one preferred case, the anti-cancer agent is MK-663 (etoricoxib), see international publication number WO 1998/03484.

[0298] The structure of etoricoxib is shown below:

<img file="PL2447283T3_D0008.tif" />

[0299] In one preferred case, the anti-cancer agent is COX-189 (lumiracoxib), see International Publication Number WO 1999/11605.

[0300] The structure of lumiracoxib is shown below:

<img file="PL2447283T3_D0009.tif" />

[0301] In one preferred case, the anti-cancer agent is BMS347070, see U.S. Patent No. 6,185,651. The structure of BMS-347070 is shown below:

<img file="PL2447283T3_D0010.tif" />

[0302] In one preferred case, the anti-cancer agent is NS-398 5 (CAS 123653-11-2). The structure of NS-398 (CAS 123653-11-2) is shown below:

<img file="PL2447283T3_D0011.tif" />

[0303] In one preferred case, the anti-cancer agent is RS 57067 (CAS 17932-91-3). The structure of RS 57067 (CAS 17932-91-3) is shown below:

<img file="PL2447283T3_D0012.tif" />

[0304] In one preferred case, the anti-cancer agent is 4-methyl2- (3,4-dimethylphenyl) -1- (4-sulfamoylphenyl) -1H-pyrrole. The structure of 4-methyl-2- (3,4-dimethylphenyl) -1- (4-sulfamoylphenyl) -1H-pyrrole is shown below:

<img file="PL2447283T3_D0013.tif" />

[0305] In one preferred case, the anti-cancer agent is 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole. The structure of 2- (4-ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole is shown below:

<img file="PL2447283T3_D0014.tif" />

[0306] In one preferred case, the anti-cancer agent is meloxamate. The structure of meloxicam is shown below:

<img file="PL2447283T3_D0015.tif" />

[0307] Other inhibitors useful as anti-cancer agents used in combination with the antibodies of the present disclosure and in the pharmaceutical compositions described herein include aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit the prostaglandin-forming enzyme (cyclooxygenase I and II), resulting in lower levels of prostaglandins. include, but are not limited to, Salsalat (Amigesic), Diflunizal (Dolobid), Ibuprofen (Motrin), Ketoprofen (Orudis), Nabumeton (Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn), Diclofenac (Voltaren), Indomethacin (Indocin), Sulindak (Clinoril), Tolmetin (Tolectin), Etodolak (Lodine), Ketorolak (Toradol), Oxaprozin (Daypro) and their combinations. Preferred COX-I inhibitors include ibuprofen (Motrin), nuprin, naproxen (Aleve), indomethacin (Indocin), nabumetone (Relafen) and combinations thereof. [0308] Targeted agents used in combination with the anti-ALK-1 antibody or antigen-binding portion thereof and with pharmaceutical compositions containing them as described herein include EGFr inhibitors such as Iressa (gefitinib, AstraZeneca), Tarceva (erlotinib or OSI- 774, OSI Pharmaceuticals Inc.), Erbitux (cetuximab, Imclone Pharmaceuticals, Inc.), EMD-7200 (Merck AG), ABX-EGF (Amgen Inc. and Abgenix Inc.), HR3 (Cuban Government), IgA antibodies (University of Erlangen-Nuremberg), TP-38 (IVAX), EGFR fusion proteins, EGF vaccine, anti-EGFr immunoliposomes (Hermes Biosciences Inc.) and combinations thereof.

[0309] Preferred EGFr inhibitors include Iressa, Erbitux, Tarceva and combinations thereof.

[0310] The present disclosure also relates to anti-cancer agents selected from pan erb receptor inhibitors or ErbB2 receptor inhibitors such as CP724,714 (Pfizer, Inc.), CI-1033 (canertinib, Pfizer, Inc.), Herceptin (trastuzumab, Genentech Inc. .), Omitarg (2C4, pertuzumab, Genentech Inc.), TAK-165 (Takeda), GW-572016 (lonafarnib, GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth), PKI166 (Novartis), dHER2 (HER2, Corixa and GlaxoSmithKline vaccine), APC8024 (HER2 Vaccine, Dendreon), anti-HER2 / neu dual specificity antibody (Decof Cancer Center), B7.her2.IgG3 (Agensys), AS HER2 (Research Institute for Rad Biology &amp; Medicine), trifunctional dual specificity antibodies (University in Munich) and mAB AR-209 (Aronex Pharmaceuticals Inc.) and mAB 2B-1 (Chiron) and combinations thereof. Preferred erb selective anti-cancer 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 Omitarg and combinations thereof.

[0311] Additional erbB2 inhibitors include those of 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 on January 22, 1998), WO 97/13760 (published on April 17, 1997), WO 95/19970 (published on July 27, 1995), United States Patent No. 5587458 (filed December 24, 1996) and U.S. Patent 5,877,305 (filed March 2, 1999). For additional ErbB2 receptor inhibitors useful in the present disclosure, see U.S. Patent Nos. 6,464,449 and 6,284,764 and International Publication No. WO 2001/98277.

[0312] In addition, other anti-cancer agents may be selected from Sorafenib (Onyx Pharmaceuticals Inc.; BAY-43-9006), Genasense (Augerosen, Genta), Panitumumab (Abgenix / Amgen), Zevalin (Schering), Bexxar (Corixa / GlaxoSmithKline), Abarelix, Alimta, EPO 906 (Novartis), diskodermolid (XAA-296), ABT-510 (Abbott), Neovastat (Aeterna), enzastaurine (Eli Lilly), combrestatin A4P (Oxigene), ZD-6126 (AstraZeneca , flavopiridol (Aventis), CYC-202 (Cyclacel), AVE63

8062 (Aventis), DMXAA (Roche / Antisoma), Thymitaq (Eximias), Temodar (temozolomide, Schering Plow) and Revilimd (Celegene) and combinations thereof.

[0313] Other anti-cancer agents may be selected from the following agents, CyPat (cyproterone acetate), Histerelin (histrelin acetate), Plenaixis (abarelix as depot), Atrasentan (ABT-627), Satraplatin (JM-216), thalidomide (Thalidomide), Theratope, Temilifene (DPPE), ABI-007 (paclitaxel), Evista (raloxifene), Atamestan (Biomed-777), Xyotax (paclitaxel polyglutamate), Targetin (bexarotin) and combinations thereof.

[0314] In addition, anti-cancer agents may be selected from the following agents, Trizaone (tirapazamine), Aposyn (exisulind), Nevastat (AE-941), Ceplene (histamine dihydrochloride), Orathecin (rubitecan), Virulisine, Gastrimmune (G17DT), DX-8951f (Exatecane mesylate), Onconase (ranpirnase), BEC2 (mitumoab), Xcytrin (gadolinium motexafine) and combinations thereof.

[0315] Further anti-cancer agents may be selected from the following agents, CeaVac (CEA), NeuTrexin (trimetrexate glucuronate) and combinations thereof. Additional anti-cancer agents can be selected from the following agents, OvaRex (oregowomab), Osidem (IDM-1) and combinations thereof. Additional anti-cancer agents can be selected from the following agents, Advexin (ING 201), Tirazone (tirapazamine), and combinations thereof. Additional anti-cancer agents can be selected from the following agents, RSR13 (efaproxiral), Cotara (1311 chTNT 1 / b), NBI-3001 (IL-4) and combinations thereof. Additional anti-cancer agents can be selected from the following agents: Canvaxin, GMK vaccine, PEG Interon A, Taxoprexin (DHA / paclitaxel) and combinations thereof. Other preferred anti-cancer agents include the MEK1 / 2 PD325901 inhibitor from Pfizer, the MEK inhibitor ARRY-142886 from Array Biopharm, the CDK2 inhibitor BMS-387,032 from Bristol Myers, the CDK inhibitor PD0332991 from Pfizer and AXD-5438 from AstraZeneca and combinations thereof. In addition, mTOR inhibitors such as CCI-779 (Wyeth) and rapamycin derivatives RAD001 (Novartis) and AP23573 (Ariad), HDAC SAHA inhibitors (Merck Inc./Aton Pharmaceuticals) and combinations thereof can also be used. Additional anti-cancer agents include the aurora 2 VX-680 inhibitor (Vertex), the Chk1 / 2 XL844 inhibitor (Exilixis).

[0316] In combination with the anti-ALK-1 antibody or antigen-binding portion thereof as described herein, and their pharmaceutical compositions as described herein, the following cytotoxic agents can also be used, e.g., one or more selected from the group including epirubicin (Ellence), doxetaxel (Taxotere), paclitaxel, Zinecard (dexrazoxane), rituximab (Rituxan), imatinib mesylate (Gleevec) and combinations thereof.

[0317] The disclosure also contemplates using the antibodies or antigen-binding portions thereof of the present disclosure together with hormone therapy, including but not limited to exemestane (Aromasin, Pfizer Inc.), leuprorelin (Lupron or Leuplin, TAP / Abbott / Takeda ), anastrozole (Arimidex, Astrazeneca), goserelin (Zoladex, AstraZeneca), doxercalciferol, fadrozole, formestane, tamoxifen citrate (tamoxifen, Nolvadex, AstraZeneca), Casodex (AstraZeneca), Abarelix (Praecis) Trelstar and their connections.

[0318] The disclosure also relates to hormone therapy agents such as antiestrogens, including but not limited to fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole (Femara, Novartis), antiandrogens such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex ® propionanilide (4'-cyano-3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3 '- (trifluoromethyl), bicalutamide) and combinations thereof.

[0319] In addition, the disclosure provides the antibodies of the present disclosure alone or in combination with one or more adjunctive therapy products, e.g., a product selected from the group consisting of Filgrastim (Neupogen), ondansetron (Zofran), Fragmin, Procrit, Aloxi, Emend, or their combinations.

[0320] Particularly preferred cytotoxic agents include Camptosar, Erbitux, 1ressa, Gleevec, Taxotere and combinations thereof.

[0321] The following topoisomerase 1 inhibitors, camptothecin, irinotecan HCl (Camptosar), edotecarin, oratecine (Supergen), exatecan (Daiichi), BN-80915 (Roche) and combinations thereof can be used as anti-cancer agents. An especially preferred topoisomerase 11 inhibitor is epirubicin (Ellence).

[0322] The antibodies of the disclosure can be used with anti-cancer agents, alkylating agents, anti-metabolites, antibiotics, plant-derived anti-cancer agents, camptothecin derivatives, tyrosine kinase inhibitors, other antibodies, interferons and / or biological response modifying agents.

[0323] Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan, mitobronitol, carbocon, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, altretamine, AP-5280, apazone 52 , brostalicin, bendamustine, carmustine, estramustine, photemustine, glufosfamide, ifosfamide, KW-2170, mafosphamide and mitolactol; Alkylating platinum coordination compounds include, but are not limited to, cisplatin, paraplatin (carboplatin), eptaplatin, lobaplatin, nedaplatin, eloxatin (oxaliplatin, Sanofi) or satrplatin and combinations thereof. An especially preferred alkylating agent is Eloxatin (oxaliplatin).

[0324] Antimetabolites include, but are not limited to, methotrexate, 6mercaptopurine riboside, mercaptopurine, 5-fluorouracil (5-FU) alone or in combination with leucovorin, tegafur, UFT, doxifluridine, carmofur, cytarabine, oxphosphate, cytarabine 1, Alimta (disodium salt of pemetrexed, LY231514, MTA), Gemzar (gemcitabine, Eli Lilly), fludarabine, 5-azacytidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethinylcytidine, arabinozymosin, cytosine melphalan, nelarabine, nolatrexed, ocphosphate, pemetrexed disodium salt, pentostatin, pelitrexol, raltitrexed, triapine, trimethrexate, vidarabine, vincristine, vinorelbine; or for example one of the preferred antimetabolites disclosed in European Patent Application No. 239362, such as N- (5- [N- (3,4-dihydro-2-methyl-4-oxoquinazolin-6-methylmethyl) -N-methylamino] -2 -thienoyl) -L-glutamine and combinations thereof.

[0325] Antibiotics include? Antibiotic intercalates agents, but are not limited to: aclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, bleomycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, galarubicin, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebekkamycin, stimalamer, streptozocin, valrubicin, zinostatin and combinations thereof.

[0326] Plant-derived anti-tumor substances include, for example, those selected from among mitosis inhibitors, for example vinblastine, doxetaxel (taxa), paclitaxel and combinations thereof.

[0327] Cytotoxic topoisomerase inhibitors include one or more agents selected from the group consisting of aclarubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, inotnotecan, HCl (edptotecarcinotine) , exatecan, gimatecan, lurtotecan, mitoxantrone, pirarubicin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan and combinations thereof.

[0328] Preferred cytotoxic topoisomerase inhibitors include one or more agents selected from the group consisting of camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan HCl (Camptosar), edotecarin, epirubicin (Ellence, SN, etop, 38) their connections.

[0329] Immune agents include interferons and many other immunosuppressants. 1-interferons include interferon alfa, interferon alfa-2a, interferon alfa-2b, interferon beta, interferon gamma-1a, interferon gamma-1b (Actimmune) or interferon gamma-n1 and combinations thereof. Other agents include filgrastim, lentinan, sisophylan, The65 raCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, limograstimod, lixinimimod, , molgramostim, OncoVAX-CL, sargramostim, tasonermin, tecleukin, thymalazine, tositumomab, Virulizine, Z-100, epratuzumab, mitumomab, oregowomab, pemtumomab (Y-muHMFG1), and Provenge (Dendreon) and their combinations.

[0330] Biologic response modifiers are agents that modify the defense mechanisms of living organisms or biological responses, such as the survival, growth or differentiation of cells from tissues, directing them towards antitumor activity. Such agents include crestin, lentinan, sisophiran, picibanil, ubenimex and combinations thereof.

[0331] Other anti-cancer agents include alitretinoin, ampligen, atrasentan bexarotene, bortezomib. Bosentan, calcitriol, exisulind, finasteride, photemustine, ibandronic acid, miltefosine, mitoxantrone, l-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazarotene, Telazemic, Telcemta , tretinoin and combinations thereof.

[0332] Other anti-angiogenic compounds include acitretin, fenretinide, thalidomide, zoledronic acid, angiostatin, aplidine, cilengide, combretastatin A-4, endostatin, halofuginone, rebimastat, removab, Revlimid, squalamine and their combination. [0333] Platinum coordination compounds include, but are not limited to, cisplatin, carboplatin, nedaplatin, oxaliplatin and combinations thereof.

[0334] Camptothecin derivatives include, but are not limited to, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan and combinations thereof.

[0335] Other anti-cancer agents include mitoxantrone, 1-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pentostatin, tretinoin and combinations thereof.

[0336] Antitumor agents capable of stimulating antitumor responses, such as anti-CTLA-4 antibodies (cytotoxic 4 T-cell antigen) and other agents capable of blocking CTLA-4, such as MDX-010 (Medarex) and CTLA- compounds can also be used 4 disclosed in US Patent No. 6,682736; and antiproliferative agents, such as other farnesyl protein transferase inhibitors, e.g., farnesyl protein transferase inhibitors. For additional specific CTLA-4 antibodies that can be used in this disclosure, see United States Provisional Patent Application 60/113647 (filed December 23, 1998), United States Patent No. 6682736. For example, another CTLA-4 antibody that can be used used in accordance with the present disclosure is ticilimumab, which has the monoclonal antibody sequence 11.2.1 in US Patent 6,682736.

[0337] Specific IGF1R antibodies that can be used in the present disclosure, see International Patent Application No. WO 2002/053596.

[0338] For specific CD40 antibodies that can be used in the present disclosure, see International Patent Application No. WO 2003/040170.

[0339] Gene therapy agents such as TNFerade (GeneVec), which expresses TNFalpha in response to radiation therapy, can also be used as anti-cancer agents. [0340] In one instance of the present disclosure, statins can be used in combination with the anti-ALK-1 antibody or antigen-binding portion thereof as described herein, and pharmaceutical compositions thereof. Statins (HMG-CoA reductase inhibitors) can be selected from the group consisting of Atorvastatin (Lipitor, Pfizer Inc.), Provastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin and Niacin (Advicor, Kos Pharmaceuticals), their derivatives and combinations.

[0341] In a preferred case, the statin is selected from the group consisting of Atorvastatin and Lovastatin, derivatives and combinations thereof.

[0342] Other agents useful as anti-cancer agents include Caduet.

[0343] For any method 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 thereof with at least one additional therapeutic agent, the anti-ALK-1 antibody may be conjugated with or derivatized with one of the additional therapeutic agents. The at least one additional therapeutic agent may also be administered separately or in a non-derivatized or unconjugated manner. When the at least one additional therapeutic agent is not derivatized or conjugated to the antibody, it may be administered in the same pharmaceutical formulation as the antibody or may be administered as a separate formulation.

Treatment of vision loss [0344] The compounds of the invention and the pharmaceutical compositions containing them are useful for the treatment of acute vision loss due to age-related macular degeneration and other diseases affecting the posterior segment of the eye, such as choroidal neovascularization, diabetic retinopathy, glaucoma, and retinopathy similar.

[0345] For example, the compounds of the invention may be used to form a drug depot at the back of the eye and may include one or more pharmaceutically active agents in addition to one or more pharmaceutically inactive excipients as described herein. Examples of pharmaceutically active agents useful in the compositions of the invention include anti-infective agents including, but not limited to, antibiotics, anti-viral and anti-fungal agents; antiallergic agents and mast cell stabilizers; steroidal and nonsteroidal anti-inflammatory agents (such as nepafenac); cyclooxygenase inhibitors, including, without limitation, Cox I and Cox II inhibitors; combinations of anti-infective and anti-inflammatory agents; decongestants, anti-glaucoma agents including, but not limited to, adrenergic drugs, beta-blockers, beta-adrenergic agonists, parasympathomimetic agents, cholinesterase inhibitors, carbonic anhydrase inhibitors and prostaglandins; combinations of anti-glaucoma agents; antioxidants; dietary supplements; drugs for the treatment of cystoid macular edema including, but not limited to, nonsteroidal anti-inflammatory drugs; medicines for the treatment of age-related macular degeneration (AMD), including non-exudative (dry) and exudative (wet) AMD, including but not limited to angiogenesis inhibitors, including angiogenesis inhibitors that inhibit protein kinase receptors, including kinase receptors protein like VEGF receptors; and dietary supplements; medications for the treatment of herpes and eye infections caused by CMV; drugs for the treatment of proliferative vitreoretinopathy, including without limitation antimetabolites and fibrinolytics; wound healing modulators including, without limitation, growth factors, antimetabolites, neuroprotective drugs, including without limitation eliprodil; and angiostatic steroids for the treatment of diseases or conditions of the posterior segment of the eye 26, including without limitation of age-related macular degeneration (AMD), including non-exudative (dry) and exudative (wet) AMD, choroidal neovascularization, retinopathy, retinitis, membrane inflammation angioedema, macular edema and glaucoma. For additional information on such angiostatic steroids, see US Patent Nos. 5,679,666 and 5,770,592. Nepafenac is a nonsteroidal anti-inflammatory drug for the treatment of cystoid macular edema.

[0346] When administered to the eye, the compound of the present disclosure is provided in a pharmaceutically acceptable ophthalmic carrier such that the compound is kept in contact with the surface of the eye for a time sufficient to allow the compound to penetrate through the cornea and / or sclera and internal parts eye, including, for example, the anterior chamber of the eye, posterior chamber of the eye, vitreous, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid / retina and sclera. The pharmaceutically acceptable ophthalmic carrier may be an ointment, vegetable oil or encapsulation material. The compound of the disclosure can also be injected directly into the vitreous or aqueous humor.

[0347] In addition, the compound can also be administered by well-known acceptable methods, such as Tenon capsule injections and / or subconjunctival injections. As is well known in the field of ophthalmology, the macula consists primarily of retinal suppositories and is the area of ​​maximum visual acuity on the retina. Tenon's purse or Tenon's membrane lies on the sclera. The conjunctiva covers a short segment of the posterior part of the eye to the limbus (conjunctiva of the eyeball) and curls up (upper vault) or down (lower vault) covering the inner areas of the upper eyelid or lower eyelid, respectively. The conjunction is on Tenon's purse. Tenon's sclera and capsule form the outer surface of the eyeball. For the treatment of eye diseases such as age-related macular degeneration (AMD), including exudative (dry) and exudative (wet) AMD, choroidal neovascularization, retinopathies (such as diabetic retinopathy, premature retinopathy), diabetic macular edema, inflammation retinitis, uveitis, cystoid macular edema (CME), glaucoma and other diseases or conditions of the back of the eye, it is preferred to apply a depot to a specific amount of the ophthalmically acceptable pharmaceutically active agent directly on the outer surface of the sclera and under the Tenon capsule. In addition, in cases of age-related macular degeneration (AMD), including non-exudative (dry) and exudative (wet) AMD and CME, it is best to apply the depot directly to the outer surface of the sclera, under the Tenon capsule, and above the macula in general.

[0348] The compounds may be formulated as depot preparations. Such long acting formulations can be administered by implantation (e.g. subcutaneously or intramuscularly), intramuscular injection or the aforementioned injection under the Tenon capsule or intravitreal. Alternatively, the active ingredient may be in the form of a powder to be reconstituted prior to use in a suitable vehicle, e.g., sterile pyrogen-free water.

[0349] In particularly preferred instances of the disclosure, the compounds may be prepared for topical administration in physiological saline (combined with any preservatives and antimicrobial agents commonly used in eye preparations) and administered as eye drops. The solution or suspension can be prepared in pure form and administered several times a day. Alternatively, the present compositions, prepared as described above, can also be administered directly into the cornea.

[0350] In preferred cases, the composition is prepared using a mucoadhesive polymer that binds to the cornea. Thus, for example, the compounds may be formulated using appropriate polymeric or hydrophobic materials (e.g. in the form of an emulsion in a pharmaceutically acceptable oil) or ion exchange resins or in the form of sparingly soluble derivatives, e.g. sparingly soluble salts. [0351] The pharmaceutical carrier for hydrophobic compounds is a co-solvent system containing benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer and an aqueous phase. The cosolvent system may be a VPD cosolvent system. VPD is a 3% w / v solution benzyl alcohol, 8% w / v non-polar surfactant polysorbate 80 and 65% w / v polyethylene glycol 300, made up to volume with anhydrous ethanol. The VPD co-solvent system (VPD: 5W) contains VPD diluted 1: 1 with a 5% dextrose in water solution. This cosolvent system dissolves hydrophobic compounds well and induces low toxicity after systemic administration.

Of course, the proportions of the cosolvent system can vary significantly without affecting its solubility and toxicity. In addition, the nature of the components of the cosolvent system may be different: for example, other low-toxicity non-polar surfactants may be used instead of polysorbate 80; the polyethylene glycol fraction may be different; other biocompatible polymers e.g. polyvinylpyrrolidone can replace polyethylene glycol, and other sugars or polysaccharides can replace dextrose.

[0352] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be used. Liposomes and emulsions are known examples of carriers or excipients for the administration of hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide may also be used, although usually at the cost of greater toxicity. In addition, 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 systems have been developed and are known to those skilled in the art. Sustained-release capsules may, depending on their chemical properties, release the compounds for several weeks or even more than 100 days. Depending on the chemical properties and biological stability of the therapeutic agent, additional strategies for protein stabilization may be employed.

[0353] The pharmaceutical compositions may also contain 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.

[0354] Each composition may be formulated for administration to a subject. The subject of the present disclosure is preferably a mammal, or more preferably a human.

[0355] The pharmaceutical preparations described herein may further comprise a therapeutic agent selected from the group consisting of an anti-cancer agent, anti-inflammatory agent, antibacterial agent, antiviral agent, angiogenic agent and anti-angiogenic agent. Examples of such agents are disclosed herein. [0356] For example, the anti-cancer 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; bizelesin; bleomycin sulfate; brechinar 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; desaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; Droloxifene Citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elzamitrucynę; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; ezorubicin hydrochloride; estramustine; sodium estramustine phosphate; etanidazole; ethiodized oil I 131; etoposide; etoposide phosphate; etoprine; Fadrozole Hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; foscidone, fostriecin sodium; gemcitabine; Gemcitabine Hydrochloride; Au 198 gold; hydroxyurea; idarubicin hydrochloride; ifosfamide; imofozynę; interferon alfa-2a; interferon alfa-2b; alpha-n1 interferon; alpha-n3 interferon; interferon beta-Ia; gamma-Ib interferon; iproplatin; Irinotecan Hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; Mechloretamine Hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mityndomid; mitocarcin; mitocromin; mitogillin; mitomalcine; mitomycin; mitosper, mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; Puromycin Hydrochloride; pyrazofurin; riboprine; rogletimid; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogerman hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium chloride Sr 89; sulofenur, talizomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; testolactone; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan hydrochloride; toremifene citrate; trestolone acetate; tricyribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; winepidine sulfate; winglicinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.

[0357] Anti-angiogenic agents are any agents that inhibit angiogenesis, both disclosed herein and known in the art. In preferred cases, the anti-angiogenic agent is an anti-VEGF agent, such as Macugen^™(Eyetech, New York, NY); or anti-VEGF antibody.

[0358] Pharmaceutical compositions can be formulated by standard techniques using one or more suitable carriers, excipients and diluents. See, e.g., Remington's Pharmaceutical Sciences, (19 Ed. Williams and Wilkins, 1995). [0359] Formulations suitable for parenteral administration include aqueous and non-aqueous preparations isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspension systems designed to direct the compound to blood components or to one or more organs. The formulations may be provided in closed containers with single or multiple doses, for example ampoules or vials. For ocular preparations, unit doses are preferred because there are no preservatives in the formulation. Other parenteral formulations may use preservatives that allow the use of multiple dose containers.

[0360] Solutions and suspensions may be prepared for immediate injection from sterile powders. Parenteral and intravenous forms may also contain minerals and other materials to suit the type of injection or delivery system chosen. [0361] Particular methods of parenteral administration contemplated in this disclosure include ocular and intravitreal administration. Pharmaceutical preparations for ocular and intravitreal administration include phosphate buffered saline (PBS) and balanced isotonic saline (BSS) with or without excipients such as manitol or sorbitol as protein stabilizers.

[0362] Suitable carriers for parenteral solutions are usually water, a suitable oil, saline, aqueous dextrose (glucose) solution or similar sugar solutions and glycols such as propylene glycol or polyethylene glycols. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffering substances. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, alone or in combination are suitable stabilizing agents. Citric acid salts or sodium EDTA are also used. In addition, parenteral solutions may contain preservatives such as benzalkonium chloride, methyl or propylparaben or chlorobutanol. Suitable pharmaceutical carriers are described in Remington, cited above.

[0363] In each of the present cases, the pharmaceutical composition or preparation may be lyophilized.

[0364] In each of the present cases, the pharmaceutical preparations preferably 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.

[0365] In certain instances, the treatment methods disclosed herein further comprise administering to a subject suffering from an angiogenic condition one or more therapeutic agents selected from the group consisting of anti-cancer agents, anti-viral agents, anti-inflammatory agents, anti-bacterial agents, or anti-angiogenic agents. [0366] Such combination therapies can be achieved either by administering to a subject the composition described herein together with the additional therapeutic agent (s) or by administering the composition and therapeutic agent (s) described herein (s) as two separate pharmaceutical preparations. In cases where more than one composition / therapeutic agent is administered to a subject, lower doses of the composition and / or therapeutic agents may be used, due to the synergistic effect of both active ingredients.

[0367] Antitumor agents that can be administered to a subject include, but are not limited to: aclarubicin; 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; bizelesin; bleomycin sulfate; brechinar 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; desaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; Droloxifene Citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elzamitrucynę; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; ezorubicin hydrochloride; estramustine; sodium estramustine phosphate; etanidazole; ethiodized oil I 131; etoposide; etoposide phosphate; etoprine; Fadrozole Hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; foscidone, fostriecin sodium; gemcitabine; Gemcitabine Hydrochloride; Au 198 gold; hydroxyurea; idarubicin hydrochloride; ifosfamide; imofozynę; interferon alfa-2a; interferon alfa-2b; alpha-n1 interferon; alpha-n3 interferon; interferon beta-Ia; gamma-Ib interferon; iproplatin; Irinotecan Hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; Mechloretamine Hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mityndomid; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper, mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; Puromycin Hydrochloride; pyrazofurin; riboprine; rogletimid; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogerman hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium chloride Sr 89; sulofenur, talizomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; testolactone; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan hydrochloride; toremifene citrate; trestolone acetate; tricyribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; winepidine sulfate; winglicinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. [0368] Antimicrobial agents that can be administered to a subject include, but are not limited to, penicillins, aminoglycosides, macrolides, monobactams, rifamycins, tetracyclines, chloramphenicol, clindamycin, lincomycin, imipenem, fusidic acid, novobiocin, fosfomycin, fosfomycin capreomycin, colistimethate, colistin, gramicidin, minocycline, doxycycline, vanomycin, bacitracin, kanamycin, gentamycin, erythromycin and cephalosporins.

[0369] Anti-inflammatory agents that can be administered to a subject include, but are not limited to, NSAIDS, e.g., aspirin (salicylamide), sodium salicylamide, indoprofen, indomethacin, indomethacin sodium trihydrate, Bayer ™, Bufferin ™, Celebrex ™, diclofenac, Ecotrin ™, diflunisal, fenoprofen, naproxen, sulindac, Vioxx ™), corticosteroids or corticotropin (ACTH), colchicine and anecortaw acetate.

[0370] Antiviral agents that can be administered to a subject include, but are not limited to, α-methyl-P-adamantanomethylamine, 1, -D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9- [2-hydroxyethoxy] methylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, CD4, 3'-azido-3'-deoxythymidine (AZT), 9- (2-hydroxyethoxymethyl) guanine (acycloviric acid), 1-adamantanamine, T-peptide and 2 ', 3'-dideoxycytidine.

[0371] In vivo administration of the compositions of the present disclosure to the target cell can be achieved using various techniques well known to those skilled in the art.

[0372] For example, the compositions of the disclosure may be administered systemically or topically by methods known in the art (e.g., oral, ocular, intravenous (iv), intradermal, intramuscular, transdermal, transmucosal, enteral, parenteral, inhalant, rectal or local) in preparations unit dose and containing traditional pharmaceutically acceptable carriers, adjuvants and vehicles.

[0373] The term ocular administration as used herein includes intravitreal, retinal, and the like administration.

[0374] The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-bridged, infusion techniques or intraperitoneal techniques. Suppositories for rectal administration can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycols, which are solid at ordinary temperatures but are liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

[0375] The dosage regimen for treating a disorder or disease by the compositions of this disclosure is based on a variety of factors, including type of disease, age, weight, sex, medical condition of the patient, severity of the condition, route of administration, and the particular compound employed. Thus, the dosage regimen can be very diverse, but it can be routinely determined using standard methods.

[0376] For systemic administration, the anti-ALK-1 antibody or antigen binding portion thereof of the present disclosure and / or one or more additional therapeutic agents is preferably administered at a dose 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, 40, 50, 75, 100 or 150 mg / kg body weight. In other cases, polypeptides (preferably dimers or homodimers) and / or small molecules described 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.

[0377] For topical administration, the anti-ALK-1 antibody or antigen binding portion thereof of the present disclosure and / or one or more additional therapeutic agents are preferably administered in a dose of at least 50 μ &amp; 100 μg, 150 μg, 200 gg, 250 gg , 300 gg, 350 gg, 400 gg, 450 gg, 500 gg, 550 gg, 600 gg, 650 μg or 700 μg. In other cases, polypeptides (preferably dimers or homodimers) and / or small molecules described herein are administered topically at a dose of 50-1000 μ &amp; more preferably 100-800 μg, more preferably 200-500 μg or more preferably 300-400 μg per site. [0378] For example, for administration to the skin of the anti-ALK-1 antibody or antigen-binding portion thereof of the present disclosure and / or peptidomimetics and / or one or more additional therapeutic agents is preferably administered at a dose of 501,000 μg / cm², more preferably 100-800 μg / cm², or more preferably 200-500 μg / cm². In another example for ocular administration, the polypeptides and / or peptidomimetics and / or small molecules of the present disclosure are administered at a dose of 50-1000 μg / eye, more preferably 100-800 μg / eye or more preferably 200-500 μg / eye.

[0379] The pharmaceutical compositions preferably contain the active ingredient (e.g. anti-ALK-1 antibody) in an effective amount, i.e. in an amount effective to achieve a therapeutic or prophylactic benefit. The actual effective amount for a particular administration will depend on the condition being treated and the route of administration. Determining the effective amount is within the skill of those in the art, particularly in light of the present disclosure.

[0380] Preferably, an effective amount of the active ingredient, e.g. anti-ALK-1 antibody is from about 0.0001 mg to about 500 mg of active agent per kilogram of patient weight, more preferably from about 0.001 to about 250 mg of active agent per kilogram of patient weight, even more preferably from about 0.01 mg to about 100 mg of active agent per kilogram of patient weight, more preferably from about 0.5 mg to about 50 mg of active agent per kilogram of patient weight, and most preferably from about 1 mg to about 15 mg of active agent per kilogram of patient weight.

[0381] In terms of percentages given, the formulations of the present disclosure will preferably contain an active agent, e.g., anti-ALK-1 antibody in an amount of from about 0.0001 to about 10% by weight, more preferably from about 0.001 to about 1% by weight, more preferably from about 0.05 to about 1% by weight, or more preferably from about 0.1% by weight up to about 0.5 wt.

Gene Therapy [0382] Nucleic acid molecules that encode the antibodies or antibody portions of the present disclosure can be administered to a patient in need thereof by gene therapy. Therapy can be carried out in vivo or ex vivo. In a preferred case, the patient is administered nucleic acid molecules encoding both the heavy chain and the light chain. In a more preferred case, the nucleic acid molecules are administered in such a way that they are permanently integrated into the B lymphocyte chromosomes, since these cells are specialized in the production of antibodies. In a preferred case, B cell precursors are transfected or infected ex vivo and then re-implanted in a patient in need. In another case, B-cell precursors or other cells are infected in vivo with a virus that is known to infect the cell 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 in vivo or ex vivo, the level of antibody expression can be monitored by taking samples from the treated patient using any immunoassay known in the art or discussed herein.

[0383] In a preferred case, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain or antigen-binding portion thereof of an anti-ALK-1 antibody and expressing that nucleic acid molecule. In another case, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the light chain or antigen-binding portion thereof of an anti-ALK-1 antibody and expressing that nucleic acid molecule. In a more preferred method, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding a heavy chain or antigen binding portion thereof and an isolated nucleic acid molecule encoding a light chain or antigen binding portion thereof of an anti-ALK-1 antibody according to the disclosure and expression of these nucleic acid molecules. The gene therapy method may also include the step of administering another therapeutic agent, such as any of the agents discussed previously in connection with the combination therapy.

Method of searching for ALK-1 antagonists and agonists [0384] In one instance, the present disclosure provides a method of determining whether a substance inhibits regulation leading to stimulation of a specific downstream specific target gene for ALK-1, Id1, such as, for example, the Taqman Id1 test described in Example 12 . This method involves contacting a sample of cells that express Id1 with this substance and determining whether Id1 expression has been inhibited, wherein the reduced level of Id1 expression in the sample of cells contacted with the substance compared to the control cell sample indicates that this substance inhibits Id1 expression . In one particular case, the substance is an antibody that binds to the extracellular domain of ALK-1. Otherwise, the substance is a small molecule. According to the disclosure, cells may innate express both ALK-1 and Id1, such as the HUVEC cells described in Example 12, or may have been transformed or transfected using DNA encoding one or both of them. Id1 expression can be determined e.g. using the Taqman Id1 test described in Example 12.

[0385] Conversely, activators or agonists can also be tested and used according to the same procedures.

[0386] In order that this disclosure may be better understood, the following examples are set forth. Examples [0387] In the following examples and preparations, "MW" means molecular weight; "His-Tag" means a C-terminal polyhistidine tag (6xHis) for rapid purification on a nickel chelating resin and detection using an anti-His (Ckoniec) antibody; "BSA" means bovine serum albumin; "EDTA" means ethylenediaminetetraacetic acid; "DMSO" means dimethyl sulfoxide; "MOPS" means 3- (Nmorpholine) propanesulfonic acid; "MES" means 2- (N-morpholino) ethanesulfonic acid; "PBS" means phosphate buffered saline; "DPBS" means phosphate buffered Dulbecco's saline; "HEMA" means 2-hydroxyethyl methacrylate; "DMEM" means modified Dulbecco's Eagle medium; "FBS" means fetal building serum; "NEAA" means non-essential amino acids; "HEPES" means N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; and "DMF" means dimethylformamide.

Example 1. Preparation of the ALK-1 immunogen [0388] ECD ALK-1 was cloned from the full-length human ORF clone for ALK-1 (Invitrogen, clone ID IOH21048) by PCR using a 5'-ACGGCCCAGCCGGCCGACCCTGTGAAGCCGTCT compatible primer (SEQ ID NO: 96) and inverse 5'-ACTAAGCTTTTAATGATGATGATGATGATGCTGGCCATCTGTTCCCG (SEQ ID NO: 97). The PCR product was purified, treated with SfiI and HindIII restriction enzymes, and cloned into the SfiI / HindIII site of the mammalian expression vector pSecTag2 / Hygro (Invitrogen Inc., Catalog No. V910-20). The clone was used for transient transfection of 293T cells using Fugene 6 transfection reagent (Roche Applied Science, Catalog No. 1814443) according to the manufacturer's instructions. The cell culture supernatant containing the secreted target protein was harvested 72 hours after 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 was eluted from the resin using a buffer containing 20 mM Tris pH 8.0, 300 mM imidazole and 300 mM sodium chloride. 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 buffer was exchanged for PBS or 10 mM HEPES, pH 7.4, plus 150 mM sodium chloride by dialysis and the protein was concentrated to 0.2-1 mg / ml with a final purity&gt; 90%, assessed on Coomassie blue stained SDS PAGE gel. His-Tag ECD ALK-1 protein was strongly glycosylated at an observed MW of 26 KDa, compared to 11 KDa of the theoretical MW of this protein. His-Tag ECD ALK-1 protein (SEQ ID NO: 98) was used to generate hybridomas producing anti-ALK-1 antibody as described in Example 2.

Human ECD ALK-1 protein from His-Tag:

gene sequence (the part marked with lowercase letters is a secretory signal):

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgcggcccagccggccGACCCTGTGAAGC

CGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGG

GGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCT

GCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGC

GACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGGCACCCAACCTCCTTCGGAGCAG

CCGGGAACAGATGGCCAGCATCATCATCATCATCAT (SEQ ID NO: 99) protein sequence:

DPVKPSRGPLVTCTCESPHCKGPTCRGAWCTWLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHY CCDSHLCNHNVSLVLEATQPPSEQPGTDGQHHHHHH (SEQ ID NO: 98)

Example 2. Production of hybridomas producing anti-ALK-1 antibody [0389] XENOMOUSE® mice eight to ten weeks old were immunized in 10 μg hind paw pads / recombinant human ALK-1 / Fc chimera mouse (R&amp;D Systems, Inc., catalog number 370 -AL) or the ECD ALK-1 protein from His-Tag described in Example 1. This dose was repeated five to seven times over a period of three to five weeks. Three or four days before the fusion, mice were given the last injection of immunogen in PBS. Lymphocytes from lymph nodes of immunized mice were fused to the non-secretory myeloma cell line P3-X63-Ag8.653 by cell electrophusion (ATCC Cat. No. CRL 1580), and these combined cells were subjected to HA-DMEM selection as previously described (DMEM / 15 % FBS / 1% 200 mM L-glutamine / 1% 100X no essential amino acid / 1% 100X Pen / Strep / 10 U / ml IL-6/1 vial / liter OPI medium supplemented with 0.5x HA (Azaserin-Hypoxanthine, Sigma, catalog number A9666)). A set of hybridomas were recovered, all of which secreted ALK-1 specific human IgG2 antibodies. [0390] An ELISA assay was used to detect antibody binding. The immunulogen was coated onto an Immulon 96 well microtiter plate (NUNCImmuno plate^™ with MaxiSorp surface^™, Nalge Nunc International, Cat # 439454) at 4 μg / ml in 50 mM sodium bicarbonate buffer overnight at 4 ° C. Plates were washed and then blocked with PBS containing 0.1% Tween-20 and 0.5% bovine serum albumin. Antibodies were added to blocked ELISA plates, incubated for one hour and washed with PBS with Tween-20. Binding was detected using anti-human IgG-horseradish peroxidase antibodies (Pierce, catalog number 31420) followed by the addition of ABTS (Pierce, catalog number 37615). Colorimetric measurements were carried out at 405 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices).

[0391] Twenty-five hybridomas were selected for further study. They were cloned into individual cells by limiting dilutions and 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; 1.151.1; 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.

[0392] The LN 15916 mouse hybridoma cell line (hybridoma 1.12.1) was deposited under the conditions of the Budapest Treaty at the American Cell Cultures Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 on June 21, 2005. Hybridomas 1.12. 1 is assigned the following access number PTA-6808. Example 3. Anti-ALK-1 antibody sequences [0393] To analyze the structure of the antibodies produced according to the disclosure, nucleic acids encoding heavy and light chain fragments were cloned from hybridomas producing anti-ALK-1 monoclonal antibodies. Cloning and sequencing were carried out by standard methods.

[0394] Poly (A)⁺ mRNA was isolated using a Fast-Track kit^™ (Invitrogen) of about 2 X 10⁵ hybridoma cells for each ALK-1 antibody. CDNA was synthesized from mRNA using random primers. This cDNA obtained using random primers was amplified by PCR using primers specific for variable domains from the human VH or Vk family in combination with primers specific for the human Cy2 constant region or the Ck constant region to amplify the antibody variable region encompassing all framework regions (FR) and complementarity determining regions (CDRs). Nucleic acid sequences encoding human heavy and light kappa transcripts from hybridomas producing anti-ALK-1 antibodies were obtained by direct sequencing of both strands of PCR products. Sequences were analyzed using proprietary Abgenix software and publicly available information about the sequence of human VH and Vk genes, "V BASE sequence directory", Tomlinson et al., MRC Center for Protein Engineering, Cambridge, UK). Identical results could be obtained using publicly available sequence alignment software by a specialist in the field using MacVector and Geneworks. [0395] In particular, the full-length anti-ALK-1 1.12.1 antibody was cloned into expression vectors as follows: Poly (A)⁺ mRNA was isolated using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized from mRNA using the Advantage RT-for-PCR kit (BD Biosciences) using oligo primers (dT). cDNA for clone 1.12.1 obtained using oligo (dT) primers was amplified using the primers listed in Table 2. Amplification was performed using Pfu Ultra polymerase (Stratagene) and PTC-200 DNA Engine (MJ Research) in the following cycles: 3 ' at 95 ° C; 25x (20 "at 95 ° C, 30" at 52 ° C, 1'20 "at 72 ° C); 10 'at 72 ° C. Clone sequences were confirmed using Grills 16^th BDTv3.1 / dGTP (Applied Biosystems Inc.) and 3730xl DNA analyzer (Applied Biosystems Inc.). In the VH 1.12.1 cloning process, a silent mutation in the eighth codon transforming "GGC" into "GGT" was introduced. All sequences were analyzed by alignment with "V BASE sequence directory" (Tomlinson, et al, J. Mol. Biol., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-860 (1994); EMBO J., 14, 4628-4638 (1995).)

Table 2

<td colspan="3">Heavy and light chain amplification primers used for cloning 1.12.1 fully</td>

<td colspan="3">length</td>

<td>Starter name</td><td>Starter sequence</td><td>SEQ ID WELL</td>

<td colspan="3">Heavy and light chain amplification primers used for cloning 1.12.1 fully</td>

<td colspan="3">length</td>

<td>Starter name</td><td>Starter sequence</td><td>SEQ ID WELL</td>

<td> 4-61</td><td>5 'tcttcaagcttgatatctctagaagccgccaccAT GAAACACCTGT GGTTCTTCCTCC 3'</td><td> 105</td>

<td>G1 / 2 FL R</td><td>5 'ttctctgatcagaattcctaCTATTTACCCGGAGACAGGGAGAGGC 3'</td><td> 106</td>

<td>A11</td><td>5 'tcttcaagcttcccgggagccgccaccATGGAAACCCCAGCGCAGCTT 3'</td><td> 107</td>

<td>K_FL_R</td><td>5 'ttctttgatcagaattctcaCTAACACTCTCCCCTGTTGAAGCTCTTTG 3'</td><td> 108</td>

<td colspan="3">Non-hybridizing rules are shown in lower case</td>

Example 4. Gene utilization analysis and CDR analysis [0396] Based on the nucleic acid sequence and predicted amino acid sequence of antibodies, gene utilization was identified for each antibody chain. Table 3 shows the use of antibody genes consistent with the disclosure for selected hybridoma clones.

Table 3

<td colspan="8">The use of heavy and light chain genes</td>

<td>Clone</td><td colspan="4">Heavy germline chain</td><td colspan="2">Light kappa chain from the entry line</td><td>nucleation</td>

<td></td><td>SEQ ID NO:</td><td>Vh</td><td>dh</td><td>jh</td><td>SEQ ID NO:</td><td>vk</td><td>jk</td>

<td> 1.11.1</td><td> 9</td><td> 3-33</td><td> 6-19</td><td>JH3B</td><td> 11</td><td>L¹</td><td>JK4</td>

<td> 1.12.1</td><td> 103</td><td> 4-31</td><td> 6-19</td><td>JH4B</td><td> 126</td><td>A27</td><td>JK5</td>

<td> 1.13.1</td><td> 13</td><td> 4-61</td><td> 6-19</td><td>JH4B</td><td> 15</td><td>A27</td><td>JK5</td>

<td> 1.14.1</td><td> 17</td><td> 4-61</td><td> 6-19</td><td>JH4B</td><td> 19</td><td>A27</td><td>JK5</td>

<td> 1.151.1</td><td> 21</td><td> 4-31</td><td> 3-3</td><td>JH3B</td><td> 23</td><td>B3</td><td>JK1</td>

<td> 1.162.1</td><td> 25</td><td> 4-31</td><td></td><td>JH3B</td><td> 27</td><td>A27</td><td>JK5</td>

<td> 1.183.1</td><td> 29</td><td> 4-59</td><td> 6-19</td><td>JH4B</td><td> 31</td><td>L2</td><td>JK3</td>

<td> 1.31.1</td><td></td><td> 4-31</td><td> 6-19</td><td>JH4B</td><td></td><td>A27</td><td>JK5</td>

<td> 1.8.1</td><td> 33</td><td> 4-31</td><td> 3-3</td><td>JH3B</td><td> 35</td><td>B3</td><td>JK1</td>

<td> 1.9.1</td><td> 37</td><td> 3-11</td><td> 3-22</td><td>JH6B</td><td> 39</td><td>A2</td><td>JK1</td>

<td> 4.10.1</td><td> 41</td><td> 3-15</td><td> 3-22</td><td>JH4B</td><td> 43</td><td>A3</td><td>JK4</td>

<td> 4.24.1</td><td> 45</td><td> 4-31</td><td> 5-12</td><td>JH6B</td><td> 47</td><td>A27</td><td>JK5</td>

<td> 4.38.1</td><td> 49</td><td> 4-31</td><td> 4-23</td><td>JH4B</td><td> 51</td><td>B3</td><td>JK1</td>

<td> 4.58.1</td><td> 53</td><td> 4-31</td><td> 4-23</td><td>JH4B</td><td> 55</td><td>A27</td><td>JK5</td>

<td> 4.62.1</td><td> 57</td><td> 4-31</td><td> 5-12</td><td>JH6B</td><td> 59</td><td>A27</td><td>JK5</td>

<td> 4.68.1</td><td> 61</td><td> 4-31</td><td> 2-2</td><td>JH5B</td><td> 63</td><td>A27</td><td>JK5</td>

<td> 4.72.1</td><td> 65</td><td> 4-31</td><td> 5-12</td><td>JH6B</td><td> 67</td><td>A27</td><td>JK5</td>

<td> 5.13.1</td><td> 69</td><td> 4-31</td><td></td><td>JH3B</td><td> 71</td><td>A27</td><td>JK4</td>

<td colspan="8">The use of heavy and light chain genes</td>

<td>Clone</td><td colspan="4">Heavy germline chain</td><td colspan="2">Light kappa chain from the entry line</td><td>nucleation</td>

<td></td><td>SEQ ID NO:</td><td>Vh</td><td>dh</td><td>jh</td><td>SEQ ID NO:</td><td>vk</td><td>jk</td>

<td> 5.34.1</td><td> 73</td><td> 4-31</td><td></td><td>JH6B</td><td> 75</td><td>A1</td><td>JK1</td>

<td> 5.53.1</td><td> 77</td><td> 3-15</td><td> 1-1</td><td>JH4B</td><td> 79</td><td>B2</td><td>JK4</td>

<td> 5.56.1</td><td> 81</td><td> 3-11</td><td> 6-19</td><td>JH6B</td><td> 83</td><td>A2</td><td>JK1</td>

<td> 5.57.1</td><td> 85</td><td> 3-11</td><td> 3-10</td><td>JH6B</td><td> 87</td><td>A2</td><td>JK1</td>

<td> 5.59.1</td><td> 89</td><td> 3-11</td><td> 6-6</td><td>JH6B</td><td> 91</td><td>A2</td><td>JK1</td>

[0397] Mutagenesis in the Vh (M29I) and Vk (D19A) regions of clone 1.12.1 was performed using the primers listed in Table 4 and the QuickChange kit (Stratagene) according to the manufacturer's instructions. The sequences of the mutated variants were confirmed and cloned into expression vectors using standard procedures.

Table 4

<td></td><td colspan="2">Mutagenic oligonucleotides (5 'to 3' sequences):</td>

<td>Starter</td><td>Sensible</td><td>antisense</td>

<td>1.12.1 (D19A)</td><td>CTCCAGGGGAAAGAGCCACCCTCTCCTGTAGG (SEQ ID NO: 109)</td><td>CCTACAGGAGAGGGTGGCTCTTTCCCCTGGAG (SEQ ID NO: 110)</td>

<td>1.12.1 (M29I)</td><td>GGTGGCTCCATCAGCAGTGGTGAATACTAC (SEQ ID NO: 111)</td><td>GTAGTATTCACCACTGCTGATGGAGCCACC (SEQ ID NO: 112)</td>

<td colspan="3">Mutations are highlighted in bold and underlined.</td>

[0398] Nucleic acid molecules encoding the heavy chain variable domain (SEQ ID NO: 5) and the light chain variable domain (SEQ ID: 7) of antibody 1.12.1 (M29I / D19A) were deposited under the terms of the Budapest Treaty at the American Cell Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 on July 14, 2005. Deposits were assigned the following access 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.

[0399] Several specific human anti-ALK-1 antibodies shared a common pattern in the heavy chain variable domain CDR1. These leader molecules use segments 4-31 or 4-61 of the heavy chain V gene. The FR1 and CDR1 sequences corresponding to these heavy antibody chains are shown in Table 4A, aligned with the germline sequence. The dash (-) in the alignment means the residue identical to the germline. In all cases, the GYYWS pattern (SEQ ID NO: 136) at the end of CDR1 underwent somatic mutations giving a new sequence pattern in which the G residue is converted to an acid residue (D or E) and the final S residue is converted to N in 9 out of 12 examples. Sequence diversity in other VH regions indicates that these are probably independent somatic mutation events leading to the same sequence pattern at the VH CDR1 end.

Table 4A

Sequence patterns in the heavy chain of the anti-ALK-1 antibody

<td>Clone</td><td>Gen V Line</td><td>Gen D embryonic</td><td>Gen J.</td><td>FR1 QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 122)</td><td>CDR1 GGSISSGGYYWS (SEQ ID NO: 123)</td>

<td> 5.34.1</td><td>VH4-31</td><td>- NA -</td><td>JH6B</td><td></td><td>------- D-_N</td>

<td> 4.58.1</td><td>VH4-31</td><td>D4-23</td><td>JH4B</td><td></td><td> -------_D—_N</td>

<td> 4.38.1</td><td>VH4-31</td><td>D4-23</td><td>JH4B</td><td></td><td> -------_D----</td>

<td> 5.13.1</td><td>VH4-31</td><td>- NA -</td><td>JH3B</td><td></td><td> -------_D--- N</td>

<td> 1.162.1</td><td>VH4-31</td><td>- NA -</td><td>JH3B</td><td> --------------------1----</td><td> -------_E----</td>

<td> 4.72.1</td><td>VH4-31</td><td>D5-12</td><td>JH6B</td><td></td><td>E ------- ----</td>

<td> 4.24.1</td><td>VH4-31</td><td>D5-12</td><td>JH6B</td><td></td><td> ------_ND---at</td>

<td> 4.62.1</td><td>VH4-31</td><td>D5-12</td><td>JH6B</td><td></td><td> -------_D--- N</td>

<td> 1.31.1</td><td>VH4-31</td><td>D6-19</td><td>JH4B</td><td></td><td> -------_D---_N</td>

<td> 1.12.1</td><td>VH4-31</td><td>D6-19</td><td>JH4B</td><td></td><td> ---_M--- e ---_N</td>

<td></td><td>Line</td><td>embryonic</td><td></td><td>QVQLQESGPGLVKPSETLSLTCTVS (SEOIDNO: 124)</td><td>GGSVSSGGYYWS (SEQ ID NO: 125)</td>

<td> 1.13.1</td><td>VH4-61</td><td>D6-19</td><td>JH4B</td><td>-H ----------------------</td><td> -------_D--- N</td>

<td> 1.14.1</td><td>VH4-61</td><td>D6-19</td><td>JH4B</td><td></td><td> -------_D---_N</td>

Example 5. Preparation of Fab 1.12.1 molecules [0400] The Fab fragment from 1.12.1 (M29I / D19A) was prepared by digesting IgG1 1.12.1 (M29I / D19A) with papain. Full length IgG1 1.12.1 (M29I / D19A) purified on Protein A was incubated with papain (VWR) in a ratio of 1:50 (papain: protein) in a buffer containing 30 mM sodium phosphate (pH 7.0), 2 mM EDTA and 2 mM cysteine ​​at 37 ° C for 2-3 hours. The digested mixture was then applied to a Protein A minicolumn to remove undigested full length protein and Fc fragment. Unbound Fab was collected in the leak. A particle size size exclusion chromatography column (Superdex 200, Amersham Pharmacia Biotech) was then used to further purify the Fab protein and to exchange the buffer with PBS. Endotoxin was removed by applying the protein solution to a Detoxi gel (PIERCE) and then to a Vivapure Mini Q ion exchange column (VivaScience). The protein was filtered using a 0.2 Pm syringe filter and endotoxin levels were tested using the LAL pyrogen kit (Cambrex). The finally purified protein had a concentration of 2-3 mg / ml, with an endotoxin level &lt;0.1 EU / mg and a purity&gt; 95%. The Fab 1.12.1 fragment (M29I / D19A) has a molecular weight of 47347 under non-reducing conditions as shown by electrospray mass spectrometry. Edman N-terminal sequencing analysis confirmed the N-terminal sequence of the light chain as EIVLTQSPG (SEQ ID NO: 113) and the heavy chain as QVQLQESG (SEQ, respectively)

ID NO: 114).

Example 6. Determination of avidity values ​​for fully human monoclonal anti-ALK-1 antibodies by surface plasmon resonance (SPR) using BIACORE ™ [0401] Avidity measurements of purified anti-ALK-1 antibodies by surface plasmon resonance using a BIACORE device^™ 3000 was carried out as follows using the manufacturer's protocols.

[0402] For performing kinetic analysis, recombinant human ALK-1 / Fc fusion protein (hALK-1 / Fc) and ALK-1 / Fc fusion cynomolgus macaque (cALK-1 / Fc) were immobilized on separate flow cells on a CM5 BIAcore sensor chip using routine amine coupling. Surfaces were prepared using 10 mM acetate buffer, pH 5.0 as immobilization buffer, achieving protein densities of 300 and 150 RU for hALK-1 / Fc and cALK-1 / Fc fusion proteins, respectively. Deactivation of unreacted N-hydroxysuccinimide esters was carried out using 1 M ethanolamine hydrochloride, pH 8.5. Antibody samples were prepared in wash buffer at concentrations ranging from 0.125 to 2 nM (0 nM solution containing wash buffer alone was included as a zero reference). Samples were randomized and injected in duplicate for 10 minutes through all 4 flow chambers using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as wash buffer. The observed association rates were independent of the flow rate from 1 to 100 gl / min, indicating no restrictions on transported masses. A flow rate of 25 μl / min was used to determine the avidity value. Dissociation of antibodies was monitored for 10 minutes, the surface was regenerated by a 12 second injection of 100 mM H3PO4 (25 μl / min). The raw data was processed using the Scrubber software package (© BioLogic Software) and analyzed using the CLAMP software package (© BioLogic Software). Multiple data sets from a single surface, six data sets at a time, were simultaneously globally fitted to a simple 1: 1 Langmuir binding model using the common value of the variable Rmax. Table 5 lists avidity values ​​for exemplary anti-ALK-1 antibodies of the present disclosure. The data presented indicate that the antibodies produced according to the disclosure have high affinities and strong binding constants for human ALK-1.

Table 5

<td colspan="4">Determination of avidity values ​​using surface plasmon resonance (BIA-</td>

<td colspan="4">core)</td>

<td>Clone</td><td>HALK1 / Fc aviability (pM)</td><td>koff hALK-1 / Fc (1 / s)</td><td>CALK1 / Fc aviability (pM)</td>

<td>1.12.1 (M29I / D19A)</td><td> &lt;6,8</td><td>&lt;5.0 x 10-⁶</td><td> 27</td>

<td> 1.14.2</td><td> 76</td><td>5.6 x 10-⁵</td><td> 280</td>

<td> 1.27.3</td><td> 2,9</td><td>1.9 x 10-⁵</td><td> 60</td>

<td> 1.31.1</td><td> &lt;13</td><td>&lt;5.0 x 10-⁶</td><td> 150</td>

<td> 1.162.1</td><td> 18</td><td>1.1 x 10-⁵</td><td> 62</td>

<td> 1.183.2</td><td> 220</td><td>3.1 x 10-⁵</td><td> 1800</td>

<td colspan="4">Determination of avidity values ​​using surface plasmon resonance (BIA-</td>

<td colspan="4">core)</td>

<td>Clone</td><td>HALK1 / Fc aviability (pM)</td><td>koff hALK-1 / Fc (1 / s)</td><td>CALK1 / Fc aviability (pM)</td>

<td> 4.24.2</td><td> 70</td><td>4.4 x 10-⁵</td><td> 430</td>

<td> 4.38.1</td><td> 100</td><td>4.0 x 10⁵</td><td> 150</td>

<td> 4.58.2</td><td> 40</td><td>1.6 x 10-⁵</td><td> 130</td>

<td> 4.62.1</td><td> 9,6</td><td>7.6 x 10-⁶</td><td> 19</td>

<td> 4.68.2</td><td> 86</td><td>3.8 x 10-⁵</td><td> 320</td>

<td> 4.72.2</td><td> 73</td><td>3.4 x 10-⁵</td><td> 280</td>

<td> 5.13.3</td><td> 91</td><td>6.3 x 10-⁵</td><td> 190</td>

<td colspan="4">1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine) .</td>

Example 7. Determination of affinity constant (Kd) variants of fully human monoclonal anti-ALK-1 1.12.1 antibody by surface plasmon resonance (SPR) method using BIACORE ™ [0403] Affinity measurements of purified anti-ALK-1 antibodies by surface plasmon resonance with using the BIACORE device^™ 3000 was carried out as follows using protocols from the manufacturer.

[0404] For kinetic analyzes, variants of the fully human monoclonal anti-ALK-1 1.12.1 antibody were immobilized on the dextran layer on a CM5 biosensor chip using amine coupling. Surfaces were prepared using 10 mM acetate buffer, pH 5.0 as immobilization buffer, achieving protein densities of 3500-4800 RU. Deactivation of unreacted N-hydroxysuccinimide esters was carried out using 1M ethanolamine hydrochloride, pH 8.5. Monomeric ALK-ECD samples were prepared in wash buffer at concentrations ranging from 2.63 to 640 nM (a 0 nM solution containing wash buffer alone was included as the zero reference). Samples were randomized and each injected for 2 minutes through all 4 flow chambers using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as wash buffer. A flow rate of 25 μl / min was used to determine the affinity constants. Dissociation of monomeric ALK-ECD was monitored for 10 minutes, the surface was regenerated by a 12 second injection of 100 mM H3PO4 (25 μl / min). The raw data was processed using the Scrubber software package (© BioLogic Software) and analyzed using the CLAMP software package (© BioLogic Software). Data were globally fitted to a Langmuir simple 1: 1 binding model. Table 6 lists the results of affinity measurements for variants of human anti-monoclonal antibody

ALK-1 1.12.1 according to the present disclosure.

Table 6

<td colspan="4">Determination of the affinity constant Kd of the mAb 1.12.1 variant by surface res</td>

<td colspan="4">plasmon zonance (BIAcore)</td>

<td>Antibody</td><td>Association Speed (M^-1 s^-1)</td><td>Speed ​​of dissociation (s-¹)</td><td>kd (NM)</td>

<td colspan="4">Determination of the affinity constant Kd of the mAb 1.12.1 variant by surface res</td>

<td colspan="4">plasmon zonance (BIAcore)</td>

<td> 1.12.1</td><td>1.9 x 10³</td><td>7.4 x 10 -⁵</td><td> 39</td>

<td>1.12.1 (RWT)</td><td>2.2 x 10³</td><td>5.8 x 10-⁵</td><td> 26</td>

<td>1.12.1 (D19A)</td><td>2.6 x 10³</td><td>4.4 x 10-⁵</td><td> 17</td>

<td>1.12.1 (M29I)</td><td>2.4 x 10³</td><td>9.1 x 10-⁵</td><td> 38</td>

<td>1.12.1 (M29I / D19A) (1) *</td><td>2.2 x 10³</td><td>9.5 x 10-⁵</td><td> 43</td>

<td>1.12.1 (M29I / D19A) (2) *</td><td>2.3 x 10³</td><td>8.4 x 10-⁵</td><td> 37</td>

<td colspan="4">* 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 has been isolated from a hybridoma. 1.12.1 (rWT) refers to the variant mAb 1.12.1 that was the expressed recombinant mAb. 1.12.1 (M29I) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which methionine at position 29 in the heavy chain was replaced with isoleucine. 1.12.1 (D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which aspartic acid at position 19 in the light chain was replaced with alanine. 1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine).</td>

Example 8. Determination of affinity constants (Kd) of exemplary fully human monoclonal anti-ALK-1 monoclonal antibodies by surface plasmon resonance (SPR) using BIACORE ™ [0405] Affinity measurements (KD and koff) of purified anti-ALK-1 antibodies by surface resonance method plasmonic using the BIACORE device^™

3000 was carried out as follows using manufacturer's protocols.

[0406] For kinetic analysis, affinity purified mAbs were immobilized on a dextran layer on a CM5 biosensor chip using amine coupling. Surfaces were prepared using 10 mM acetate buffer, pH

5.0 as immobilization buffer achieving protein densities of 200-400 RU. Inactivation of unreacted N-hydroxysuccinimide esters was carried out using 1 M ethanolamine hydrochloride, pH 8.5. Monomer samples were prepared ALKECD were prepared in wash buffer at concentrations ranging from 3.125-400 nM (0 nM solution containing wash buffer alone was included as a zero reference). Samples were randomized and injected in duplicate for 2 minutes through all 4 flow chambers using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as wash buffer. The observed association rates were independent of the flow rate from 1 to 100 μl / min, indicating no restrictions on transported masses. A flow rate of 25 μl / min was used to determine the affinity constants. Dissociation of monomeric ALKECD was monitored for 10 minutes, the surface was regenerated by a 12 second injection of 100 mM H3PO4 (25 μl / min). The raw data was processed using the Scrubber software package (© BioLogic Software) and analyzed using the CLAMP software package (© BioLogic Software). Data were globally fitted to a Langmuir simple 1: 1 binding model. Table 7 lists the results of affinity measurements for exemplary anti-ALK-1 antibodies of the present disclosure:

Table 7

<td colspan="4">Determination of the Kd affinity constant for exemplary monoclonal antibodies</td>

<td colspan="4">surface plasmon resonance (BIAcore)</td>

<td>has b</td><td>Association Speed (M^-1 s^-1)</td><td>Speed ​​of dissociation (s^-1)</td><td>kd (NM)</td>

<td>1.12.1 (M29I / D19A)</td><td>3.3 x 10⁴</td><td>8.2 x 10 "⁴</td><td> 25</td>

<td> 1.31.1</td><td>3.2 x 10⁴</td><td>1.9 x 10 "⁴</td><td> 6</td>

<td> 4.72.1</td><td>3.2 x 10⁴</td><td>2.5 x 10 "⁵</td><td> 0,8</td>

<td>Fab 1.12.1 (M29I / D19A)</td><td>3.8 x 10⁴</td><td>8.2 x 10 "⁴</td><td> 22</td>

[0407] The monomeric ALK-ECD used to obtain the data in Example 8 was a different preparation than that used to obtain the data in Example 7.

[0408] 1.112.1 (M29I / D19A) refers to the variant mAb 1.12.1 which was the expressed recombinant mAb containing the 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 heavy chain replaced with isoleucine).

[0409] Fab 1.12.1 (M29I / D19A) refers to a Fab mAb 1.12.1 fragment (M29I / D19A) generated by digestion with IgG1 1.12.1 (M29I / D19A) using papain.

Example 9. Determination of epitope selectivity of anti-ALK-1 antibodies [0410] Cross-competition testing was performed using a BIACORE device^™ 3000 (Biacore International AB, Uppsala, Sweden and Piscataway, NJ), according to the manufacturer's protocols.

[0411] The recombinant human ALK-1 / FC chimera was immobilized on a dextran layer on a CM5 biosensor chip using amine coupling. The chips were prepared using 10 mM acetate buffer, pH 5.0 as immobilization buffer, achieving 940 RU protein densities. Inactivation of unreacted N-hydroxysuccinimide esters was carried out using 1 M ethanolamine hydrochloride, pH 8.5.

[0412] The purified mAb was diluted to a concentration of 50 nM in HBS-EP wash buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Polysorbate 20). The primary antibody was selected and then injected through the flow chamber for 60 seconds at a rate of 10 g / min. After the injection was completed, a second antibody was selected and injected through the same flow chamber for 600 seconds at a rate of 10 μl / min. The sensor surface was regenerated by injecting 100 mM H3PO4 (25 μl / min) for 12 seconds.

[0413] After regeneration, the primary antibody was re-injected through the flow chamber for 600 seconds at a rate of 10 Pl / min. After the injection was completed, another secondary antibody was selected and injected through the same flow chamber for 600 seconds at a rate of 10 μl / min. After using the whole set of 14 antibodies as secondary antibodies, a new primary antibody was selected and the procedure with the new primary antibody was repeated. These procedures were carried out until all possible combinations of primary and secondary antibodies were injected through the flow chamber. Secondary antibody binding was considered to have occurred if the overall response observed after injection of both antibodies exceeded that observed for both possible thresholds. Threshold values ​​were determined using the same antibody as both primary and secondary antibody. Table 8 shows the response matrix based on the observed binding: - no secondary antibody binding, x means binding was observed (response was higher than single antibody thresholds). Grouping of clones that showed the same activity patterns provides two different epitope drawers with 1.11.1 in one drawer and all other antibodies in the other drawer.

Table 8. Response matrix for epitope shaping using the BIAcore method

<td rowspan="2">Primary mAb</td><td colspan="14">Secondary mAb</td>

<td> 1.9.1</td><td> 1.11.1</td><td>1.12.1 (Mz? Roi9A)</td><td> 1 27.3</td><td> 1.31 1</td><td> 1 162,1</td><td> 1.183.2</td><td> 4.24.2</td><td> 4.38.1</td><td> 4.58.2</td><td> 4.62.1</td><td colspan="2"> 4.68.2 4.72</td><td> 5.13</td>

<td> 1.9.1</td><td></td><td>X</td><td></td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td></td><td> -</td>

<td> 1.11.1</td><td></td><td></td><td>X</td><td> *</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>1.12.KM29I / D19A)</td><td></td><td>X</td><td>- * - «j</td><td>• and</td><td></td><td> -</td><td> -</td><td> -</td><td></td><td> -</td><td> -</td><td> -</td><td></td><td> -</td>

<td> 1.27.3</td><td></td><td>X</td><td> -</td><td></td><td></td><td> -</td><td> -</td><td> •</td><td></td><td> -</td><td> -</td><td> -</td><td></td><td></td>

<td> 1.31.1</td><td></td><td>X</td><td></td><td> -</td><td></td><td></td><td> -</td><td></td><td></td><td> -</td><td> -</td><td> -</td><td> -</td><td> •</td>

<td> 1.162.1</td><td></td><td>X</td><td> -</td><td> -</td><td></td><td></td><td> -</td><td></td><td> -</td><td> -</td><td></td><td> -</td><td> -</td><td> -</td>

<td> 1.183.2</td><td></td><td>X</td><td> -</td><td> -</td><td></td><td></td><td></td><td></td><td> -</td><td></td><td></td><td></td><td> -</td><td> -</td>

<td> 4.24,2</td><td></td><td>X</td><td></td><td> -</td><td></td><td> -</td><td> -</td><td></td><td> -</td><td></td><td></td><td></td><td> -</td><td></td>

<td> 4.38.1</td><td> -</td><td>X</td><td> -</td><td> -</td><td></td><td></td><td> -</td><td></td><td>abovementioned</td><td> -</td><td></td><td> -</td><td> -</td><td></td>

<td> 4.582</td><td></td><td>X</td><td> -</td><td> -</td><td></td><td> -</td><td> -</td><td></td><td> -</td><td></td><td></td><td> -</td><td> -</td><td></td>

<td> 4.62.1</td><td></td><td>X</td><td> -</td><td> -</td><td></td><td> -</td><td></td><td></td><td> -</td><td> -</td><td> ' *· ·’</td><td> -</td><td> -</td><td></td>

<td> 4.68 2</td><td></td><td>X</td><td> -</td><td> -</td><td></td><td> -</td><td> -</td><td></td><td> -</td><td> -</td><td></td><td></td><td> -</td><td></td>

<td> 4.72</td><td></td><td>X</td><td> -</td><td></td><td></td><td> -</td><td></td><td></td><td> -</td><td> -</td><td> -</td><td> -</td><td></td><td></td>

<td> 5.13</td><td></td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>'G.</td>

Example 10. Isolation of cynomolgus ALK-1 gene [0414] The cynomolgus ALK-1 gene ("Cyno") was taken from Cyno lung tissue. Based on the published gene sequence for human ALK-1 (register in Genebank L17075), primers for full-length ALK-1 cynomolgus PCR amplification were designed. mRNA was prepared from frozen cynomolgus lung tissue (approximately 1 g) using the mRNA purification kit (Ambion, Catalog No. 1915) according to the manufacturer's instructions. 200 ng mRNA was reverse transcribed and amplified by PCR using the OneStep RT-PCR kit (Qiagen, Catalog No. 210210) using the gene specific oligo: 5'-AGCGGGGCCCAGAGGGACCATG (Seq ID NO: 115) (compliant) and 5'-CAGAAAGGAATCAGGGCG ID NO: 116) (reverse) at a connection temperature of 61 ° C. The appropriate size RT-PCR product (~ 1.5 Kb) was excised and purified from a 0.9% agarose gel after electrophoresis, then TOPO-TA was cloned into the pCR4-TOPO vector (Invitrogen, Catalog No. K4575-01). The insert was sequenced to obtain the ORF nucleotide sequence for ALK-1 cynomolgus macaque. The translated nucleotide sequence and predicted amino acid sequence are shown in SEQ ID NOs: 93 and 94, respectively. While the cytoplasmic part of the gene encodes identical protein sequences in Cyno and human, there are 5 amino acid differences and 1 amino acid difference in the transmembrane domain of the protein in the extracellular domain (ECD, which covers positions 22-118). The ECD sequence identity between human and Cyno is 94.8%. The ECD alignment of humans and primates is shown in Figure 2.

[0415] A pair of primers (compatible: 5'-GATTAAGGCCTTGGGCTCCCCCAGGAAA (Seq ID NO: 117) and inverse: 5'-GGGCL4TTGAATCACTTTAGGCTTCTCTGGACTqT NO: 118 'primer pair was used for PCR amplification of the cynomolgus ALK-1 gene by PCR.

Example 11. Determination of cell surface binding and cross hybridization traits for primates by flow cytometry (FACS) [0416] To create cell lines with ALK-1 overexpression that can be used to test binding affinity for ALK-1 using flow cytometry (FACS) ), full-length human ALK-1 genes, Tin and rat were cloned into pcDNA5 / FRT / To TOPO vectors from Invitrogen (catalog number K6510-20) and transfected with 293 Flp-In T-Rex host cells (Invitrogen, catalog number R780-07). Selections were made using hygromycin to obtain terminal stable cell lines. Overexpression of the corresponding full-length ALK-1 proteins was achieved by induction with tetracycline (2 pg / ml) at 37 ° C / 5% CO2 for 24 hours.

[0417] Anti-ALK-1 mAbs were tested for their binding affinity for ALK-1 on the cell surface using the FACS assay using 293 stable cells overexpressing ALK-1 proteins. Cells were separated using trypsin-EDTA and washed with changed PBS-SA. After portioning into 96 well plates, cells were blocked with serum and incubated with various concentrations of specific mAb for one hour at 4 ° C. Cells were then washed and incubated with an anti-human κ secondary antibody conjugated to an R-PE phosphor prior to analysis using a FACSCalibur flow cytometer (BD Biosciences). 10,000 events were collected for each sample without any gates. Table 9 shows the geometric mean from the histogram for each sample plotted as a function of mAb concentration and the KD for each mAb calculated after fitting in a two-stage equilibrium model. Examples of equivalent FACS experiments for humans and primates are shown in Figure 3.

Table 9

<td colspan="3">Average binding affinity (Kd) results of anti-ALK-1 monoclonal antibodies</td>

<td colspan="3">with ALK-1 from the surface of human cells or Cyno as measured by FACS</td>

<td rowspan="2">Antibody</td><td colspan="2">Kd (nM)</td>

<td>human</td><td>tin</td>

<td> 1.12.1</td><td> 6,7</td><td> 2,0</td>

<td> 1.27.1</td><td> 3,7</td><td> 2,2</td>

<td> 1.162.1</td><td> 5,6</td><td> 3,0</td>

<td> 4.38.1</td><td> 9,3</td><td> 3,4</td>

<td> 4.58.1</td><td> 14,0</td><td> 6,7</td>

<td> 4.72.1</td><td> 6,4</td><td> 3,8</td>

<td> 5.13.1</td><td> 3,2</td><td> 1,6</td>

<td> 1.31.1</td><td> 3,2</td><td> 1,7</td>

<td> 4.24.1</td><td> 7,6</td><td> 3,1</td>

<td> 4.62.1</td><td> 2,3</td><td> 0,78</td>

<td> 4.68.1</td><td> 8,4</td><td> 9,0</td>

[0418] In addition, the FACS test showed that 1.12.1 has very limited cross-affinity in the rat (KD&gt; 100 nM) and is predicted to have very low cross-affinity in mice given 74% and 68% identity ECD sequences between rat / human and mouse / human ALK-1, respectively.

[0419] The FACS test was also used to determine KD for recombinant mAb 1.12.1 variants. The results shown in Table 10 indicate similar binding affinity for the recombinant antibody.

Table 10

<td colspan="3">Average binding affinity results (Kd) of variants 1.12.1 (M29I / D19A) with ALK-1 with</td>

<td colspan="3">human or Cyno cell surface measured by FACS</td>

<td rowspan="2">Antibody</td><td colspan="2">Kd (nM)</td>

<td>human</td><td>tin</td>

<td> 1.12.1</td><td> 6,7</td><td> 2,0</td>

<td>1.12.1 (RWT)</td><td> 5,9</td><td> 6,0</td>

<td>1.12.1 (M29I)</td><td> 6,0</td><td> 3,3</td>

<td>1.12.1 (D19A)</td><td> 5,7</td><td> 3,8</td>

<td>1.12.1 (M29I / D19A)</td><td> 7,2</td><td> 3,4</td>

<td>Fab 1.12.1 (M29I / D19A)</td><td> 0,77</td><td>no data</td>

<td colspan="3">1.12.1 refers to the mAb 1.12.1 variant that has been isolated from a hybridoma. 1.12.1 (rWT) refers to the variant mAb 1.12.1 that was the expressed recombinant mAb. 1.12.1 (M29I) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which methionine at position 29 in the heavy chain was replaced with isoleucine. 1.12.1 (D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing a single specific amino acid mutation in which aspartic acid at position 19 in the light chain was replaced with alanine. 1.12.1 (M29I / D19A) refers to the variant mAb 1.12.1, which was an expressed recombinant mAb containing two specific amino acid mutations (methionine at position 29 in the heavy chain replaced by isoleucine and aspartic acid at position 19 in the light chain replaced by alanine) . Fab 1.12.1 (M29I / D19A) refers to a Fab mAb 1.12.1 fragment (M29I / D19A) generated by digestion with IgG1 1.12.1 (M29I / D19A) using papain.</td>

Example 12. Tagman test for Id1 [0420] HUVEC cells (Biowhittaker, cat. No. CC-2519) were plated on 24 well plates, 12,000 cells / well in 600 μl HUVEC complete medium (EGM-2 Bullet kit, Biowhittaker, cat. No. CC-3162) and allowed to grow overnight. The next day, cells usually showed 50% confluence. Full medium was removed and 200 Pl starvation medium (EBM-2 with 0.2% FBS only) was added. Cells were incubated for 2 hours. The cells were then treated with 40 μl antibody solution in PBS. Lyophilized Ab was reconstituted in sterile PBS. Finally, the cells were treated with 1% FBS / basal medium (final concentrations) for 30 minutes, the medium was removed and while lysed in 400 μl RTL buffer (Rneasy 96 kit, Qiagen, catalog number 74182) according to the manufacturer's instructions. RNA was then prepared using the RNeasy kit (according to the manufacturer's instructions). RNA was eluted and quantified using the RiboGreen® RNA Quantitation Kit (Molecular probes, catalog number R-11490). Equal amounts of total RNA were used for real-time PCR analysis to detect Id1 RNA expression (ABI 7900 device). PCR was carried out using the Tagman One Step PCR Master Mix Kit (ABI, Cat No. 4309169) and the primer sequences / ID1 probes listed below. PCR was performed in 40 cycles under the following attachment and amplification conditions: 95 ° C, 15 seconds; 60 ° C, 1 min.

TagMan probe: CPG-5'-6-FAM and 3'-TAMRA coupling.

Name: ID1 probe

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

Taqman PCR starters:

Name: ID1-F

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

Name: ID1-R

Sequence: 5 'TTCCGAGTTCAGCTCCAACTG 3' (Seq ID NO: 121) [0421] Examples of Id1 titrations for the leader molecule 1.12.1 (M29I / D19A) (including sequence variants 1.12.1) and the Fab derivative are shown in Figures 4 and 5.

[0422] A summary of the mean IC50 values ​​from this test are shown in Table 11. All IC50 determinations were performed in triplicate.

Example 13. Smad1 phosphorylation detected using an Odyssey infrared imaging system from LI-COR Biosciences (using a 24-well plate) [0423] HUVEC cells (Biowhittaker, Cat. No. CC-2519) were plated into 24-well plates, 18,000 cells / well in 600 μl HUVEC complete medium (EGM-2 Bullet kit, Biowhittaker, cat. no. CC-3162) and allowed to grow overnight. The next day, cells usually showed 50% confluence. The complete medium was removed and 200 μl of hunger medium was added (Hunger medium: EBM-2 with 0.2% FBS only). Cells were incubated for 2 hours. The cells were then treated with 40 Pl 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 while lysed in 80 μl 1.1X sample buffer (Invitrogen, cat # NP0007). Smad1 phosphorylation was determined by Western Blotting using the X Cell Surelock Mini-Cell &amp; Blot Module (Invitrogen, catalog number EI0002). Phosphorylated Smad1 was detected using rabbit anti-phosphorSmad1 antibody (Cell Signaling, catalog number 9511), which is then detected using IRDye ™ 800 Conjugated Anti-RABBIT IgG (Rockland Immunochemicals, catalog number 611-732-127). The amount of phosphorylated Smad1 was quantified using an Odyssey infrared imaging system (Li-Cor). For normalization, actin (Santa Cruz, no. Sc8432) was used (anti-mouse Alex 680, Molecular Probe, cat. No. A-21058). A summary of the mean IC50 values ​​from this test are shown in Table 11. All IC50 determinations were performed in triplicate.

Table 11

<td>Clone</td><td>ID1 Taqman IC50 nM</td><td>pSmad1 Western IC50 nM</td>

<td> 1.11.1</td><td>no data</td><td>no data</td>

<td>1.12.1 (M29I / D19A)</td><td> 16</td><td> 18</td>

<td> 1.13.1</td><td> 100</td><td> 87</td>

<td> 1.27.1</td><td> 82</td><td> 70</td>

<td> 1.29.1</td><td> 94</td><td> 82</td>

<td> 1.31.1</td><td> 24</td><td> 21</td>

<td> 1.162.1</td><td> 75</td><td> 15</td>

<td> 1.183.1</td><td> 58</td><td> 17</td>

<td> 4.24.1</td><td> 100</td><td> 82</td>

<td> 4.38.1</td><td> 87</td><td> 52</td>

<td> 4.58.1</td><td> 14</td><td> 15</td>

<td> 4.62.1</td><td> 24</td><td> 34</td>

<td> 4.68.1</td><td> 141</td><td> 110</td>

<td>Clone</td><td>ID1 Taqman IC50 nM</td><td>pSmad1 Western IC50 nM</td>

<td> 4.72.1</td><td> 21</td><td> 35</td>

<td> 5.13.1</td><td> 30</td><td> 68</td>

Example 14. Properties of internalization of anti-ALK-1 monoclonal antibodies [0424] FACS was used to monitor the temporal course of surface remaining on the ALK-1 receptor cell as well as the neutralizing antibody. ALK1 remaining on the cell surface is monitored using a marker antibody that is capable of binding to ALK-1 on the cell surface, however recognizing a different epitope than the neutralizing antibody. Mouse anti-human ALK-1 ECD mAb (R&amp;D sytems, catalog number AF310) was identified and used as a marker antibody in the study.

[0425] The time course of internalization was examined using the HUVEC and HUAEC endothelial cell lines. Cells were cultured at 37 ° C and 5% CO 2 in 24 well plates containing 200 μl complete culture medium per well. At each of 11 time points over 48 hours, 2 μl of a 1 mg / ml antibody solution was added to one well and mixed (final neutralizing antibody concentration is 10 μg / ml). The plate is then placed back in a 37 ° C incubator up to the 0 o'clock time point when the plate is placed on ice to stop the internalization process. At this point, marker antibody (up to a final concentration of 10 μg / ml) was added to the wells and incubated on ice for one hour. The cells were then washed with PBS, separated by trypsination and transferred to a 96 well plate. Cells were then washed, blocked and treated with secondary antibodies carrying various phosphors to depict both neutralizing antibody and receptor ALK-1 remaining on the cell surface. Samples were evaluated using a FACSCalibur flow cytometer, counting 3000-5000 events / sample. The geometric mean of each sample in the specified fluorescence channel was calculated and plotted as a function of time. The data was fitted to the modified radioactive decay equation to obtain the half-life (t1 / 2) of internalization as well as the percentage of neutralizing antibody or ALK-1 receptor remaining on the cell surface after stabilization of internalization. As shown in Figure 6, mAb 1.12.1 (M29I / D19A) is internalized at the same rate and to the same extent as the ALK-1 receptor from the cell surface. The half-life of internalization 1.12.1 (M29I / D19A) is ~ 2 hours. Equilibrium was reached when 50% of the antibody was internalized. A polyclonal antibody purchased from R&amp;D systems (catalog number AF370) internalizes at t1 / 2 equal to one hour and achieves stabilization at internalization of ~ 70% of the receptor (Figure 6). Similar internalization properties were observed for other human anti-ALK-1 mAbs of the disclosure (not shown).

Example 15. Development of chimney SCID mice with human foreskin [0426] The surgical procedure previously published by HC Yan, et al. Has been significantly modified. "Human / Severe Combined Immunodeficient Mouse Chimeras, An Experimental In Vivo Model System to Study the Regulation of Human Endothelial CellLeukocyte 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 from the National Disease Research Institute and Cooperative Human Tissue Network, fragments of the human foreskin were cut off unhealthy areas and transferred to RPMI media (Cellgro / Mediatech, cat. No. MT-15-040-CV supplemented with penicillin and streptamycin (Gibco / Life Tech No. 15070-063) (add 5 ml pen / strep stock solution to 500 ml RPMI). Using a scalpel and cutting on a Petri dish, the skins were cut to an oval shape approximately 8 x 13 mm in size, cleaned of shredded ends and connective tissue, and stored on wet ice before surgery. Appropriate volume (4 μl / gram animal) of 100 mg / ml ketamine solution (Ketaset^TR, Fort Dodge Animal Health) / 1 mg / ml medetomidine (Pfizer Animal Health - Dormitor) was injected intraperitoneally in the abdomen of scid mice (i.e. at an angle of 45 °, under the skin, but not very deep inward). After dormancy, mice were introduced into the eye glidant and ketoprofen (10 mg / kg, Fort Dodge Animal Health) was injected subcutaneously and the skin was shaved at the surgical site. The operating region was surgically removed three times with Clorahexiderm (Butler, Chclo-Scrub 40, cat. No. WAB20109) followed by alcohol in circular motions starting from the center of the surgical site and then outwards avoiding moving from a dirty area back to clean. Mice were transferred to the prepared surgical hood and placed on a water-heated base (Gaymar Industries, catalog number TP500 T / Pump), which was kept at 37 ° C. Mice were then anesthetized with isofluorane for the duration of the operation. Dorsal mice were covered with a surgical sheet with a notch revealing the surgical site. The mouse skin was lifted with forceps and an oval piece of skin tissue was cut off with one move using curved scissors. The right size human foreskin was placed on the mice. Human and mouse skin were sewn together using the Ethilon seam (Ethicon Cat. No. 697H.), Starting from the top of the oval, then down, then the furthest part from the right and left. More sutures were made in between to connect the tissues more tightly. Around 8 stitches were made around the skin at equal distances from each other. A sterile saline syringe was used during surgery to hydrate the skin / surgical wound of the mice when they became dry. Bandaid was applied to the wound. A transparent wrapper was wrapped loosely around the bandage (3M Tegaderm^™). The wrapper was cut to such a size that it covered an area slightly wider than the Bandaid dressing. Mice were then given Atipamezol (50-100 gl, Pfizer Animal Health - Antisedan) and allowed to recover in a heated cage for 5 - 10 min. The dressing and bandage were removed after 7-10 days, and by day 15 most of the skin looked like a scab. Complete healing occurred between 21-28 days, after which the skins were ready to inoculate the cancer cells. Figure 8 shows an example of histological analysis (H and E staining) of a section of transplanted skin after surgery. The histological structure of transplanted skin closely resembles the features of human skin transplanted to the 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: Mol. Cancer. Ther. 2 (8): 729, 2003. he: layer of human epidermis; hd: a layer of human dermis.

Example 16. Collagen model in the SCID mouse chimera with human foreskin [0427] Stock collagen I (Cat. No. 354236, Becten-Dickinson) was diluted to 4 mg / ml with 0.02 N acetic acid and kept on ice before implantation. Acid collagen solution (8 parts) was mixed with 10X M199 (Sigma, catalog number M9163) (1 part) and human plasma fibronectin (catalog number) 354008, Becten-Dickinson) to achieve a final Fn concentration of 90 gl / ml; NaOH (1.0 N) was added to bring the pH to ~ 7.2. The collagen / Fn mixture was kept on ice until use. The implantation mixture was prepared using the above collagen / Fn mixture with the addition of an angiogenesis inhibitor of interest and with or without the addition of human large vessel endothelial cells (HMVEC) (Cascade Biologics, cat. No. C-010-5C). HMVEC cells were prepared in the form of 6 x 10⁶ cells / ml in PBS. 50-100 μl of implanted bourgeois were injected intradermally into the foreskin in a chimeric scid mouse. 7-14 days later collagen plugs were collected, embedded in OCT compound (cat. No. 4583, Sajura Finetek, CA) and immediately frozen for immunohistochemical analysis. The collagen plug in the foreskin was identified using the Trichrome kit (cat. No. KC1641, Mater Tech, CA) as the blue staining shown in Figure 9 (A). Human vessels were identified by staining of human P-CAM using anti-human CD-31 antibody (Clone 13.3, Vector Laboratories) (Figure 9 (B)). Table 12 summarizes the staining and quantification of human vessels in a collagen model in the SCID mouse human foreskin chimera.

Table 12. Summary of results with the collagen model

<td>Matrix</td><td>HMVEC in the matrix</td><td>Experimental procedure (Rx)</td><td>Days Rx</td><td>points final survey</td><td>Counting human vessels (1 x 10³)</td><td>% control (human dishes)</td>

<td>1.6 mg / ml collagen</td><td>lack</td><td>lack</td><td> 4</td><td rowspan="6">coloration human CD-31</td><td> 0,036 ± 0,001</td><td> 40</td>

<td>1.6 mg / ml collagen</td><td>7 x 10³</td><td>lack</td><td> 4</td><td> 0,071 ± 0,022</td><td> 78</td>

<td>1.6 mg / ml collagen</td><td>1.4 x 10⁴</td><td>lack</td><td> 4</td><td> 0,063 ± 0,016</td><td> 69</td>

<td>2.4 mg / ml collagen</td><td>lack</td><td>lack</td><td> 4</td><td> 0,091 ± 0,056</td><td> 100</td>

<td>2.4 mg / ml collagen</td><td>7 x 10³</td><td>lack</td><td> 4</td><td> 0,067 ± 0,049</td><td> 74</td>

<td>2.4 mg / ml collagen</td><td>1.4 x 10⁴</td><td>lack</td><td> 4</td><td> 0,062 ± 0,047</td><td> 68</td>

<td>3.0 mg / ml collagen</td><td>8.8 x 10³</td><td>lack</td><td> 4</td><td rowspan="3">coloration human CD-31</td><td> 54 ± 9</td><td> 100</td>

<td>3.0 mg / ml collagen</td><td>8.8 x 10³</td><td>Isotype 100 control antibody μg / ml mixed in the gel</td><td> 4</td><td> 52 ± 13</td><td> 96</td>

<td>3.0 mg / ml collagen</td><td>8.8 x 10³</td><td>Antibody 1.12.1 (M29I / D19A) 100 μg / ml mixed in the gel</td><td> 4</td><td> 15 ± 3</td><td> 28</td>

<td>3.0 mg / ml collagen</td><td>lack</td><td>lack</td><td> 4</td><td rowspan="2">coloration human CD-31</td><td> 0,112 + 0,026</td><td> 100</td>

<td>5.0 mg / ml collagen</td><td>lack</td><td>lack</td><td> 4</td><td> 0,031 + 0,012</td><td> 28</td>

<td>3.0 mg / ml collagen</td><td>lack</td><td>Isotype 100 control antibody gg / ml injection id.</td><td> 4</td><td rowspan="2">coloration human CD-31</td><td> 75 ± 15</td><td> 100</td>

<td>3.0 mg / ml col</td><td>lack</td><td>Antibody</td><td> 4</td><td> 39 ± 11</td><td> 52</td>

<td>Matrix</td><td>HMVEC in the matrix</td><td>Procedure experimental (Rx)</td><td>Days Rx</td><td>points final survey</td><td>Counting human vessels (1 x 10³)</td><td>% control (human dishes)</td>

<td>gene</td><td></td><td>1.12.1 (M29I / D19A) 100 gg / ml injection id.</td><td></td><td rowspan="2"></td><td></td><td></td>

<td>3.0 mg / ml collagen</td><td>lack</td><td>Antibody 1.14.1 100 gg / ml injection id.</td><td> 4</td><td> 44 ± 28</td><td> 59</td>

Example 17. M24met tumor model in the SCID mouse human foreskin chimney [0428] In these studies, mice with an implant between 5-10 weeks after surgery were typically used. The M24met cell line was described by Mueller and co-workers in "Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells", Cancer Research, 55 (8): 1629-32, 1995. The M24met cell suspension was prepared as follows: M24met cells with 80% confluency were washed, trypsinized with trypsin / EDTA (Gibco, catalog number 25200-056) and harvested in PRMI medium (Cellgro / Mediatech, catalog number MT-15-040 -CV) supplemented with 10% FBS (Cellgro / Mediatech, catalog number AKD-11775) and 2 mM L-glutamine (Cellgro / Mediatech, catalog number 25-005-CI). Cells were centrifuged at 600 rpm for 5 min, resuspended in sterile PBS. Cell counts were estimated using a Coulter Counter (Beckman Coulter, Model Z2). The cells were centrifuged at 600 rpm for 5 min and resuspended in a collagen-Fn mixture (3 mg / ml) to obtain a 4x10 suspension⁷ cells / ml for implantation.

[0429] For inoculation 2x10⁶ the above cells were injected (50 μl 4x10⁷ cells / ml) intradermally to human skin of transplanted mice. On day 5-7 after implantation, tumors will be palpable and the mice are randomly divided into control and experimental groups prior to dosing. The control group was defined in such a way that the animals do not receive any doses or receive a vehicle dose in which the anti-ALK-1 antibody or the isotype dose of the corresponding IgG2 anti-KLH human monoclonal antibody (Pfizer Inc) has been reconstituted. The experimental group was determined in such a way that the animals receive a dose of anti-ALK-1 antibody 1.12.1 (M29I / D19A).

Example 18. Double immunofluorescence (IF) staining of human and mouse CD-31 [0430] Frozen tissue sections were air dried and fixed at -20 ° C in acetone (Fisher, cat. No. A16S-4) for 10 min. The samples were air-dried again and washed in PBS three times for 5 min each. Samples were blocked in 5% rabbit serum (Vector Laboratories, catalog number S-5000) in PBS for 30 min at room temperature. The primary antibody mixture was prepared in 5% rabbit serum with anti-human CD-31 antibody (Santa Cruz, catalog number SC1505) and anti-mouse CD-31 (Pharmingen, Clone Mec1 3.3, catalog number 01951A) at a dilution of 1: 100 and 1: 150. The above antibody mixture was added to tissue samples for one hour at RT. The blocks were washed in PBS three times for 5 min each before incubation with the secondary antibody mixture for one hour at RT. A mixture of secondary antibodies was prepared in PBS / 0.05% Tween-20 (Sigma, catalog number P1379), rabbit anti-goat antibody from Texas Red (Jackson Labs, catalog number 305-075-003) and rabbit anti-rat antibody from FITC (Jackson Labs, cat. No. 312-095-003). Antibodies were diluted 1:50 when frozen antibodies were used or 1: 100 when fresh antibodies were used. Sections were washed again in PBS three times 5 min before embedding in Vectashield (Hard Set, DAPI embedding medium, Vector Lab, CA, catalog number H-1500). Sections were kept in the dark and 4 ° C until image analysis. Imaging analysis was performed using an Olympus BX60 fluorescence microscope and photos were taken using an Olympus microfire color digital camera. Pictures were taken from 3-5 hot spots / section, from one section / animal, from 4-7 animals / group and the areas of the vessels indicated by positive anti-human CD-31 staining were quantified by three people using Image Pro Plus v4.5 (MediaCybernetics). The pharmacodynamic endpoint (group average) was expressed as a percentage of inhibition of human CD-31 compared to the control group or as the total area of ​​human vessels. Statistical significance was determined in the ANOVA test. Figure 10 shows an immunofluorescent image of human (red) and mouse (green) vessels in an M24met tumor in a human chimeric SCID mouse foreskin.

Example 19. Immunohistochemical (IHC) staining of human CD-31 [0431] Frozen tissue sections were air dried and fixed at -20 ° C in acetone for 10 min. The samples were air-dried again and washed in PBS twice for 5 min. Samples were incubated in 0.075% H2O2 / methanol (Fisher Cat. No. A433-4) for 15 min and washed in PBS three times for 5 min each. Samples were blocked in 5% rabbit serum / PBS for 30 min and coated with 1: 100 anti-human CD-31 antibody (Santa Cruz, cat. No. SC1505) in 5% rabbit serum for one hour at RT. Samples were washed in PBS twice for 5 min and coated with 1: 200 rabbit anti-goat antibody (Vector Labs, cat. No. BA-5000) in 5% rabbit serum for 35 min at RT. The sections were then washed in PBS twice for 5 min and freshly prepared streptavidin (Vector Labs, ABC Elite kit, cat. No. PK-6100) was added. Sections were again washed in PBS two times for 5 min and then developed in diaminobenzidine (DAB) (Vector Labs, catalog number SK-4100). Sections were washed in PBS twice for 5 min and then in Mayers hematoxylin (Sigma, cat. No. HHS-32) for 5 seconds. The samples were well rinsed in diH2O and immersed twice in a dilute (5 ml stock solution in 1L diH2O) ammonium hydroxide solution (Sigma, cat. No. A-6899) ​​and washed again in diH2O. The samples were then dehydrated in 70%, 90% and then 100% alcohol (Harleco, catalog number 65347/85) after 1 minute each and finally in xylene (JT Baker, catalog number 516.09). Sections were embedded in Cytoseal 60 (Stephens Scientific, catalog no. 8310-4) and covered with coverslips for imaging analysis. Figure 11 shows an IHC image of human vessels (brown) in an M24met tumor in a human chimeric SCID mouse foreskin.

Example 20. Therapeutic treatment with anti-ALK-1 antibody 1.12.1 (M29I / D19A) [0432] For treatment, dosing was carried out subcutaneously (sc) or intravenously (iv). Typically, one dose of 1.12.1 antibody (M29I / D19A) was administered in each study. A second dose of anti-ALK-1 antibody, if necessary, was administered on days 9 or 10. Multiple dose levels, e.g. 1, 5, 10, 50 mg / kg, have sometimes been administered to test the dose-dependent inhibition of human vascular growth. Animals were monitored daily and tumors were measured three times / week using a caliper. By day 14-17 tumors had a size between 250-350 mm³ and were removed from the mice, embedded in OCT and frozen for IF or IHC analysis. Figure 12 shows exemplary immunofluorescent images of human (red) and mouse (green) vessels in control and treated with 1.12.1 (M29I / D19A) (10 mg / kg) M24met tumors in a human foreskin scid chimera. Dose-dependent inhibition of human tumor vessel growth by 1.12.1 (M29I / D19A) in the human foreskin SCID chimera model is shown in Figure 13 and a summary of related studies is shown in Table 13.

Table 13

<td colspan="8">Summary of in vivo model properties and inhibition of human vascular growth in tumor</td>

<td colspan="8">Zach M24met in the SCID chimera model</td>

<td colspan="5">Protocol parameters</td><td colspan="2">Endpoints</td><td rowspan="2">General thoughts</td>

<td>Bump</td><td>Bow</td><td>Dose</td><td>Road</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day survey</td>

<td>MCF-7</td><td>lack</td><td>not applicable</td><td>no established</td><td>not established</td><td>not marked</td><td> 19</td><td>Tumors were implanted intradermally. Tested with and without implant from estradiol and collagen. The tumors grew slowly and showed low expression of human CD31</td>

<td>M24met</td><td>lack</td><td>no established</td><td>no established</td><td>not established</td><td>not marked</td><td> 19</td><td>Tumors were implanted intradermally. Tested with and without collagen / FN matrix. A larger tumor growth was found with the matrix, all subsequent studies will include the additive matrix. Tumors showed good human staining CD31</td>

<td>M24met (small)</td><td>lack</td><td>no established</td><td>no established</td><td>not established</td><td>not marked</td><td> 9</td><td>Tumor size &lt;100 mm³. Little human CD31</td>

<td>M24met (average)</td><td>lack</td><td>no established</td><td>no established</td><td>not established</td><td>not marked</td><td> 12</td><td>Tumor size &lt;100-200 mm³. A bit human</td>

<td colspan="8">Summary of in vivo model properties and inhibition of human vascular growth in tumor</td>

<td colspan="8">Zach M24met in the SCID chimera model</td>

<td colspan="5">Protocol parameters</td><td colspan="2">Endpoints</td><td rowspan="2">General thoughts</td>

<td>Bump</td><td>Bow</td><td>Dose</td><td>Road</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day survey</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>CD31</td>

<td>M24met (big)</td><td>lack</td><td>no established</td><td>no established</td><td>not established</td><td>not marked</td><td> 12</td><td>Tumor size &lt;200 mm³. Big tumors M24met have higher numbers of stained human vessels, future tests will be carried out with larger tumors</td>

<td rowspan="2">M24met</td><td>Non-specific human IgG</td><td>10 mg / kg</td><td>IV</td><td rowspan="2">2 doses (days 5 and 9)</td><td> 0</td><td rowspan="2"> 15</td><td rowspan="2">First screening. 1.12.1 (M29I / D 19A) showed a significant reduction in staining of human CD31. No tumor growth inhibition was observed.</td>

<td>1.12.1 (M2 9I / D19A)</td><td>10 mg / kg</td><td>IV</td><td> 42</td>

<td rowspan="2">M24met</td><td>Non-specific human IgG</td><td>10 mg / kg</td><td>IV</td><td rowspan="2">2 doses (days 5 and 10)</td><td> 0</td><td rowspan="2"> 14</td><td rowspan="2">Second screening. Confirmed the results of GW-366. No tumor growth inhibition was observed.</td>

<td>1.12.1 (M2 9I / D19A)</td><td>10 mg / kg</td><td>IV</td><td> 40</td>

<td>M24met</td><td>Non-specific human IgG</td><td>10 mg / kg</td><td>SC</td><td rowspan="2">2 doses (days 5 and 10)</td><td> 0</td><td rowspan="2"> 14</td><td rowspan="2">First dose-dependent activity test 1.12.1 (M29I / D 19A) against human</td>

<td></td><td>1.12.1 (M2 9I / D19A)</td><td>10 mg / kg</td><td>SC</td><td> 43</td>

<td colspan="8">Summary of in vivo model properties and inhibition of human vascular growth in tumor</td>

<td colspan="8">Zach M24met in the SCID chimera model</td>

<td colspan="5">Protocol parameters</td><td colspan="2">Endpoints</td><td rowspan="3">General thoughts CD31. A dose-dependent effect has been observed. PK shows that one dose will be enough.</td>

<td>Bump</td><td>Bow</td><td>Dose</td><td>Road</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day survey</td>

<td></td><td></td><td></td><td></td><td rowspan="3"></td><td></td><td rowspan="3"></td>

<td></td><td>1.12.1 (M2 9I / D19A)</td><td>1 mg / kg</td><td>SC</td><td> 50</td><td rowspan="2">for significant CD31 reduction. No tumor growth inhibition was observed.</td>

<td></td><td>1.12.1 (M2 9I / D19A)</td><td>0.1 mg / kg</td><td>SC</td><td> 20</td>

<td rowspan="6">M24met</td><td>No dose</td><td>0 mg / kg</td><td>no established</td><td>not established</td><td>ND</td><td rowspan="6"> 16</td><td rowspan="6">Second dose-dependent activity test 1.12.1 (M29I / D 19A) to human CD31. A clear dose-dependent effect relative to CD31. No tumor growth inhibition was observed.</td>

<td>IgG matched isotype</td><td>10 mg / kg</td><td>SC</td><td rowspan="5">One dose (5. day)</td><td> 0</td>

<td>Non-specific human IgG</td><td>10 mg / kg</td><td>SC</td><td>ND</td>

<td>1.12.1 (M2 9I / D19A)</td><td>1 mg / kg</td><td>SC</td><td> 24</td>

<td>1.12.1 (M2 9I / D19A)</td><td>5 mg / kg</td><td>SC</td><td> 59</td>

<td>1.12.1 (M2 9I / D19A)</td><td>10 mg / kg</td><td>SC</td><td> 72</td>

<td rowspan="6">M24met</td><td>IgG matched isotype</td><td>10 mg / kg</td><td>SC</td><td rowspan="6">One dose (5. day)</td><td> 0</td><td rowspan="6"> 14</td><td rowspan="6">Final test with a wide range of doses for 1.12.1 (M29I / D 19A). The study showed good dose-dependent effects. Sigmoid data matching</td>

<td>1.12.1 (M2 9I / D19A)</td><td>1 mg / kg</td><td>SC</td><td> 33</td>

<td>1.12.1 (M2 9I / D19A)</td><td>3 mg / kg</td><td>SC</td><td> 41</td>

<td>1.12.1 (M2 9I / D19A)</td><td>5 mg / kg</td><td>SC</td><td> 60</td>

<td>1.12.1 (M2 9I / D19A)</td><td>7.5 mg / kg</td><td>SC</td><td> 60</td>

<td>1.12.1 (M2</td><td> 10</td><td>SC</td><td> 73</td>

<td colspan="8">Summary of in vivo model properties and inhibition of human vascular growth in tumor</td>

<td colspan="4">Zach M24met in the SCID chimera model</td><td></td><td></td><td></td><td></td>

<td colspan="2">Protocol parameters</td><td></td><td></td><td></td><td colspan="2">Endpoints</td><td></td>

<td>Bump</td><td>Bow</td><td>Dose</td><td>Road</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day survey</td><td>General thoughts</td>

<td></td><td>9I / D19A)</td><td>mg / kg</td><td></td><td></td><td></td><td></td><td>curve from</td>

<td></td><td>1.12.1 (M2 9I / D19A)</td><td>50 mg / kg</td><td>SC</td><td></td><td> 70</td><td></td><td>say the dose it gives IC50 93 nM. No tumor growth inhibition was observed.</td>

Example 21. In vivo EC50 determination [0433] Chimerom SCRE mice with human foreskin were intradermally implanted with M24met cells and treated (sc) with anti-ALK-1 1.12.1 antibody (M29I / D19A) at 1, 3, 5, 7.5, 10 and 50 mg / kg or isotype matched anti-human KLH antibody (10 mg / kg). As a result of the experiment, the area of ​​human vessels in each tumor was quantified as described above. Anti-ALK-1 1.12.1 (M29I / D19A) concentrations in mouse plasma were measured using the following method: serum samples from mice were analyzed for the concentration of anti-ALK-1 1.12.1 antibody (M29I / D19A) by ELISA (enzyme-linked immunosorbent assay) ). ELISA plates were coated with 10 µg / ml goat specific anti-human IgG Fc antibody (Pierce, cat # 31123) in PBS, incubated overnight at 4 ° C and then blocked with StartBlock blocking buffer (Pierce, cat. No. 37542) at room temperature for an hour. Serum samples were diluted 100 and 1000-fold in StartBlock blocking buffer prior to analysis. Two sets of serum standards were prepared as blank dilutions of 100 and 100 fold. Standards and diluted serum samples were incubated on the plate for an hour. Bound anti-ALK-1 1.12.1 antibody (M29I / D19A) was detected using horseradish peroxidase-labeled (HRP) goat anti-human IgG (Fab-specific) antibody (Sigma, Cat. No. A0293). The substrate used was 3.3 ', 5.5'-tetramethylbenzidine (Sigma, catalog number T8665). Absorbance was read at 450 nm using a Vmax plate reader (Molecular Devices, Menlo Park, CA). The standard curve was fitted using nonlinear regression. The detection limit for this test was 10 ng / ml anti-ALK-1 1.12.1 antibody (M29I / D19A).

[0434] The concentration of anti-ALK-1 1.12.1 (M29I / D19A) antibody in the plasma of SCID mice is shown in Figure 15.

[0435] Figure 15 shows the estimated EC50 for 1.12.1 (M29I / D19A) in the M24met chimera model of SCID foreskin. The area of ​​human vessels was plotted versus mean plasma PK over the entire study period (14 days) for each experimental group. The fitted curve was generated in the Sigmoidal Dose Dependent program from Graphpad (Prizm). An EC50 of 93 ng / ml was obtained from the fit curve (EC50 is defined as the plasma concentration required for a 50% reduction in the area of ​​human vessels in the control group).

[0436] Preferred points of the present disclosure are disclosed below and designated as E1 to E24.

E 1. A monoclonal antibody or antigen binding portion that binds to ALK-1 comprising a first variable domain comprising SEQ ID NO: 6 and a second variable domain comprising SEQ ID NO: 8.

E 2. The monoclonal antibody of E 1, comprising the amino acid sequence of SEQ ID NO: 2 heavy chain and containing the amino acid sequence of SEQ ID NO: 4 light chain.

E 3. The monoclonal antibody of E 1, comprising the amino acid sequence of SEQ ID NO: 100 heavy chain and comprising the amino acid sequence of SEQ ID NO: 102 light chain.

E 4. The monoclonal antibody according to E 1, comprising a heavy chain comprising SEQ ID NO: 6 and a light chain comprising SEQ ID NO: 8.

E 5. The monoclonal antibody according to E 1 or 4, wherein the monoclonal antibody is selected from IgG1 or IgG2.

E 6. A human monoclonal antibody or antigen binding portion thereof that binds to ALK-1 and which has at least one additional property selected from the group consisting of:

a) binds to the extracellular domain of ALK-1 primates at an avidity value of 5 nM or less, measured by surface plasmon resonance;

b) binds to the extracellular domain of human ALK-1 at an avidity value of 250 pM or less as measured by surface plasmon resonance;

c) has a dissociation rate constant (koff) for human ALK-1 equal to 5 x 10^-3 s^-1 or lower, measured by surface plasmon resonance;

d) binds to ALK-1 primates with a KD of 50 nM or less, measured by flow cytometry;

e) has a KD (rodent) / KD (primate) that is greater than 1.5;

f) has an IC 50 of 150 nM or lower, measured on the basis of their ability to inhibit regulation leading to stimulation of a specific downstream target gene for ALK-1, Id1;

g) has an IC 50 of 150 nM or less, measured on the basis of their ability to inhibit Smad1 phosphorylation determined by Western Blot;

h) inhibits human vascular angiogenesis in SCID mice transplanted with human foreskin tissue in which human M24met melanoma tumor cells are intradermally implanted as determined by IHC analysis using a human CD-31 signaling assay by at least 40% compared to sample control;

i) inhibits human vascular angiogenesis in SCID mice transplanted with human foreskin tissue in which collagen is intradermally implanted as determined by IHC analysis using human CD-31 signaling assay by at least 50% compared to the control sample;

j) competes for binding to ALK-1 with an antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (M29I / D19A); 1.12.1 (M29I); 1.12.1 (D19A); 1.12.1 (RWT); 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;

k) cross-competes for binding to ALK-1 with an antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (M29I / D19A); 1.12.1 (M29I);

1.12.1 (D19A); 1.12.1 (RWT); 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;

l) binds to the same ALK-1 epitope as the antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (M29I / D19A); 1.12.1 (M29I); 1.12.1 (D19A);

1.12.1 (RWT); 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;

m) binds to ALK-1 at substantially the same KD as the antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (M29I / D19A); 1.12.1 (M29I); 1.12.1 (D19A); 1.12.1 (RWT); 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; and

n) binds to ALK-1 with substantially the same koff as the antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (M29I / D19A); 1.12.1 (M29I); 1.12.1 (D19A); 1.12.1 (RWT); 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.

E 7. The antibody or antigen binding portion thereof of E 6, comprising a VH domain that is at least 90% identical in amino acid sequence to any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104.

E 8. The antibody or antigen binding portion thereof of E 6, comprising a VL domain that is at least 90% identical in amino acid sequence to any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127.

E 9. The antibody or antigen binding portion thereof according to E 6, wherein the VH domain is selected from any one of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence that differs from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, at least one conservative amino acid substitution, wherein the sequence with at least one conservative amino acid substitution is identical in amino acid sequence to at least 90% with the unsubstituted sequence, and the VL domain is independently selected from any of SEQ ID NO: 8 ; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or a sequence that differs from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence.

E 10. The antibody or antigen binding portion thereof according to E 6, wherein said antibody or portion is selected from the group consisting of:

(a) the antibody or portion that comprises the VH CDR1, CDR2 and CDR3 sequences independently selected from the heavy chain CDR1, CDR2 or CDR3 sequences respectively in any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence that differs from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence; and (b) the antibody or portion that comprises the VR CDR1, CDR2 and CDR3 sequences independently selected from the light chain CDR1, CDR2 and CDR3 sequences respectively found in any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127 or se99 a sequence that differs from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence.

E 11. The antibody or antigen binding portion according to E 10, wherein said antibody or portion is selected from the group consisting of:

(a) an antibody or portion that comprises the VH CDR1, CDR2 and CDR3 sequences found in any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence that differs from any of SEQ ID NO: 6; 10; 14; 18; 22; 26; thirty; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence; and (b) an antibody or portion that comprises the VL CDR1, CDR2 and CDR3 sequences found in any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or a sequence that differs from any of SEQ ID NO: 8; 12; 16; twenty; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence.

E 12. The antibody or worship according to E 10 or 11, selected from the group consisting of:

a) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 6 or different from SEQ ID NO: 6 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 8 or different from SEQ ID NO: 8 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

b) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 10 or different from SEQ ID NO: 10 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 12 or different from SEQ ID NO: 12 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

c) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 14 or different from SEQ ID NO: 14 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 16 or different from SEQ ID NO: 16 at least one conservative amino acid substitution, said sequence with

100 at least one conservative amino acid substitution is identical in amino acid sequence to at least 90% with the unsubstituted sequence;

d) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 18 or different from SEQ ID NO: 18 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 20 or different from SEQ ID NO: twenty at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

e) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 22 or different from SEQ ID NO: 22 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 24 or different from SEQ ID NO: 24 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

f) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 26 or different from SEQ ID NO: 26 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 28 or different from SEQ ID NO: 28 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

g) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 30 or different from SEQ ID NO: 30 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 32 or different from SEQ ID NO: 32 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

h) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 34 or different from SEQ ID NO: 34 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 36 or different from SEQ ID NO: 36 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being identical

101 in terms of amino acid sequence at least 90% with unsubstituted sequence;

i) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 38 or different from SEQ ID NO: 38 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 40 or different from SEQ ID NO: 40 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

j) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 42 or different from SEQ ID NO: 42 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 44 or different from SEQ ID NO: 44 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

k) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 46 or different from SEQ ID NO: 46 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 48 or different from SEQ ID NO: 48 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

l) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 50 or different from SEQ ID NO: 50 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 52 or different from SEQ ID NO: 52 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

m) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 54 or different from SEQ ID NO: 54 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 56 or different from SEQ ID NO: 56 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

102

n) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 58 or different from SEQ ID NO: 58 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 60 or different from SEQ ID NO: 60 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

o) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 62 or different from SEQ ID NO: 62 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 64 or different from SEQ ID NO: 18 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

p) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 66 or different from SEQ ID NO: 66 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 68 or different from SEQ ID NO: 68 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

g) the antibody or antigen-binding portion thereof that comprises a VH domain as shown in SEQ ID NO: 70 or different from SEQ ID NO: 70 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 72 or different from SEQ ID NO: 72 at least one conservative amino acid substitution, wherein said sequence with at least one conservative amino acid substitution is identical in amino acid sequence to at least 90% with the unsubstituted sequence;

r) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 74 or different from SEQ ID NO: 74 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 76 or different from SEQ ID NO: 76 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

s) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 78 or different from SEQ ID NO: 78 by at least one conservative

103 amino acid substitution, wherein the sequence with at least one conservative amino acid substitution is identical in amino acid sequence to at least 90% with the unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 80 or different from SEQ ID NO: 80 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

t) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 82 or different from SEQ ID NO: 82 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 84 or different from SEQ ID NO: 84 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

u) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 86 or different from SEQ ID NO: 86 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 88 or different from SEQ ID NO: 88 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

v) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 90 or different from SEQ ID NO: 90 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 92 or different from SEQ ID NO: 92 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

w) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 104 or different from SEQ ID NO: 104 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 127 or different from SEQ ID NO: 127 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence;

x) an antibody or portion thereof that contains a VH domain as shown in SEQ ID NO: 6 or different from SEQ ID NO: 6 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 127 or different from SEQ ID NO: 127 at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution being at least 90% identical in amino acid sequence to the unsubstituted sequence; and

y) the antibody or portion thereof that contains the VH domain as shown in SEQ ID NO: 104 or different from SEQ ID NO: 104 with at least one conservative amino acid substitution, said sequence with at least one conservative amino acid substitution is identical in terms of amino acid sequence at least 90% with unsubstituted sequence, and the VL domain as shown in SEQ ID NO: 8 or different from SEQ ID NO: 8 at least one conservative amino acid substitution, wherein the sequence with at least one conservative amino acid substitution is at least 90% identical in amino acid sequence to the unsubstituted sequence,

E 13. The antibody or antigen binding portion thereof according to E 5, which antibody or antigen binding portion comprises a heavy chain that utilizes the human VH 4-31, VH 3-11, VH 3-15, VH 3-33, VH 4-61 gene or VH 4-59, or in which at least one conservative amino acid substitution occurs in the human VH 4-31, VH 3-11, VH 3-15, VH 3-33, VH 4-61 or VH 4-59 gene, wherein the sequence with at least one conservative amino acid substitution is identical in amino acid sequence to at least 90% with the unsubstituted sequence.

E 14. The antibody or antigen binding portion thereof according to E 6, which antibody or antigen binding portion comprises a light chain that utilizes the human Vk A27, Vk A2, Vk A1, Vk A3, Vk B3, Vk B2, Vk L1 or Vk L2 gene, or in which at least one conservative amino acid substitution occurs in the human Vk A27, Vk A2, Vk A1, Vk A3, Vk B3, Vk B2, Vk L1 or Vk L2 gene, wherein the sequence with at least one conservative amino acid substitution is identical in amino acid sequence to at least 90% with the unsubstituted sequence.

E 15. The antibody according to any of E 6-14, which is or is derived from an IgG, IgM, IgE, IgA, or IgD molecule.

E 16. An isolated nucleic acid molecule comprising a nucleotide sequence as set out in any of SEQ ID NOs: 1, 3, 5, 7, 95, 101, 103, 126, 128 or 129.

E 17. An isolated nucleic acid molecule comprising a nucleotide sequence as set out in any of SEQ ID NOs: 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 or 91.

E 18. Hybridoma deposited with ATCC under accession number PTA-6808.

E 19. The antibody produced by the hybridoma of E 18, or antigen binding portion thereof.

E 20. The antibody or antigen binding portion thereof that binds to ALK-1, wherein the antibody or antigen binding portion comprises an amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 2;

b) SEQ ID NO: 4;

c) SEQ ID NO: 6;

d) SEQ ID NO: 8;

e) SEQ ID NO: 100;

105

f) SEQ ID NO: 102;

g) SEQ ID NO: 104;

h) SEQ ID NO: 127;

i) the VH amino acid sequence encoded by the nucleotide sequence of the insert present in the clone deposited at ATCC under accession number PTA-6864; and

j) the VL amino acid sequence encoded by the nucleotide sequence of the insert present in the clone deposited at ATCC under accession number PTA-6865.

E 21. The monoclonal antibody or antigen binding portion thereof according to E 20, comprising the heavy chain amino acid sequence and the light chain amino acid sequence selected from the group consisting of:

a) the amino acid sequence of SEQ ID NO: 2 heavy chain and the amino acid sequence of SEQ ID NO: 102 light chain;

b) the amino acid sequence of SEQ ID NO: 100 heavy chain and the amino acid sequence of SEQ ID NO: 4; and

c) the amino acid sequence of SEQ ID NO: 100 heavy chain and the amino acid sequence of SEQ ID NO: 102 light chain.

E 22. A pharmaceutical composition comprising the antibody or antigen binding portion thereof of any of E 1-15 or 19-21 and a physiologically acceptable carrier.

E 23. A method of inhibiting angiogenesis in a mammal in need thereof, comprising the step of administering to said mammal a therapeutically effective amount of the antibody or antigen binding portion according to any one of E 1-15 or 19-21 or the pharmaceutical composition according to E 22.

E 24. A human monoclonal antibody or antigen binding portion thereof that specifically bind to ALK-1, wherein the antibody or portion comprises a heavy chain variable domain comprising the CDR1 amino acid sequence selected from the group consisting of:

(a) the amino acid sequence CDR1 comprising SEQ ID NO: 136, wherein G in the first position is replaced by D and S in the 5 position is replaced by N; and (b) the amino acid sequence CDR1 comprising SEQ ID NO: 136, wherein G in the first position is replaced by E and S in the 5 position is replaced by N.

LIST OF SEQUENCES [0437] &lt;110&gt; Amgen Fremont Inc. Pfizer Inc &lt;120&gt; HUMAN MONOCLONAL ANTIBODIES AGAINST KINASE SIMILAR TO ACTIVINE-1 RECEPTOR &lt;130&gt; P31976EP-D1-PCT &lt;140&gt; Not yet assigned &lt;141&gt; 2009-09-06 &lt;150&gt; EP06824915.0 &lt;151&gt; 2009-09 -06 &lt;160&gt; 136 &lt;170&gt; PatentIn version 3.3 &lt;210&gt; 1 &lt;211&gt; 1332

106 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 1 caggtgcagc tgcaggagtc gggtccagga acctgcactg tctctggtgg ctccatcagc cagcacccag ggaagggcct ggagtggatt tacaacccgt ccctcaagag tcgagttacc tccctgaagc tgagctctgt gactgccgcg tcagtggctg ggtttgacta ctggggccag accaagggcc catcggtctt ccccctggcg gcggccctgg gctgcctggt caaggactac tcaggcgctc tgaccagcgg cgtgcacacc tactccctca gcagcgtagt gaccgtgccc tgcaacgtag atcacaagcc cagcaacacc tgtgtcgagt gcccaccgtg cccagcacca cccccaaaac ccaaggacac cctcatgatc gtggacgtga gccacgaaga ccccgaggtc gtgcataatg ccaagacaaa gccacgggag agcgtcctca ccgtcgtgca ccaggactgg tccaacaaag gcctcccagc ccccatcgag cgagaaccac aggtgtacac cctgccccca agcctgacct gcctggtcaa aggcttctac aatgggcagc cggagaacaa ctacaagacc ttcttcctct acagcaagct caccgtggac tcatgctccg tgatgcatga ggctctgcac tctccgggta aa &lt;210&gt; 2 &lt;211&gt; 444 &lt;212&gt; PRT &lt;213&gt; Human ctggtgaagc agtggtgaat gggtacatct atatcagtag gacacggccg ggaaccctgg ccctgctcca ttccccgaac ttcccggctg tccagcaact aaggtggaca cctgtggcag tcccggaccc cagttcaact gagcagttca ctgaacggca aaaaccatct tcccgggagg cccagcgaca acacctccca aagagcaggt aaccactaca cttcacagac actactggaa attacagtgg acacgtctaa tgtattactg tcaccgtctc ggagcacctc cggtgacggt tcctacagtc tcggcaccca agacagttga gaccgtcagt ctgaggtcac ggtacgtgga acagcacgtt aggagtacaa ccaaaaccaa agatgaccaa tcgccgtgga tgctggactc ggcagcaggg cacagaagag cctgtccctc ctggatccgc gagtacctac gaaccagttc tgcgagagag ctcagcctcc cgagagcaca gtcgtggaac ctcaggactc gacctacacc gcgcaaatgt cttcctcttc gtgcgtggtg cggcgtggag ccgtgtggtc gtgcaaggtc agggcagccc gaaccaggtc gtgggagagc cgacggctcc gaacgtcttc cctctccctg

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<img file="PL2447283T3_D0016.tif" />

108

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 Cheese Val Phe Leu Phe 225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 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

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

Cheese Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 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 Cheese Asp Ile Ala Val Glu Trp Glu Cheese 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 I 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 &lt;210&gt; 3 &lt;211&gt; 645 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 3

109

<td>gaaattgtgt</td><td>tgacgcagtc tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgta</td><td>gggccagtca gagtgtcagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct cctcatctat</td><td>ggtacatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg gtctgggaca</td><td>gacttcaccc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta ttactgtcag</td><td>cagtatggta</td><td>gctcgccgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat taaacgaact</td><td>gtggctgcac</td><td>catctgtctt</td><td>catcttcccg</td><td> 360</td>

<td>ccatctgatg</td><td>agcagttgaa atctggaact</td><td>gcctctgttg</td><td>tgtgcctgct</td><td>gaataacttc</td><td> 420</td>

<td>tatcccagag</td><td>aggccaaagt acagtggaag</td><td>gtggataacg</td><td>ccctccaatc</td><td>gggtaactcc</td><td> 480</td>

<td>laggagagtg</td><td>tcacagagca ggacagcaag</td><td>gacagcacct</td><td>acagcctcag</td><td>cagcaccctg</td><td> 540</td>

<td>acgctgagca</td><td>aagcagacta cgagaaacac</td><td>aaagtctacg</td><td>cctgcgaagt</td><td>cacccatcag</td><td> 600</td>

<td>ggcctgagct</td><td>cgcccgtcac aaagagcttc</td><td>aacaggggag</td><td>agtgt</td><td></td><td> 645</td>

&lt;210&gt; 4 &lt;211&gt; 215 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="2"> &lt;400&gt; 4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>How much</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td> |</td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>Thr</td><td>val</td><td>ala</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>ala</td><td>Pro</td><td>Cheese</td><td>val</td><td>phe</td><td>How much</td><td>phe</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>Glu</td><td>Gin</td><td>Leu</td><td>lys</td><td>Cheese</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

110

<td rowspan="2">Gly Thr 130</td><td rowspan="2">ala</td><td rowspan="2">Val cheese</td><td colspan="2">Val Cys Leu Leu Asn Asn Phe Tyr</td><td rowspan="2">Pro</td><td rowspan="2">Arg</td><td rowspan="2">Glu</td>

<td> 135</td><td> 140</td>

<td>Ala Lys</td><td>val</td><td>Gin Trp</td><td>Lys Val</td><td>Asp Asn Ala Leu Gin Ser</td><td>Gly</td><td>own</td><td>Cheese</td>

<td> 145</td><td></td><td></td><td> 150</td><td> 155</td><td></td><td></td><td> 160</td>

<td>Gin Glu</td><td>Cheese</td><td>Val Thr</td><td>Glu Gin</td><td>Asp Ser Lys Asp Ser Thr</td><td>Tyr</td><td>Cheese</td><td>Leu</td>

<td></td><td></td><td> 165</td><td></td><td> 170</td><td></td><td> 175</td><td></td>

<td>Cheese Cheese</td><td>Thr</td><td>Leu Thr</td><td>Leu Cheese</td><td>Lys Ala Asp Tyr Glu Lys</td><td>His</td><td>lys</td><td>val</td>

<td></td><td></td><td> 180</td><td></td><td> 185</td><td> 190</td><td></td><td></td>

<td>Tyr Ala</td><td>Cys</td><td>Glu Val</td><td>Thr his</td><td>Gin Gly Leu Cheese Ser Pro</td><td>val</td><td>Thr</td><td>lys</td>

<td></td><td> 195</td><td></td><td></td><td> 200 205</td><td></td><td></td><td></td>

<td>Phe cheese</td><td>own</td><td>Arg Gly</td><td>Glu Cys</td><td></td><td></td><td></td><td></td>

<td> 210</td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 5 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 5

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggtccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccatcagc</td><td>agtggtgaat</td><td>actactggaa</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct</td><td>ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagtacotac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag</td><td>tcgagttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagag</td><td> 300</td>

<td>tcagtggctg</td><td>ggtttgacta</td><td>ctggggccag</td><td>ggaaccctgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 6 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 6

<td>Gin 1</td><td>val</td><td>Gin</td><td>Leu</td><td>Gin 5</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>Gly 10</td><td>Leu</td><td>val</td><td>lys</td><td>Pro</td><td>Cheese 15</td><td>Gin</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese 25</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>Gly</td>

<td>Glu</td><td>Tyr</td><td>Tyr 35</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg 40</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>How much</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

55 60

111

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

Cheese Leu Lys Leu Cheese Cheese 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 &lt;210&gt; 7 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 7

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgta</td><td>gggccagtca</td><td>gagtgtcagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>ggtacatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcaccc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcag</td><td>cagtatggta</td><td>gctcgccgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 8 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 8

Glu Ile 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 Thr 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 Cheese Ser Pro 85 90 95

Ile Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 &lt;210&gt; 9 &lt;211&gt; 361

112 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 9

<td>caggtgcagc</td><td>tggtggagtc</td><td>tgggggaggc</td><td>gtggtccagc</td><td>ctgggaggtc</td><td>cctgagactc</td><td> 60</td>

<td>tcctgtgcag</td><td>cgtctggatt</td><td>caccttcagt</td><td>agtcatggca</td><td>tgtactgggt</td><td>ccgccaggct</td><td> 120</td>

<td>ccaggcaagg</td><td>ggctggagtg</td><td>ggtggcagct</td><td>atatggtatg</td><td>atggaagtaa</td><td>taaatactat</td><td> 180</td>

<td>M &gt;&gt; (**</td><td></td><td>attCaCCutC</td><td>tccuCTaCraCu</td><td>9¼ + · 4 · «JR 9 9 ^ T9 9 aa&gt; and» waayaa</td><td>/ • 1 9 / R / R / ·, + · Λ + »9 + · \ .avys.uy u-ua.</td><td> 240</td>

<td>ctgcaaatga</td><td>atagcctgag</td><td>agccgaggac</td><td>acggctgtgt</td><td>attactgtgc</td><td>gagagatcag</td><td> 300</td>

<td>gagcagtggc</td><td>ccgatgtttt</td><td>tgatatctgg</td><td>ggccaaggga</td><td>caatggtcac</td><td>cgtctcttca</td><td> 360</td>

g 361 &lt;210&gt; 10 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 10</td><td colspan="2" rowspan="3">Glu Cheese</td><td rowspan="3">Gly</td><td rowspan="3">Gly</td><td colspan="7" rowspan="2">Gly Val Val Gin Pro Gly Arg</td>

<td rowspan="2">Gin 1</td><td colspan="2" rowspan="2">Val Gin</td><td rowspan="2">Leu</td><td rowspan="2">val 5</td>

<td colspan="3"> 10</td><td colspan="4"> 15</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>ala</td><td>ala</td><td>Cheese</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Cheese</td><td>Cheese</td><td>His</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Underworld</td><td>Tyr</td><td>Trp</td><td>val</td><td>Arg</td><td>Gin</td><td>ala</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Trp</td><td>val</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>ala</td><td>ala</td><td>How much</td><td>Trp</td><td>Tyr</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>own</td><td>lys</td><td>Tyr</td><td>Tyr</td><td>ala</td><td>Asp</td><td>Cheese</td><td>val</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>lys</td><td>Gly</td><td>Arg</td><td>phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>own</td><td>Cheese</td><td>lys</td><td>own</td><td>Thr</td><td>Leu</td><td>Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td>AND</td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>Gin</td><td>Underworld</td><td>own</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>ala</td><td>Glu</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>ala</td><td>Arg</td><td>Asp</td><td>Gin</td><td>Glu</td><td>Gin</td><td>Trp</td><td>Pro</td><td>Asp</td><td>val</td><td>phe</td><td>Asp</td><td>How much</td><td>Trp</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Underworld</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 11 &lt;211&gt; 322 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 11

113

<td>gacatccaga</td><td>tgacccagtc</td><td>tccatcctca</td><td>ctgtctgcat</td><td>ctgtaggaga</td><td>cagagtcacc</td><td> 60</td>

<td>atcacttgtc</td><td>gggcgagtca</td><td>gggcattaga</td><td>aattatttag</td><td>cctggtttca</td><td>gcagaaacca</td><td> 120</td>

<td>gggaaagccc</td><td>ctaagtccct</td><td>gatctatggt</td><td>gcatccagtt</td><td>tgcaaagtgg</td><td>ggtcccatca</td><td> 180</td>

<td>aagttcagcg</td><td>gcagtggatc</td><td>tgggacagat</td><td>ttcactetca</td><td>ccatcagcag</td><td>cctgcagcct</td><td> 240</td>

<td>gaagattttg</td><td>caacttatta</td><td>ctgccaacag</td><td>tataatagtt</td><td>acccgctcac</td><td>tttcggcgga</td><td> 300</td>

<td>gggaccaagg</td><td>tggagatcaa</td><td>ac</td><td></td><td></td><td></td><td> 322</td>

&lt;210&gt; 12 &lt;210&gt; 12 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="4"> &lt;400&gt; 12</td><td rowspan="3">Thr 5</td><td rowspan="3">Gin</td><td colspan="3" rowspan="3">Cheese Pro Cheese</td><td colspan="6" rowspan="2">Cheese Leu Cheese Ala Cheese Val</td><td rowspan="3">Gly</td>

<td colspan="2" rowspan="2">Asp Ile 1</td><td rowspan="2">Gin</td><td rowspan="2">Underworld</td>

<td colspan="5"> 10</td><td> 15</td>

<td>Asp</td><td>Arg</td><td>val</td><td>Thr</td><td>How much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Gly</td><td>How much</td><td>Arg</td><td>own</td><td>Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>ala</td><td>Trp</td><td>phe</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>lys</td><td>ala</td><td>Pro</td><td>lys</td><td>Cheese</td><td>Leu</td><td>How much</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td><td>Cheese</td><td>lys</td><td>phe</td><td>Cheese</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>own</td><td>Cheese</td><td>Tyr</td><td>Pro</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>phe</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>lys</td><td>val</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td><td></td>

100 105 &lt;210&gt; 13 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 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 gaoacggcct tgtattactg tgcgagagaa 300 tcagtggccg cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355 &lt;210&gt; 14

114 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 14</td><td colspan="2" rowspan="3">Glu Cheese</td><td rowspan="3">Gly and</td><td rowspan="3">Pro</td><td colspan="7" rowspan="2">Gly Leu Val Lys Pro Cheese Glu</td>

<td rowspan="2">Gin 1</td><td colspan="2" rowspan="2">Val His</td><td rowspan="2">Leu</td><td rowspan="2">Gin 5</td>

<td colspan="3"> 10</td><td colspan="4"> 15</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>vjtl</td><td>Cheese 25</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>val</td><td>Cheese thirty</td><td>Cheese</td><td>Gly</td>

<td>Asp</td><td>Tyr</td><td>Tyr 35</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg 40</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gly</td><td>lys 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>How much 50</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr 55</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>own 60</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td>Leu 65</td><td>lys</td><td>Cheese</td><td>Arg</td><td>How much</td><td>Thr 70</td><td>How much</td><td>Cheese</td><td>How much</td><td>Asp</td><td>Thr 75</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>own 85</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala 90</td><td>Asp</td><td>Thr</td><td>ala</td><td>Leu</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu 100</td><td>Cheese</td><td>val</td><td>ala</td><td>ala</td><td>phe 105</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin 110</td><td>Gly</td><td>Thr</td>

<td>Leu</td><td>val</td><td>Thr 115</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 15 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 15

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtattagc</td><td>agtaggtact</td><td>tagcctggta</td><td>ccagcaggaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>ggtgcatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcactc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcaa</td><td>cactatggta</td><td>gctcaccgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 16 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 16

115

<td>Glu 1</td><td>How much</td><td>val</td><td>Leu Thr 5</td><td>Gin Ser Pro Gly</td><td>Thr Leu 10</td><td>Leu cheese</td><td>Cheese</td><td>Pro 15</td><td>Gly</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr Leu</td><td>Cys Arg Ala cheese</td><td>Gin cheese</td><td>Ile cheese</td><td>Cheese</td><td>Cheese</td><td>Arg</td>

<td></td><td></td><td></td><td> 20</td><td> 25</td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>ala</td><td>Trp Tyr</td><td>Gin Gin Glu Pro</td><td>Gly Gin</td><td>Ala Pro</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td> 40</td><td></td><td> 45</td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>Ala Ser</td><td>Ser Arg Ala Thr</td><td>Gly Ile</td><td>Pro Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td> 60</td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Gly cheese</td><td>Thr Asp Phe Thr</td><td>Leu Thr</td><td>How much cheese</td><td>Arg</td><td>Leu</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>Phe Ala</td><td>Val Tyr Tyr</td><td>Gin His</td><td>Tyr Gly</td><td>Cheese</td><td>Cheese</td><td>Pro</td>

<td></td><td></td><td></td><td> 85</td><td></td><td> 90</td><td></td><td></td><td> 95</td><td>and</td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly Gin</td><td>Gly Thr Arg Leu</td><td>Glu Ile</td><td>lys</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 100</td><td> 105</td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 17 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 17

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggcccagga</td><td>ctggtgaagc</td><td>cttcggagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccgtcagc</td><td>agtggtgatt</td><td>actactggaa</td><td>ctggatccgg</td><td> 120</td>

<td>cagcccccag</td><td>ggaagggact</td><td>ggagtggatt</td><td>gggtatatct</td><td>attacagtgg</td><td>gagcaccaac</td><td> 180</td>

<td>tacaacccct</td><td>ccctcaagag</td><td>tcgagtcacc</td><td>atatcagtag</td><td>acacgtccaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>Gacc ^ ctgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagaa</td><td>30 (j</td>

<td>gcagtgtccg</td><td>cctttgacta</td><td>ctggggccag</td><td>ggaaccctgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 18 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 18

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

116

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Gly</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Tyr</td><td>Tyr</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>How much</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>own</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu</td><td>ala</td><td>val</td><td>Cheese</td><td>ala</td><td>phe</td><td>ASp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Leu</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 &lt;210&gt; 19 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 19

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtgttagc</td><td>agtacctact</td><td>tagcctggca</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>ggtgtatcca</td><td>gcagggccag</td><td>tggcgtccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcactc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcag</td><td>cagtatggta</td><td>gttcaccgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 20 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="2"> &lt;400&gt; 20</td><td rowspan="2">Leu</td><td rowspan="2">Thr Gin 5</td><td rowspan="2">Cheese Pro</td><td rowspan="2">Gly</td><td rowspan="2">Thr 10</td><td rowspan="2">Leu</td><td rowspan="2">Leu cheese</td><td rowspan="2">Cheese</td><td rowspan="2">Pro 15</td><td rowspan="2">Gly</td>

<td>Glu 1</td><td>Ile Val</td>

<td>Glu</td><td>Arg Ala</td><td>Thr twenty</td><td>Leu Cheese</td><td>Cys Arg</td><td>ala 25</td><td>Cheese</td><td>Gin</td><td>Val cheese</td><td>Cheese thirty</td><td>Cheese</td><td>Thr</td>

<td>Tyr</td><td>Leu Ala</td><td>Trp</td><td>His Gin</td><td>Gin Lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Ala Pro</td><td>Arg</td><td>Leu</td><td>Leu</td>

117

Gly Val Pro Asp Arg Phe Cheese

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td>

<td>How much</td><td>Tyr 50</td><td>Gly</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Arg 55</td><td>ala</td>

<td>Gly 65</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr 70</td><td>Asp</td><td>phe</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala 85</td><td>val</td><td>Tyr</td><td>Tyr</td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly 100</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td>

Thr Leu Thr Ile Ser Arg Leu Glu 75 80

Cys Gin Gin Tyr Gly Cheese Ser Pro 90 95

Leu Glu Ile Lys 105 &lt;210&gt; 21 &lt;211&gt; 370 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 21 caggtgcagc acctgcactg cagcacccag tacaacccgt tccctgaagc gggcgatttt gtctcctcag tgcaggagtc tctctgggtgc tctctgggg

CCCu tgagttctgt tggagtggtc gggcccagga ctccatcagc ggagtggatt gactgccgcg tgatgttttt ctggtgaagc agtggtggtc gggtacatct gacacggccg gatatctggg cttcacagac actactggag attacagtgg acacgtctaa tatattactg gccaagggac cctgtccctc ctggatccgc gagcacctac yuaCCuyttC tgcgagagcg aatggtcacc

120

180 at λ n

Λλ "3 V

300

360

370 &lt;210&gt; 22 &lt;211&gt; 123 &lt;212&gt; PRT &lt;213&gt; Human

<td>&lt;400&gt; 22 Gin Val Gin Leu 1</td><td>Gin 5</td><td>Glu Cheese Gly</td><td>Pro Gly Leu 10</td><td>val</td><td>lys</td><td>Pro</td><td>Cheese 15</td><td>Gin</td>

<td>Thr Leu Ser Leu 20</td><td>Thr</td><td>Cys Thr Val</td><td>Gly Gly 25 cheese</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>Gly</td>

<td>Gly His Tyr Trp 35</td><td>Cheese</td><td>Trp Ile Arg 40</td><td>Gin His Pro</td><td>Gly</td><td>LyS 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp Ile Gly Tyr 50</td><td>How much</td><td>Tyr Tyr Cheese 55</td><td>Gly Ser Thr</td><td>Tyr 60</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td>Leu Lys Ser Arg 65</td><td>val</td><td>Thr Ile Ser 70</td><td>Val Asp Thr 75</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe 80</td>

118

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>ala</td><td>Arg</td><td>ala</td><td>Gly</td><td>Arg</td><td>phe</td><td>Leu</td><td>Glu</td><td>Trp</td><td>Cheese</td><td>Asp</td><td>val</td><td>phe</td><td>Asp</td><td>How much</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Underworld</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td>

115 120 &lt;210&gt; 23 &lt;211&gt; 340 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 23

<td>gacatcgtga</td><td>tgacccjagtc tccagactcc ctggctgtgt ctctgggcga gagggcdacc</td><td> 60</td>

<td>atcaactgca</td><td>agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct</td><td> 120</td>

<td>tggtaccagc</td><td>agaaaccagg gcagcctcct aagctgctca tttactgggc atctacccgg</td><td> 180</td>

<td>gaatccgggg</td><td>tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc</td><td> 240</td>

<td>atcagcagcc</td><td>tgcaggctga agatgtggca gtttattact gtcagcaata ttatgatact</td><td> 300</td>

<td>cctccgacgt</td><td>tcggccaagg gaccaaggtg gaaatcaaac</td><td> 340</td>

&lt;210&gt; 24 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 24

<td>Asp 1</td><td>How much</td><td>val</td><td>Underworld</td><td>Thr 5</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Asp</td><td>Cheese 10</td><td>Leu</td><td>ala</td><td>val</td><td>Cheese</td><td>Leu 15</td><td>Gly</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr twenty</td><td>How much</td><td>own</td><td>Cys</td><td>lys</td><td>Cheese 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Leu thirty</td><td>Tyr</td><td>Cheese</td>

<td>Cheese</td><td>own</td><td>own 35</td><td>lys</td><td>own</td><td>Tyr</td><td>Leu</td><td>ala 40</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys 45</td><td>Pro</td><td>Gly</td><td>Gin</td>

<td>Pro</td><td>Pro 50</td><td>lys</td><td>Leu</td><td>Leu</td><td>How much</td><td>Tyr 55</td><td>Trp</td><td>ala</td><td>Cheese</td><td>Thr</td><td>Arg 60</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>val</td>

<td>Pro 65</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly 70</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr 75</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr 80</td>

<td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin 85</td><td>ala</td><td>Glu</td><td>Asp</td><td>val</td><td>ala 90</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin 95</td><td>Gin</td>

<td>Tyr</td><td>Tyr</td><td>Asp</td><td>Thr 100</td><td>Pro</td><td>Pro</td><td>Thr</td><td>phe</td><td>Gly 105</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lys</td><td>val 110</td><td>Glu</td><td>How much</td>

&lt;210&gt; 25 &lt;211&gt; 355

119 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 25

<td>caggtgcagc</td><td>tgcaggagtc gggcccagga</td><td>ctggtgaagc</td><td>cctcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>atctgtactg</td><td>tttctggtgg ctccatcagc</td><td>agtggtgaat</td><td>actactggag</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagcacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag tcgacttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagag</td><td> 300</td>

<td>gggatcggtg</td><td>cttttgatat ctggggccaa</td><td>gggacaatgg</td><td>tcaccgtctc</td><td>ttcag</td><td> 355</td>

&lt;210&gt; 26 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 26</td><td colspan="2" rowspan="2">Glu Cheese</td><td rowspan="2">Gly</td><td rowspan="2">Pro</td><td colspan="3" rowspan="2">Gly Leu Val 10</td><td rowspan="2">lys</td><td rowspan="2">Pro</td><td rowspan="2">Cheese 15</td><td rowspan="2">Gin</td>

<td>Gin 1</td><td colspan="2">Val Gin</td><td>Leu</td><td>Gin 5</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu twenty</td><td>How much</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese 25</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>Gly</td>

<td>Glu</td><td>Tyr</td><td>Tyr 35</td><td>Trp</td><td>Cheese</td><td>Trp</td><td>How much</td><td>Arg 40</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>How much 50</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr 55</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr 60</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td>Leu 65</td><td>lys</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Thr 70</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr 75</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese 85</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala 90</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu 100</td><td>Gly</td><td>How much</td><td>Gly</td><td>ala</td><td>phe 105</td><td>Asp</td><td>How much</td><td>Trp</td><td>Gly</td><td>Gin 110</td><td>Gly</td><td>Thr</td>

<td>Underworld</td><td>val</td><td>Thr 115</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 27 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 27

120

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>gccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtgttagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcctaggct</td><td>cctcatctat</td><td>ggagcatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcactc</td><td>tcaccatcat</td><td>cagactggac</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcag</td><td>cggtatggta</td><td>gctcaccgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 28 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 28</td><td rowspan="2">Gin</td><td colspan="2" rowspan="2">Cheese Pro</td><td rowspan="2">Gly</td><td rowspan="2">Thr 10</td><td colspan="3" rowspan="2">Leu Cheese Leu</td><td rowspan="2">Cheese</td><td rowspan="2">Pro 15</td><td rowspan="2">Gly</td>

<td>Glu 1</td><td>How much</td><td>val</td><td>Leu</td><td>Thr 5</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr twenty</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese thirty</td><td>Cheese</td><td>Cheese</td>

<td>Tyr</td><td>Leu</td><td>ala 35</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys 40</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro 45</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td>How much</td><td>Tyr 50</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Arg 55</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro 60</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td>Gly 65</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr 70</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr 75</td><td>How much</td><td>How much</td><td>Arg</td><td>Leu</td><td>Asp 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala 85</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin 90</td><td>Arg</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese 95</td><td>Pro</td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td>

] 100 105 | &lt;210&gt; 29 &lt;211&gt; 361 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 29 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agaggacgat 300 agcagtggct gcccctactt tgactactgg ggccagggaa ccctggtcac cgcttcctca 360 g 361 &lt;210&gt; 30 &lt;211&gt; 120

121 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 30</td><td colspan="2" rowspan="2">Glu Cheese</td><td rowspan="2">Gly</td><td rowspan="2">Pro</td><td colspan="3" rowspan="2">Gly Leu Val 10</td><td rowspan="2">lys</td><td rowspan="2">Pro</td><td colspan="2" rowspan="2">Glu Cheese 15</td>

<td>Gin 1</td><td colspan="2">Val Gin</td><td>Leu</td><td>Gin 5</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Trp</td><td>Cheese</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Trp</td><td>How much</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>own</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td><td>Leu</td><td>lys</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Leu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>lys</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>ala</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Arg</td><td>Glu</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Cys</td><td>Pro</td><td>Tyr</td><td>phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Leu</td><td>val</td><td>Thr</td><td>ala</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 31 &lt;211&gt; 322 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 31 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccagggtcct catctatggt gcatccacca gggccactgg tatcccagtc 180 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240 gaagattttg cagtttatta ctgtcagcag tataataact ggccattcac tttcggccct 300 gggaccaaag tggatatcaa ac 322 &lt;210&gt; 32 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 32

122

<td>Glu 1</td><td>How much</td><td>Val Met</td><td>Thr Gin 5</td><td>Cheese</td><td>Pro</td><td>Ala Thr 10</td><td>Leu</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Pro 15</td><td>Gly</td>

<td>Glu</td><td>Arg</td><td>Ala Thr</td><td>Leu Cheese</td><td>Cys</td><td>Arg</td><td>Ala Ser</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>own</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>ala</td><td>Trp Tyr</td><td>Gin Gin</td><td>lys</td><td>Pro</td><td>Gly Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>val</td><td>Leu</td><td>How much</td>

<td></td><td></td><td> 35</td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td>4 Fe</td><td></td><td></td><td></td>

<td>Tyr</td><td>Gly</td><td>Ala Ser</td><td>Thr Arg</td><td>ala</td><td>Thr</td><td>Gly Ile</td><td>Pro</td><td>val</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Gly</td><td>Gly cheese</td><td>Thr glu</td><td>phe</td><td>Thr</td><td>Leu Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Cheese</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Glu</td><td>Asp</td><td>Phe Ala</td><td>Val Tyr</td><td>Tyr</td><td>Cys</td><td>Gin Gin</td><td>Tyr</td><td>own</td><td>own</td><td>Trp</td><td>Pro</td><td>phe</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>phe</td><td>Gly Pro</td><td>Gly Thr</td><td>lys</td><td>val</td><td>Asp Ile</td><td>lys</td><td></td><td></td><td></td><td></td><td></td>

100 105 &lt;210&gt; 33 &lt;211&gt; 370 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 33

<img file="PL2447283T3_D0017.tif" />

&lt;210&gt; 34 &lt;211&gt; 123 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 34

123

<td colspan="2">Gin Met Gin Leu Gin</td><td rowspan="2">Glu</td><td rowspan="2">Gly Pro cheese</td><td colspan="2">Gly Leu Val Lys Pro Gin Cheese</td>

<td> 1</td><td> 5</td><td> 10</td><td> 15</td>

<td>Thr</td><td>Leu Ser Leu Thr</td><td>Cys</td><td>Thr Val Ser</td><td>Gly</td><td>Gly Cheese How much Cheese Gly Cheese</td>

<td></td><td> 20</td><td></td><td> 25</td><td></td><td> 30</td>

<td>Gly</td><td>His Tyr Trp Ser</td><td>Trp</td><td>Ile Arg Gin</td><td>His</td><td>Pro Gly Lys Gly Leu Glu</td>

<td></td><td> 35</td><td></td><td> 40</td><td></td><td> 45</td>

<td>Trp</td><td>Ile Gly Tyr Ile</td><td>Tyr</td><td>Tyr Ser Gly</td><td>Cheese</td><td>Ala Tyr Tyr Asn Pro Ser</td>

<td></td><td> 50</td><td></td><td> 55</td><td></td><td> 60</td>

<td>Leu</td><td>Lys Ser Arg Val</td><td>Thr</td><td>Ile Ser Val</td><td>Asp</td><td>Thr Ser Lys Asn Gin Phe</td>

<td> 65</td><td></td><td> 70</td><td></td><td></td><td> 75 80</td>

<td>Cheese</td><td>Leu Lys Leu Ser</td><td>Cheese</td><td>Val Thr Ala</td><td>ala</td><td>Asp Thr Ala Val Tyr Tyr</td>

<td></td><td> 85</td><td></td><td></td><td> 90</td><td> 95</td>

<td>Cys</td><td>Ala Arg Ala Gly</td><td>Arg</td><td>Phe Leu Glu</td><td>Trp</td><td>Asp Val Phe Asp Ile</td>

<td></td><td> 100</td><td></td><td> 105</td><td></td><td> 110</td>

<td>Trp</td><td>Gly Gin Gly Thr</td><td>Underworld</td><td>Val Thr Val</td><td>Cheese</td><td>Leu</td>

<td></td><td> 115</td><td></td><td> 120</td><td></td><td></td>

&lt;210&gt; 35 &lt;211&gt; 340 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 35

<td>gacatcgtga</td><td>tgacccagtc</td><td>tccagactcc</td><td>ctggctgtgt</td><td>ctctgggcga</td><td>gagggccacc</td><td> 60</td>

<td>atcaactgca</td><td>agtccagcca</td><td>gagtgtttta</td><td>tacagctcca</td><td>acaataagaa</td><td>ctacttaact</td><td> 120</td>

<td>tggtaccagc</td><td>agaaaccagg</td><td>acggcctcct</td><td>aagctgctca</td><td>tttactgggc</td><td>atctacccgg</td><td> 180</td>

<td>gaatccgggg</td><td>tccctgaccg</td><td>attcagtggc</td><td>agcgggtctg</td><td>ggacagattt</td><td>cactctcacc</td><td> 240</td>

<td>atcagcagcc</td><td>tgcaggctga</td><td>agatgtggca</td><td>gtttattact</td><td>gtcaacaata</td><td>ttataatact</td><td> 300</td>

<td>cctccgacgt</td><td>tcggccaagg</td><td>gaccaaggtg</td><td>gaaatcaagc</td><td></td><td></td><td> 340</td>

&lt;210&gt; 36 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 36

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

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

124

<td>Cheese</td><td>own</td><td>own 35</td><td>lys</td><td>own</td><td>Tyr</td><td>Leu</td><td>Thr 40</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys 45</td><td>Pro</td><td>Gly</td><td>Arg</td>

<td>Pro</td><td>Pro 50</td><td>lys</td><td>Leu</td><td>Leu</td><td>How much</td><td>Tyr 55</td><td>Trp</td><td>ala</td><td>Cheese</td><td>Thr</td><td>Arg 60</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>val</td>

<td>Pro 65</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly 70</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr 75</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr 80</td>

<td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin 85</td><td>ala</td><td>Glu</td><td>Asp</td><td>val</td><td>ala 90</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin 95</td><td>Gin</td>

<td>Tyr</td><td>Tyr</td><td>own</td><td>Thr 100</td><td>Pro</td><td>Pro</td><td>Thr</td><td>phe</td><td>Gly 105</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lys</td><td>val 110</td><td>Glu</td><td>How much</td>

Lys &lt;210&gt; 37 &lt;211&gt; 379 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 37

<td>caggtgcagc</td><td>tggtggagtc</td><td>tgggggaggc</td><td>ttggtcaagc</td><td>ctggagggtc</td><td>cctgagactc</td><td> 60</td>

<td>tcctgtgcag</td><td>cctctggatt</td><td>caccttcagt</td><td>gactactaca</td><td>tgagctggat</td><td>ccgccaggct</td><td> 120</td>

<td>ccagggaagg</td><td>ggctggagtg</td><td>ggtttcatac</td><td>attagtagta</td><td>gtggtaatac</td><td>catatactac</td><td> 180</td>

<td>gcagactctg</td><td>tgaagggccg</td><td>attcaccatc</td><td>tccagggaca</td><td>acgccaggaa</td><td>ctcactgtat</td><td> 240</td>

<td>ctgcaaatga</td><td>acagcctgag</td><td>agccgaggac</td><td>acggccgtgt</td><td>attactgtgc</td><td>gagggaagcc</td><td> 300</td>

<td>tatgatagta</td><td>gtggttacta</td><td>ctactactac</td><td>tacggtatgg</td><td>acgtctgggg</td><td>ccaagggacc</td><td> 360</td>

<td>acggtcaccg</td><td>tctcctcag</td><td></td><td></td><td></td><td></td><td> 379</td>

&lt;210&gt; 38 &lt;211&gt; 126 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 38

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

Cheese Leu Arg Leu Cheese Cys Ala Ala Cheese Gly Phe Thr Phe Cheese Asp Tyr 20 25 30

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

125

Cheese Tyr Ile Cheese Cheese Cheese Gly Asn Thr Ile Tyr Tyr Ala Asp Cheese Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ser Leu Tyr

70 75 80

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

90 95

Ala Arg Glu Ala Tyr Asp Cheese Cheese 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 &lt;210&gt; 39 &lt;211&gt; 337 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 39

<td>gatattgtga</td><td>tgacccagac</td><td>tccactctct</td><td>ctgtccgtca</td><td>cccctggaca</td><td>gccggcctcc</td><td> 60</td>

<td>atctcctgca</td><td>agtctagtca</td><td>gagcctcctg</td><td>catagtgatg</td><td>gaaagaccta</td><td>cttgtattgg</td><td> 120</td>

<td>tacctgcaga</td><td>agccaggcca</td><td>gcctccacag</td><td>ctcctgatct</td><td>atgaagtttc</td><td>caaccggttc</td><td> 180</td>

<td>tctggagtgc</td><td>cagataggtt</td><td>cagtggcagc</td><td>gggtcaggga</td><td>cagatttcac</td><td>actgacaatc</td><td> 240</td>

<td>agccgggtgg</td><td>aggctgacga</td><td>tgttggggtt</td><td>tattactgca</td><td>tgcaaagtac</td><td>acaccttcct</td><td> 300</td>

<td>tggacgttcg</td><td>gccaagggac</td><td>caaggtggaa</td><td>atcaaac</td><td></td><td></td><td> 337</td>

&lt;210&gt; 40 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 40

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

5 10 15

Gin Pro Ala Ser Ile Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser

25 30

<td>Asp</td><td>Gly</td><td>lys</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Pro</td><td>Gin</td><td>Leu</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Glu</td><td>val</td><td>Cheese</td><td>own</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

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

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

126 &lt;210&gt; 41 &lt;211&gt; 376 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 41

<td>gaggtgcagc</td><td>tggtggagtc</td><td>tgggggaggc</td><td>ttggtaaagc</td><td>ctggggggtc</td><td>ccttagactc</td><td> 60</td>

<td>tcctgtgcag</td><td>cctctggatt</td><td>cactttcagt</td><td>aacgcctgga</td><td>tgagctgggt</td><td>ccgccaggct</td><td> 120</td>

<td>ccagggaagg</td><td>ggctggagtg</td><td>ggttggccgt</td><td>attaaaagca</td><td>aaagtgatgg</td><td>tgggacaaca</td><td> 180</td>

<td>gactacgctg</td><td>cacccgtgaa</td><td>aggcagattc</td><td>accatctcaa</td><td>gagatgattc</td><td>aaaaaacacg</td><td> 240</td>

<td>ctgtatctgc</td><td>aaatgaacag</td><td>cctgaaaacc</td><td>gaggacacag</td><td>ccgtgtatta</td><td>ctgtaccaca</td><td> 300</td>

<td>gggaattact</td><td>atgatggtag</td><td>tggttattac</td><td>tcttttgact</td><td>actggggcca</td><td>gggaaccctg</td><td> 360</td>

<td>gtcaccgtct</td><td>cctcag</td><td></td><td></td><td></td><td></td><td> 376</td>

&lt;210&gt; 42 &lt;211&gt; 125 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 42

<td>Glu</td><td>val</td><td>Gin</td><td>Leu</td><td>val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Leu</td><td>val</td><td>lys</td><td>Pro</td><td>Gly gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>ala</td><td>ala</td><td>Cheese</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Cheese</td><td>Asn Ala</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td>

<td>tm</td><td>Underworld</td><td>Cheese</td><td>Tm - —c</td><td>val</td><td>Arrr · □</td><td>Gin</td><td>ala</td><td>Pro</td><td>harvest time - '- J</td><td>TjVS —Λ -</td><td>harvest time^{— —} Λ</td><td>L @ ​​u</td><td>Glu</td><td>Trp Val</td>

40 45

Gly Arg Ile Lys Ser Lys Ser Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 551 60

<td>Pro</td><td>val</td><td>lys</td><td>Gly</td><td>Arg</td><td>phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>lys</td><td>own</td><td>Thr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>Underworld</td><td>own</td><td>Cheese</td><td>Leu</td><td>lys</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Tyr</td><td>Cys</td><td>Thr</td><td>Thr</td><td>Gly</td><td>own</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>phe</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 &lt;210&gt; 43 &lt;211&gt; 337 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 43

127

<td colspan="2">gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc</td><td> 60</td>

<td colspan="2">atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg</td><td> 120</td>

<td colspan="2">tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc</td><td> 180</td>

<td colspan="2">tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc</td><td> 240</td>

<td>agcagagtgg aggctgagga t</td><td>gttggggtt tattactgca tgcaagctct acaaactcct</td><td> 300</td>

<td>cccactttcg gcggagggac c</td><td>aaggtggag atcaaac</td><td> 337</td>

&lt;210&gt; 44 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="4"> &lt;400&gt; 44</td><td colspan="2" rowspan="2">Thr Gin 5</td><td colspan="2" rowspan="2">Cheese Pro</td><td rowspan="2">Leu</td><td rowspan="2">Cheese 10</td><td rowspan="2">Leu</td><td rowspan="2">Pro</td><td rowspan="2">val</td><td rowspan="2">Thr</td><td rowspan="2">Pro 15</td><td rowspan="2">Gly</td>

<td>Asp 1</td><td>How much</td><td>val</td><td>Underworld</td>

<td>Glu</td><td>Pro</td><td>ala</td><td>Cheese twenty</td><td>How much</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>Cheese 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>Leu</td><td>Leu thirty</td><td>His</td><td>Cheese</td>

<td>own</td><td>Gly</td><td>Tyr 35</td><td>own</td><td>Tyr</td><td>Leu</td><td>Asp</td><td>Trp 40</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>lys</td><td>Pro 45</td><td>Gly</td><td>Gin</td><td>Cheese</td>

<td>Pro</td><td>Gin 50</td><td>Leu</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Leu 55</td><td>Gly</td><td>Cheese</td><td>own</td><td>Arg</td><td>ala 60</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td>

<td>Asp 65</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese 70</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp 75</td><td>phe</td><td>Thr</td><td>Leu</td><td>lys</td><td>How much 80</td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Glu</td><td>ala 85</td><td colspan="2">Glu Asp</td><td>val</td><td>Gly</td><td>val 90</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Underworld</td><td>Gin 95</td><td>ala</td>

<td>Leu</td><td>Gin</td><td>Thr</td><td>Pro 100</td><td>Pro</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gly 105</td><td>Gly</td><td>Thr</td><td>lys</td><td>val</td><td>Glu 110</td><td>How much</td><td>lys</td>

&lt;210&gt; 45 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 45

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggcccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccatcagc</td><td>agtaatgatt</td><td>actactggaa</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct</td><td>ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagcacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag</td><td>tcgagttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagaa</td><td> 300</td>

<td>tccacggacg</td><td>gtatggacgt</td><td>ctggggccaa</td><td>gggaccacgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 46 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human

128

<td colspan="5"> &lt;400&gt; 46</td><td rowspan="2">| g1u</td><td colspan="3" rowspan="2">Gly Pro cheese</td><td rowspan="2">Gly 10</td><td rowspan="2">Leu</td><td colspan="3" rowspan="2">Val Lys Pro</td><td rowspan="2">Cheese 15</td><td rowspan="2">Gin</td>

<td>Gin 1</td><td colspan="2">Val Gin</td><td>Leu</td><td>Gin 5</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese 25</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>own</td>

<td>Asp</td><td>Tyr</td><td>Tyr 35</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg 40</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>How much 50</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr 55</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr 60</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td>Leu 65</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr 70</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr 75</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese 85</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala 90</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu 100</td><td>Cheese</td><td>Thr</td><td>Asp</td><td>Gly</td><td>Underworld 105</td><td>Asp</td><td>val</td><td>Trp</td><td>Gly</td><td>Gin 110</td><td>Gly</td><td>Thr</td>

<td>Thr</td><td>val</td><td>Thr 115</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 47 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 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 &lt;210&gt; 48 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 48

129

<td colspan="2">Glu Asn Val Leu Thr</td><td rowspan="2">Gin</td><td rowspan="2">Pro Gly cheese</td><td rowspan="2">Thr Leu Ser 10</td><td rowspan="2">Leu</td><td rowspan="2">Cheese</td><td rowspan="2">Pro Gly 15</td>

<td> 1</td><td> 5</td>

<td>Glu</td><td>Arg Ala Thr Leu</td><td>Cheese</td><td>Cys Arg Ala</td><td>Gin Cheese</td><td>val</td><td>Cheese</td><td>Asn cheese</td>

<td></td><td> 20</td><td></td><td> 25</td><td></td><td></td><td> 30</td><td></td>

<td>Tyr</td><td>Leu Ala Trp Tyr</td><td>Gin</td><td>Gin Lys Pro</td><td>Gly Gin Ala</td><td>Pro</td><td>Arg</td><td>Leu Leu</td>

<td></td><td> 35</td><td></td><td> 40</td><td></td><td> 45</td><td></td><td></td>

<td>How much</td><td>Tyr Gly Ala Ser</td><td>Cheese</td><td>Gly Ala Thr</td><td>Gly Ile Pro</td><td>Asp</td><td>Arg</td><td>Phe Cheese</td>

<td></td><td> 50</td><td></td><td> 55</td><td> 60</td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese Gly Cheese Gly</td><td>Thr</td><td>Asp Phe Thr</td><td>Leu Thr Ile</td><td>Cheese</td><td>Arg</td><td>Leu Glu</td>

<td> 65</td><td></td><td> 70</td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu Asp Phe Ala</td><td>val</td><td>Tyr Tyr</td><td>Gin His Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese Pro</td>

<td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td> 95</td>

<td>How much</td><td>Thr Phe Gly Gin</td><td>Gly</td><td>Thr Arg Leu</td><td>Glu Ile Lys</td><td></td><td></td><td></td>

<td></td><td> 100</td><td></td><td> 105</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 49 &lt;211&gt; 361 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 49

<img file="PL2447283T3_D0018.tif" />

&lt;210&gt; 50 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 50

130

<td>Gin Val 1</td><td>Gin</td><td>Leu Gin Glu 5</td><td>Gly cheese</td><td>Pro</td><td>Gly Leu 10</td><td>val</td><td>Lys Pro</td><td>Gin cheese 15</td>

<td>Thr Leu</td><td>Cheese</td><td>Leu Thr Cys</td><td>Thr Val</td><td>Cheese</td><td>Gly gly</td><td>Cheese</td><td>How much cheese</td><td>Gly cheese</td>

<td></td><td></td><td> 20</td><td></td><td> 25</td><td></td><td></td><td> 30</td><td></td>

<td>Asp Tyr</td><td>Tyr</td><td>Trp Cheese Trp</td><td>Ile Arg</td><td>Gin</td><td>His Pro</td><td>Gly</td><td>Lys Gly</td><td>Leu Glu</td>

<td></td><td> 35</td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td>

<td>Trp Ile</td><td>Gly</td><td>Tyr Ile Tyr</td><td>Tyr Ser</td><td>Gly</td><td>Ser Thr</td><td>Tyr</td><td>Tyr Asn</td><td>Pro Ser</td>

<td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td> 60</td><td></td><td></td>

<td>Leu Lys</td><td>Cheese</td><td>Atg Val Thr</td><td>How much cheese</td><td>How much</td><td>Asp Thr</td><td>Cheese</td><td>Lys Asn</td><td>Gin Phe</td>

<td> 65</td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td>Leu cheese</td><td>lys</td><td>Leu Cheese Cheese</td><td>Val Thr</td><td>ala</td><td>Ala Asp</td><td>Thr</td><td>Ala Val</td><td>Tyr Tyr</td>

<td></td><td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td> 95</td>

<td>Ala's</td><td>Arg</td><td>Glu Arg Asp</td><td>Tyr Gly</td><td>Gly</td><td>Gly Phe</td><td>Asp</td><td>Tyr Trp</td><td>Gly Gin</td>

<td></td><td></td><td> 100</td><td></td><td> 105</td><td></td><td></td><td> 110</td><td></td>

<td>Gly Thr</td><td>Leu</td><td>Val Thr Val</td><td>Cheese Cheese</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 115</td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 51 &lt;211&gt; 340 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 51

<td>gacatcgtga</td><td>tgacccagtc</td><td>tccagactcc</td><td>ctggctgtgt</td><td>ctctgggcga</td><td>gagggccacc</td><td> 60</td>

<td>atcaactgca</td><td>agtccagcca</td><td>gagtgtttta</td><td>tacagctcca</td><td>tcaataagat</td><td>ctacttagct</td><td> 120</td>

<td>tggtaccagc</td><td>agaaaccagg</td><td>acagcctcct</td><td>aagctgctca</td><td>tttactgggc</td><td>atctacccgg</td><td> 180</td>

<td>gaatccgggg</td><td>tccctgaccg</td><td>attcagtggc</td><td>agcgggtctg</td><td>ggacagattt</td><td>cactctcacc</td><td> 240</td>

<td>atcagcagcc</td><td>tgcaggctga</td><td>agatgtggca</td><td>gtttattact</td><td>gtcaccaata</td><td>ttatagtact</td><td> 300</td>

<td>ccgtggacgt</td><td>tcggccaagg</td><td>gaccaaggtg</td><td>gaaatcaaac</td><td></td><td></td><td> 340</td>

&lt;210&gt; 52 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 52

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

131

<td>Glu</td><td>Arg Ala</td><td>Thr twenty</td><td>How much</td><td>own</td><td>Cys</td><td>lys</td><td>Cheese 25</td><td>Cheese Gin Cheese Val</td><td>Leu thirty</td><td>Tyr</td><td>Cheese</td>

<td>Cheese</td><td>How much Asn 35</td><td>lys</td><td>How much</td><td>Tyr</td><td>Leu</td><td>ala 40</td><td>Trp</td><td>Tyr Gin Gin Lys 45</td><td>Pro</td><td>Gly</td><td>Gin</td>

<td>Pro</td><td>Pro Lys 50</td><td>Leu</td><td>Leu</td><td>How much</td><td>Tyr 55</td><td>Trp</td><td>ala</td><td>Cheese Thr Arg Glu 60</td><td>Cheese</td><td>Gly</td><td>val</td>

<td>That. λ</td><td>Λ «.μ Λ</td><td>nk λ.</td><td></td><td></td><td>OA</td><td></td><td></td><td>r? 1 «· - Ά λ—. ^ nk</td><td>mk</td><td>r -</td><td>mk</td>

<td>You. IN 65</td><td></td><td>ruc</td><td></td><td>«χγ 70</td><td></td><td></td><td>QCI</td><td>me nothing 75</td><td>Ilya</td><td>UCU</td><td>Ilya 80</td>

<td>How much</td><td>Cheese Cheese</td><td>Leu</td><td>Gin 85</td><td>ala</td><td>Glu</td><td>Asp</td><td>val</td><td>Ala Val Tyr Tyr 90</td><td>Cys</td><td>His 95</td><td>Gin</td>

<td>Tyr</td><td>Tyr Ser</td><td>Thr 100</td><td>Pro</td><td>Trp</td><td>Thr</td><td>phe</td><td>Gly 105</td><td>Gin Gly Thr Lys</td><td>val 110</td><td>Glu</td><td>How much</td>

&lt;210&gt; 53 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 53

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggcccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccatcagc</td><td>agtggtgatt</td><td>actactggaa</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct</td><td>ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagcacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag</td><td>tcgagttacc</td><td>atttcagtag</td><td>ccacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagag</td><td> 300</td>

<td>gctacggagg</td><td>ggtttgacta</td><td>ctggggccag</td><td>ggaaccctgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 54 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 54

Gin Val Gin Leu Gin Glu Cheese Gly Pro Gly Leu Val Lys Pro Cheese 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

132

Trp Ile Gly Tyr Ile Tyr Tyr Cheese 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

70 75 80

Cheese Leu Lys Leu Cheese Cheese Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

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 &lt;210&gt; 55 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 55

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagggccacc</td><td> 60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtgttagc</td><td>accacctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>ggtgcatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcactc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcactgta</td><td>ttactgtcag</td><td>cactatggta</td><td>cctcatcgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 56 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="3"> &lt;400&gt; 56</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>How much</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Thr</td><td>Thr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>ni '. "·</td><td>T A "</td><td>loop,</td><td>These-**</td><td>Φ "» *&gt;</td><td>ι-l "</td><td>m</td><td>Τ</td><td></td><td>/ -i ·.</td><td>Gin</td><td> » 1 -</td><td>n "4-p</td><td>AND</td><td>T</td><td>Τ</td>

<td>iJ, J.</td><td>at</td><td></td><td></td><td></td><td></td><td>.J4.il</td><td>JJJf &lt;9</td><td>JC J. V</td><td></td><td></td><td></td><td>JC 4- W</td><td>aj. s</td><td>JJ ^ u</td><td>JJ ^ U</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu</td>

133

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 100 105 &lt;210&gt; 57 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 57

<td>caggtgcagc</td><td>tgcaggagtc gggcccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg ctccatcagc</td><td>agtggtgatt</td><td>actactggaa</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagcacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag tcgagttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagaa</td><td> 300</td>

<td>tccacggacg</td><td>gtatggacgt ctggggccaa</td><td>gggaccacgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 58 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 58

<td>Gin 1</td><td>val</td><td>Gin</td><td>Leu</td><td>Gin 5</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>Gly 10</td><td>Leu</td><td>val</td><td>lys</td><td>Pro</td><td>Cheese 15</td><td>Gin</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese 25</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>Gly</td>

<td>Asp</td><td>Tyr</td><td>Tyr 35</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg 40</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>How much 50</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr 55</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr 60</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td>Leu 65</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr 70</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr 75</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese 85</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala 90</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu 100</td><td>Cheese</td><td>Thr</td><td>Asp</td><td>Gly</td><td>Underworld 105</td><td>Asp</td><td>val</td><td>Trp</td><td>Gly</td><td>Gin 110</td><td>Gly</td><td>Thr</td>

Thr Val Thr Val Ser Ser 115 &lt;210&gt; 59 &lt;211&gt; 325 &lt;212&gt; DNA

134 &lt;213&gt; Human &lt;400&gt; 59

<td>gaaagtgtgt tgacgcagtc tcctggcacc ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgca gggccagtca gagtgttagc agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg ctcccaggct cctcatatat ggtgtttcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca gtggcagtgg gtctgggaca gacttcactc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt ttgcagtgta ttactgtcag cagtatggta</td><td>gctcaccgat</td><td>caccttcggc</td><td> 300</td>

<td>Jaagggacac gactggagat taaac</td><td></td><td></td><td> 325</td>

&lt;210&gt; 60 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="12"> &lt;400&gt; 60</td><td rowspan="3">Leu</td><td rowspan="3">Cheese</td><td colspan="2" rowspan="3">Pro Gly 15</td>

<td rowspan="2">Glu 1</td><td colspan="2" rowspan="2">Val cheese</td><td colspan="8">Leu Thr Gin Ser Pro Gly Thr Leu</td><td rowspan="2">Cheese</td>

<td colspan="4"> 5</td><td colspan="4"> 10</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Pro</td>

<td>AND</td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td>AND</td><td></td><td></td><td> 95</td><td></td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td>

100 105 &lt;210&gt; 61 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 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 tcaccgtctc ctcag 355

135 &lt;210&gt; 62 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 62

<td>Gin 1</td><td>val</td><td>Gin</td><td>Leu</td><td colspan="3">Gin Glu Cheese 5</td><td colspan="4">Gly Pro Gly Lei 10</td><td colspan="4">l Val Lys Pro</td><td>Cheese 15</td><td>Gin</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese</td><td>Gly</td><td colspan="2">Gly</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Gly</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Tyr</td><td>Tyr</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>His</td><td colspan="2">Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>How much</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td colspan="2">Thr</td><td>Tyr</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td colspan="2">Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td colspan="2">Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu</td><td>Asp</td><td>How much</td><td>ala</td><td>Gly</td><td>phe</td><td>Asp</td><td colspan="2">Pro</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Leu</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 &lt;210&gt; 63 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 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 &lt;210&gt; 64 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 64

136

<td>Glu</td><td>How much</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly |</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 65 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 65

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggcccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccatcagc</td><td>agtggtgaat</td><td>actactggag</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct</td><td>ggagtggatt</td><td>gggtatatct</td><td>tttacagtgg</td><td>gagcacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag</td><td>tcgagttacc</td><td>atatcactag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagaa</td><td> 300</td>

<td>tccacggacg</td><td>gtatggacgt</td><td>ctggggccaa</td><td>gggaccacgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 66 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 66

137

<td colspan="2">Gin Val Gin Leu Gin</td><td rowspan="2">Glu Cheese</td><td rowspan="2">Gly</td><td colspan="2">Pro Gly Leu Val Lys Pro Ser</td><td rowspan="2">Gin</td>

<td> 1</td><td> 5</td><td> 10</td><td> 15</td>

<td>Thr</td><td>Leu Ser Leu Thr</td><td>Cys Thr</td><td>val</td><td>Gly cheese</td><td>Gly Cheese Ile Cheese Ser</td><td>Gly</td>

<td></td><td> 20</td><td></td><td></td><td> 25</td><td> 30</td><td></td>

<td>Glu</td><td>Tyr Tyr Trp Ser</td><td>Trp Ile</td><td>Arg</td><td>Gin His</td><td>Pro Gly Lys Gly Leu</td><td>Glu</td>

<td></td><td> 35</td><td></td><td> 40</td><td></td><td> 45</td><td></td>

<td>Trp</td><td>Ile Gly Tyr Ile</td><td>Phe Tyr</td><td>Cheese</td><td>Gly Cheese</td><td>Thr Tyr Tyr Asn Pro</td><td>Cheese</td>

<td></td><td> 50</td><td> 55</td><td></td><td></td><td> 60</td><td></td>

<td>Leu</td><td>Lys Ser Arg Val</td><td>Thr Ile</td><td>Cheese</td><td>Leu Asp</td><td>Thr Ser Lys Asn Gin</td><td>phe</td>

<td> 65</td><td></td><td> 70</td><td></td><td></td><td> 75</td><td> 80</td>

<td>Cheese</td><td>Leu Lys Leu Ser</td><td>Val cheese</td><td>Thr</td><td>Ala Ala</td><td>Asp Thr Ala Val Tyr</td><td>Tyr</td>

<td></td><td> 85</td><td></td><td></td><td> 90</td><td>⁹⁵</td><td></td>

<td>Cys</td><td>Ala Arg Glu Cheese</td><td>Thr Asp</td><td>Gly</td><td>Met Asp</td><td>Val Trp Gly Gin Gly</td><td>Thr</td>

<td></td><td> 100</td><td></td><td></td><td> 105</td><td> 110</td><td></td>

<td>Thr</td><td>Val Thr Val Ser</td><td>Cheese</td><td></td><td></td><td></td><td></td>

115 &lt;210&gt; 67 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 67

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtgttagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcggaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatatat</td><td>ggtgtatcca</td><td>gtagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcactc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcag</td><td>caatatggta</td><td>gctcaatgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 68 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 68

Glu Ile 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

138

<img file="PL2447283T3_D0019.tif" />

&lt;210&gt; 69 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 69

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggcccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccatcagc</td><td>agtggtgatt</td><td>actactggaa</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct</td><td>ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagcacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag</td><td>tcgagttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagag</td><td> 300</td>

<td>ggcctcgagg</td><td>cttttgatat</td><td>ctggggtcaa</td><td>gggacaatgg</td><td>tcaccgactc</td><td>ttcag</td><td> 355</td>

&lt;210&gt; 70 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="3"> &lt;400&gt; 70</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>val</td><td>Gin</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Leu</td><td>val</td><td>lys</td><td>Pro</td><td>Cheese</td><td>Gin</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Gly</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Tyr</td><td>Tyr</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>How much</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td>

139

70 75 80

Cheese Leu Lys Leu Cheese Cheese Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Glu Gly Leu Glu Ala Phe Asp Ile Trp Gly Gin Gly Thr 100 105 110

Met Val Thr Asp Ser Ser 115 &lt;210&gt; 71 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 71

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td> 60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtgttagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>gatgcatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>ctctgggaca</td><td>gacttcactc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ctactgtcag</td><td>cattatggta</td><td>gctcacttct</td><td>cactttcggc</td><td> 300</td>

<td>ggagggacca</td><td>aggtggagat</td><td>caaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 72 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 72

Glu Ile 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 Asp Ala Ser Cheese 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 His Tyr Gly Cheese Cheese Leu 85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 &lt;210&gt; 73 &lt;211&gt; 355 &lt;212&gt; DNA

140 &lt;213&gt; Human &lt;400&gt; 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 ctggęjgccaa gggaccacgg tcaccgtctc ctcag 35i &lt;210&gt; 74 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="6"> &lt;400&gt; 74</td><td rowspan="3">Cheese</td><td rowspan="3">Gly</td><td colspan="8" rowspan="2">Pro Gly Leu Val Lys Pro Ser Gin</td>

<td rowspan="2">Gin 1</td><td colspan="2" rowspan="2">Val Gin</td><td rowspan="2">Leu</td><td colspan="2" rowspan="2">Gin Glu 5</td>

<td colspan="2"> 10</td><td colspan="6"> 15</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu twenty</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese 25</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>Gly</td>

<td>Asp</td><td>Tyr</td><td>Tyr 35</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg 40</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys 45</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td>Trp</td><td>How much 50</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr 55</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr 60</td><td>Tyr</td><td>own</td><td>Cheese</td><td>Cheese</td>

<td>Leu 65</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr 70</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr 75</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese 85</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala 90</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr 95</td><td>Tyr</td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu 100</td><td>Gly</td><td>Gin</td><td>own</td><td>Gly</td><td>Underworld 105</td><td>Asp</td><td>val</td><td>Trp</td><td>Gly</td><td>Gin 110</td><td>Gly</td><td>Thr</td>

<td>Thr</td><td>val</td><td>Thr 115</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 75 &lt;211&gt; 340 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 75

141

<td>gatgttgtga</td><td>tgactcagtc</td><td>tccactctcc</td><td>ctgcccgtca</td><td>cccttggaca</td><td>gccggcctcc</td><td> 60</td>

<td>atctcctgca</td><td>ggtctagtca</td><td>aagcctcgta</td><td>tacagtgatg</td><td>gaaacaccta</td><td>cttgaattgg</td><td> 120</td>

<td>tttcagcaga</td><td>ggccaggcca</td><td>atctccaagg</td><td>cgcctaattt</td><td>ataaggtttc</td><td>taactgggac</td><td> 180</td>

<td>tctggggtec</td><td>cagacagatt</td><td>cagcggcagt</td><td>gggtcaggca</td><td>ctgatttcae</td><td>actgaaaatc</td><td> 240</td>

<td>agcagggtgg</td><td>aggctgagga</td><td>tgttggggtt</td><td>tattactgca</td><td>tgcaaggtac</td><td>acactggcct</td><td> 300</td>

<td>ccgtggacgt</td><td>tcggccaagg</td><td>gaccaaggtg</td><td>gaaatcaaac</td><td></td><td></td><td> 340</td>

&lt;210&gt; 76 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="3"> &lt;400&gt; 76</td><td rowspan="3">Underworld</td><td colspan="8" rowspan="2">Thr Gin Ser Pro Leu Ser Leu Pro</td><td rowspan="3">val</td><td colspan="3" rowspan="3">Thr Leu Gly 15</td>

<td rowspan="2">Asp 1</td><td rowspan="2">val</td><td rowspan="2">val</td>

<td> 5</td><td colspan="7"> 10</td>

<td>Gin</td><td>Pro</td><td>ala</td><td>Cheese twenty</td><td>How much</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>Cheese 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>Leu</td><td>val thirty</td><td>Tyr</td><td>Cheese</td>

<td>Asp</td><td>Gly</td><td>own 35</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>own</td><td>Trp 40</td><td>phe</td><td>Gin</td><td>Gin</td><td>Arg</td><td>Pro 45</td><td>Gly</td><td>Gin</td><td>Cheese</td>

<td>Pro</td><td>Arg 50</td><td>Arg</td><td>Leu</td><td>How much</td><td>Tyr</td><td>lys 55</td><td>val</td><td>Cheese</td><td>own</td><td>Trp</td><td>Asp 60</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td>

<td>Asp 65</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese 70</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp 75</td><td>phe</td><td>Thr</td><td>Leu</td><td>lys</td><td>How much 80</td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Glu</td><td>ala 85</td><td>Glu</td><td>Asp</td><td>val</td><td>Gly</td><td>val 90</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Underworld</td><td>Gin 95</td><td>Gly</td>

<td>Thr</td><td>His</td><td>Trp</td><td>Pro 100</td><td>Pro</td><td>Trp</td><td>Thr</td><td>phe</td><td>Gly 105</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lys</td><td>val 110</td><td>Glu</td><td>How much</td>

Lys | &lt;210&gt; 77 &lt;211&gt; 358 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 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 &lt;210&gt; 78

142 &lt;211&gt; 119 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 78</td><td colspan="2" rowspan="2">Glu Cheese</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">Gly 10</td><td rowspan="2">Leu</td><td rowspan="2">val</td><td rowspan="2">lys</td><td rowspan="2">Pro</td><td colspan="2" rowspan="2">Gly Gly 15</td>

<td>Glu 1</td><td colspan="2">Val Gin</td><td>Leu</td><td>val 5</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu twenty</td><td>Cheese</td><td>eys</td><td>ala</td><td>ala</td><td>Cheese 25</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Cheese thirty</td><td>own</td><td>ala</td>

<td>Trp</td><td>Underworld</td><td>Cheese 35</td><td>Trp</td><td>val</td><td>Arg</td><td>Gin</td><td>ala 40</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu 45</td><td>Glu</td><td>Trp</td><td>val</td>

<td>Gly</td><td>Arg 50</td><td>How much</td><td>lys</td><td>Cheese</td><td>lys</td><td>Thr 55</td><td>Asp</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Thr 60</td><td>Asp</td><td>Tyr</td><td>ala</td><td>ala</td>

<td>Pro 65</td><td>val</td><td>lys</td><td>Gly</td><td>Arg</td><td>phe 70</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp 75</td><td>Asp</td><td>Cheese</td><td>lys</td><td>own</td><td>Thr 80</td>

<td>Leu</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>Underworld 85</td><td>own</td><td>Cheese</td><td>Leu</td><td>lys</td><td>Thr 90</td><td>Glu</td><td>Asp</td><td>Thr</td><td>ala</td><td>val 95</td><td>Tyr</td>

<td>Tyr</td><td>Cys</td><td>Thr</td><td>Thr 100</td><td>Gly</td><td>Asp</td><td>Gly</td><td>Thr</td><td>His 105</td><td>phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly 110</td><td>Gin</td><td>Gly</td>

<td>Thr</td><td>Leu</td><td>val 115</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 79 &lt;211&gt; 322 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 79

<td>gaaacgacac</td><td>tcacgcagtc</td><td>tccagcattc</td><td>atgtcagcga</td><td>ctccaggaga</td><td>caaagtcaac</td><td> 60</td>

<td>atctcctgca</td><td>aagccagcca</td><td>agacattgat</td><td>gatgatatga</td><td>actggtacca</td><td>acagaaacca</td><td> 120</td>

<td>ggagaagctg</td><td>ctattttcat</td><td>tattcaagaa</td><td>gctactactc</td><td>tcgttcctgg</td><td>aatcccacct</td><td> 180</td>

<td>cgattcagtg</td><td>gcagcgggta</td><td>tggaacagat</td><td>tttaccctca</td><td>caattaataa</td><td>catagaatct</td><td> 240</td>

<td>gaggatgctg</td><td>catattactt</td><td>ctgtctacaa</td><td>catgataatt</td><td>tcccgctcac</td><td>tttcggcgga</td><td> 300</td>

<td>gggaccaagg</td><td>tggagatcaa</td><td>ac</td><td></td><td></td><td></td><td> 322</td>

&lt;210&gt; 80 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 80

143

<td>Glu 1</td><td>Thr Thr Leu</td><td>Thr 5</td><td>Gin</td><td>Pro Ala cheese</td><td>phe 10</td><td>Underworld</td><td>Ala cheese</td><td>Thr Pro Gly 15</td>

<td>Asp</td><td>Lys Val Asn</td><td>How much</td><td>Cheese</td><td>Cys Lys Ala</td><td>Cheese</td><td>Gin</td><td>Asp Ile</td><td>Asp Asp</td>

<td></td><td> 20</td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td>

<td>Underworld</td><td>Asn Trp Tyr</td><td>Gin</td><td>Gin</td><td>Lys Pro Gly</td><td>Glu</td><td>ala</td><td>Ala Ile</td><td>Phe Ile Ile</td>

<td></td><td> 35</td><td></td><td></td><td> 40</td><td></td><td></td><td> 45</td><td></td>

<td>Gin</td><td>Glu Ala Thr</td><td>Thr</td><td>Leu</td><td>Val Pro Gly</td><td>How much</td><td>Pro</td><td>Pro Arg</td><td>Phe Ser Gly</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td></td><td></td><td> 60</td><td></td>

<td>Cheese</td><td>Gly Tyr Gly</td><td>Thr</td><td>Asp</td><td>Phe Thr Leu</td><td>Thr</td><td>How much</td><td>Asn Asn</td><td>Ile Glu Cheese</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td> 75</td><td></td><td> 80</td>

<td>Glu</td><td>Asp Ala Ala</td><td>Tyr</td><td>Tyr</td><td>Phe Cys Leu</td><td>Gin</td><td>His</td><td>Asp Asn</td><td>Phe Pro Leu</td>

<td></td><td></td><td> 85</td><td></td><td></td><td> 90</td><td></td><td></td><td> 95</td>

<td>Thr</td><td>Phe Gly Gly</td><td>Gly</td><td>Thr</td><td>Lys Val Glu</td><td>How much</td><td>lys</td><td></td><td></td>

100 105 &lt;210&gt; 81 &lt;211&gt; 379 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 81 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac cacatactac 180 tC you tyuayyyCCy uttCaCCatC tCCayyyaCa a.CyCCaay au ytCSCtyta t 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagagajgg 300 tatagcagtg gctggtacga ggactactac tacggtatgg acgtctgggg ccaagggccc 360 acggccccg 379 &lt;210&gt; 82 &lt;211&gt; 126 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 82

144

<td rowspan="2">Gin 1</td><td rowspan="2">Val Gin</td><td rowspan="2">Leu</td><td rowspan="2">val 5</td><td colspan="2">Glu Cheese Gly Gly Gly Leu Val Lys Pro Gly</td><td rowspan="2">Gly</td>

<td> 10</td><td> 15</td>

<td>Cheese</td><td>Leu Arg</td><td>Leu</td><td>Cheese</td><td>Cys Ala Ala Ser Gly Phe</td><td>Thr Phe Ser Asp</td><td>Tyr</td>

<td></td><td></td><td> 20</td><td></td><td> 25</td><td> 30</td><td></td>

<td>Tyr</td><td>Met Ser</td><td>Trp</td><td>How much</td><td>Arg Gin Ala Pro Gly Lys</td><td>Gly Leu Glu Trp</td><td>val</td>

<td></td><td> 35</td><td></td><td></td><td> 40</td><td> 45</td><td></td>

<td>Cheese</td><td>Tyr Ile</td><td>Cheese</td><td>Cheese</td><td>Ser Gly Ser Thr Thr Tyr</td><td>Tyr Ala Asp Ser</td><td>val</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td> 60</td><td></td>

<td>lys</td><td>Gly Arg</td><td>phe</td><td>Thr</td><td>Ile Ser Arg Asp Asn Ala</td><td>Lys Lys Cheese Leu</td><td>Tyr</td>

<td> 65</td><td></td><td></td><td></td><td> 70 75</td><td></td><td> 80</td>

<td>Leu</td><td>Gin Met</td><td>asi</td><td>Cheese</td><td>Leu Arg Ala Glu Asp Thr</td><td>Ala Val Tyr</td><td>Cys</td>

<td></td><td></td><td></td><td> 85</td><td> 90</td><td> 95</td><td></td>

<td>ala</td><td>Arg Glu</td><td>Gly</td><td>Tyr</td><td>Cheese Cheese Gly Trp Tyr Glu</td><td>Asp Tyr Tyr Tyr</td><td>Gly</td>

<td></td><td></td><td> 100</td><td></td><td> 105</td><td> 110</td><td></td>

<td>Underworld</td><td>Asp Val</td><td>Trp</td><td>Gly</td><td>Gin Gly Thr Thr Val Thr</td><td>Val Cheese Ser</td><td></td>

<td></td><td> 115</td><td></td><td></td><td> 120</td><td> 125</td><td></td>

&lt;210&gt; 83 &lt;211&gt; 337 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 83

<td>gatattgtga</td><td>tgacccagac</td><td>tccactctct</td><td>ctgtccgtca</td><td>cccctggaca</td><td>gccggcctcc</td><td> 60</td>

<td>atctcctgca</td><td>agtttagtca</td><td>gagcctcctg</td><td>catagtgatg</td><td>gaaagaccta</td><td>tttgtattgg</td><td> 120</td>

<td>tacctgcaga</td><td>agccaggcca</td><td>gcctccacag</td><td>ctcctgatct</td><td>atgaagtttc</td><td>caaccggttt</td><td> 180</td>

<td>tctggagtgc</td><td>cagataggtt</td><td>cagtggcagc</td><td>gggtcaggga</td><td>cagatttcac</td><td>actgaaaatc</td><td> 240</td>

<td>agccgggtgg</td><td>aggctgagga</td><td>tgttggggtt</td><td>tattactgca</td><td>tgcaaagtat</td><td>acagcttcct</td><td> 300</td>

<td>cggacgttcg</td><td>gccaagggac</td><td>caaggtggaa</td><td>atcaaac</td><td></td><td></td><td> 337</td>

&lt;210&gt; 84 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 84

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

Gin Pro Ala Ser Ile Ser Cys Lys Phe Ser Gin Ser Leu Leu His Ser 20 25 30

145

<td>Asp</td><td>Gly</td><td>lys 35</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Trp 40</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>lys</td><td>Pro 45</td><td>Gly</td><td>Gin</td><td>Pro</td>

<td>Pro</td><td>Gin 50</td><td>Leu</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Glu 55</td><td>val</td><td>Cheese</td><td>own</td><td>Arg</td><td>phe 60</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td>

<td>Asp 65</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese 70</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp 75</td><td>phe</td><td>Thr</td><td>Leu</td><td>lys</td><td>How much 80</td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Glu</td><td>ala 85</td><td>Glu</td><td>Asp</td><td>val</td><td>Gly</td><td>val 90</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Underworld</td><td>Gin 95</td><td>Cheese</td>

<td>How much</td><td>Gin</td><td>Leu</td><td>Pro 100</td><td>Arg</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin 105</td><td>Gly</td><td>Thr</td><td>lys</td><td>val</td><td>Glu 110</td><td>How much</td><td>lys</td>

&lt;210&gt; 85 &lt;211&gt; 388 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 85

<td>caggtgcagc</td><td>tggtggagtc</td><td>tgggggaggc</td><td>ttggtcaagc</td><td>ctggagggtc</td><td>cctgagactc</td><td> 60</td>

<td>tcctgtgcag</td><td>cctctggatt</td><td>caccttcagt</td><td>gacttctaca</td><td>tgagctggat</td><td>ccgccaggct</td><td> 120</td>

<td>ccagggaagg</td><td>ggctggaatg</td><td>gatttcatac</td><td>attagtagta</td><td>gtggtagtac</td><td>catttactac</td><td> 180</td>

<td>gcagactctg</td><td>tgaagggccg</td><td>attcaccatg</td><td>tccagggaca</td><td>acgccaagaa</td><td>ctcactgtat</td><td> 240</td>

<td>ctgcaaatga</td><td>acagcctgag</td><td>agccgaggac</td><td>acggccgtgt</td><td>attattgtgc</td><td>gagagaagga</td><td> 300</td>

<td>tactatgatt</td><td>cggggagtta</td><td>ttataaggac</td><td>tacgactact</td><td>acggtatgga</td><td>cgtctggggc</td><td> 360</td>

<td>caagggacca</td><td>cggtcaccgt</td><td>ctcctcag</td><td></td><td></td><td></td><td> 388</td>

&lt;210&gt; 86 &lt;211&gt; 129 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 86

<td>Gin 1</td><td>val</td><td>Gin</td><td>Leu</td><td>val 5</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Gly 10</td><td>Leu</td><td>val</td><td>lys</td><td>Pro</td><td>Gly 15</td><td>Gly</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu twenty</td><td>Cheese</td><td>Cys</td><td>ala</td><td>ala</td><td>Cheese 25</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Cheese thirty</td><td>Asp</td><td>phe</td>

<td>Tyr</td><td>Underworld</td><td>Cheese 35</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>ala 40</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu 45</td><td>Glu</td><td>Trp</td><td>How much</td>

<td>Cheese</td><td>Tyr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>ala</td><td>Asp</td><td>Cheese</td><td>val</td>

55 60

146

Lys Gly Arg Phe Thr Met 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 Axy Glu Gly Tyr Tyr Asp Sec Gly Star. Tyc Tys Lys Asp Tyc Asp 100 105 110

Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser 115 120 125

Ser &lt;210&gt; 87 &lt;211&gt; 337 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 87

<td>gatattgtga</td><td>tgacccagac</td><td>tcoactctct</td><td>ctgtccgtca</td><td>cccctggaca</td><td>gccggcctcc</td><td> 60</td>

<td>atctcctgca</td><td>agtctagtca</td><td>gagcctcctg</td><td>catagtgatg</td><td>gaaagaccta</td><td>tttgtattgg</td><td> 120</td>

<td>tacctgcaga</td><td>agccaggcca</td><td>gcctccacag</td><td>ctcctgatct</td><td>atgaagtttc</td><td>caaccggttc</td><td> 180</td>

<td>tctggagtgc</td><td>cagataggtt</td><td>cagtggcagc</td><td>gggtcaggga</td><td>cagatttcac</td><td>actgaaaatc</td><td> 240</td>

<td>agccgggtgg</td><td>aggctgagga</td><td>tgttggggtt</td><td>tattactgca</td><td>tgcaaagtat</td><td>acagcttcct</td><td> 300</td>

<td>cggacgttcg</td><td>gccaagggac</td><td>caaggtggaa</td><td>atcaaac</td><td></td><td></td><td> 337</td>

&lt;210&gt; 88 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 88

Asp Ile Val Met Thr Gin Thr Pro 1 5

Leu Ser Leu Ser Val Thr Pro Gly 10 15

Gin Pro Ala Cheese Ile Ser Cys Lys 20

Cheese Cheese Gin Cheese Leu Leu His Cheese 25 I 30

Asp Gly Lys Thr Tyr Leu Tyr Trp 35 40

Tyr Leu Gin Lys Pro Gly Gin Pro 45

Pro Gin Leu Leu Ile Tyr Glu Val 50 55

Asn Arg Phe Cheese Gly Val Pro 60

Asp Arg Phe Cheese Gly Cheese Gly Cheese 65 70

Cheese Arg Val Glu Ala Glu Asp Val 85

Gly Thr Asp Phe Thr Leu Lys Ile 75 80

Gly Val Tyr Tyr Cys Met Gin Ser 90 95

Ile Gin Leu Pro Arg Thr Phe Gly 100

Gin Gly Thr Lys Val Glu Ile Lys 105 110

147 &lt;210&gt; 89 &lt;211&gt; 379 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 89

<td>caggtgcggc</td><td>tggtggagtc</td><td>tgggggaggc</td><td>ttggtcaagc</td><td>ctggagggtc</td><td>cctgagactc</td><td> 60</td>

<td>tcctgtgcag</td><td>cctctggatt</td><td>caccttcagt</td><td>gactactaca</td><td>tgagctggat</td><td>ccgccaggct</td><td> 120</td>

<td>ccagggaagg</td><td>ggctggagtg</td><td>ggtttcatac</td><td>attagtagta</td><td>gtggtatttc</td><td>catatactac</td><td> 180</td>

<td>gcagactctg</td><td>tgaagggccg</td><td>attcaccatc</td><td>tccagggaca</td><td>acgccaagaa</td><td>ctcactgtat</td><td> 240</td>

<td>ctgcaaatga</td><td>acagcctgag</td><td>agccgaggac</td><td>acggccgtgt</td><td>attactgtgc</td><td>gagagaagga</td><td> 300</td>

<td>tatagcagct</td><td>cgtcacatta</td><td>ctacgactac</td><td>tacggtatgg</td><td>acgtctgggg</td><td>ccaagggacc</td><td> 360</td>

<td>acggtcaccg</td><td>tctcctcag</td><td></td><td></td><td></td><td></td><td> 379</td>

&lt;210&gt; 90 &lt;211&gt; 126 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="3"> &lt;400&gt; 90</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Gin 1</td><td>val</td><td>Arg</td><td>Leu</td><td>val 5</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Gly 10</td><td>Leu</td><td>val</td><td>lys</td><td>Pro</td><td>Gly 15</td><td>Gly</td>

<td>Cheese</td><td>Leu</td><td>Arg</td><td>Leu twenty</td><td>Cheese</td><td>Cys</td><td>ala</td><td>ala</td><td>Cheese 25</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Cheese thirty</td><td>Asp</td><td>Tyr</td>

<td>Tyr</td><td>Underworld</td><td>Cheese 35</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>ala 40</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu 45</td><td>Glu</td><td>Trp</td><td>val</td>

<td>Cheese</td><td>Tyr 50</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Gly 55</td><td>How much</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Tyr 60</td><td>ala</td><td>Asp</td><td>Cheese</td><td>val</td>

<td>lys 65</td><td>Gly</td><td>Arg</td><td>phe</td><td>Thr</td><td>How much 70</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>own</td><td>ala 75</td><td>lys</td><td>own</td><td>Cheese</td><td>Leu</td><td>Tyr 80</td>

<td>Leu</td><td>Gin</td><td>Underworld</td><td>own</td><td>Cheese 85</td><td>Leu</td><td>Arg</td><td>ala</td><td>Glu</td><td>Asp 90</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr 95</td><td>Cys</td>

<td>ala</td><td>Arg</td><td>Glu</td><td>Gly 100</td><td>Tyr</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Cheese 105</td><td>His</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>Tyr 110</td><td>Tyr</td><td>Gly</td>

Met Asp

Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 &lt;210&gt; 91 &lt;211&gt; 337 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 91

148

<td>gatattgtga</td><td>tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc</td><td> 60</td>

<td>atctcctgca</td><td>agtctagtca gagcctcctg catagtgatg gaaagaccta tttgtattgg</td><td> 120</td>

<td>tacctgcaga</td><td>agccaggcca gcctccacag gtccttatct atgaagtttc caaccggttc</td><td> 180</td>

<td>tctggagtgc</td><td>cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc</td><td> 240</td>

<td>agccgggtgg</td><td>aggctgagga tgttggggtt tattactgca tgcaaagtac acagcttcct</td><td> 300</td>

<td>cggacgttcg</td><td>gccaagggac caaggtggaa atcjaaac</td><td> 337</td>

&lt;210&gt; 92 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="5"> &lt;400&gt; 92</td><td rowspan="3">Gin</td><td rowspan="3">Thr</td><td rowspan="3">Pro</td><td colspan="8" rowspan="2">Leu Ser Leu Ser Val Thr Pro Gly</td>

<td rowspan="2">Asp 1</td><td rowspan="2">How much</td><td colspan="2" rowspan="2">Val Met</td><td rowspan="2">Thr 5</td>

<td colspan="2"> 10</td><td colspan="6"> 15</td>

<td>Gin</td><td>Pro</td><td>ala</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Cheese</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>His</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Gly</td><td>lys</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Pro</td><td>Gin</td><td>val</td><td>Leu</td><td>How much</td><td>Tyr</td><td>Glu</td><td>val</td><td>Cheese</td><td>own</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>val</td><td>Pro</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>lys</td><td>How much</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Glu</td><td>ala</td><td>Glu</td><td>Asp</td><td>val</td><td>Gly</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Underworld</td><td>Gin</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> ?°</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Gin</td><td>Leu</td><td>Pro</td><td>Arg</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lys</td><td>val</td><td>Glu</td><td>How much</td><td>lys</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

&lt;210&gt; 93 &lt;211&gt; 503 &lt;212&gt; PRT &lt;213&gt; Monkey, Crab-eating macaque &lt;400&gt; 93

149

Met Thr Leu Gly Ser Pro Arg Arg Gly Leu Leu Met Leu Leu Met Ala 1 5 10 15

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 CJys 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 Ile 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

Cheese Leu Ile Leu Lys Ala Cheese Glu Gin Gly Asp Cheese 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

Cheese Val Ala Val Lys Ile Phe Cheese Cheese Arg Asp Glu Gin Cheese Trp Phe 225 230 235 240

Arg Glu Thr Glu Ile Tyr Asn Thr Val Leu Leu Arg His Asp Asn Ile 245 250 255

Leu Gly Phe Ile Ala Ser Asp Met Thr Ser Arg Asn Ser Ser Thr Gin

150

<td colspan="8"> 260</td>

<td>Leu</td><td>Trp</td><td>Leu 275</td><td>How much</td><td>Thr</td><td>His</td><td>Tyr</td><td>His 280</td>

<td>Leu</td><td>Gin 290</td><td>Arg</td><td>Gin</td><td>Thr</td><td>Leu</td><td>Glu 295</td><td>Pro</td>

<td>Cheese 305</td><td>ala</td><td>ala</td><td>Cys</td><td>Gly</td><td>Leu 310</td><td>ala</td><td>His</td>

<td>Gin</td><td>Gly</td><td>lys</td><td>Pro</td><td>ala 325</td><td>How much</td><td>ala</td><td>His</td>

<td>Leu</td><td>val</td><td>lys</td><td>Cheese 340</td><td>own</td><td>Leu</td><td>Gin</td><td>C ^ s</td>

<td>val</td><td>Underworld</td><td>His 355</td><td>Cheese</td><td>Gin</td><td>Gly</td><td>Cheese</td><td>Asp 360</td>

<td>Arg</td><td>val 370</td><td>Gly</td><td>Thr</td><td>lys</td><td>Arg</td><td>Tyr 375</td><td>Underworld</td>

<td>How much 385</td><td>Arg</td><td>Thr</td><td>Asp</td><td>Cys</td><td>phe 390</td><td>Glu</td><td>Cheese</td>

<td>phe</td><td>Gly</td><td>Leu</td><td>val</td><td>Leu 405</td><td>Trp</td><td>Glu</td><td>How much</td>

<td>How much</td><td>val</td><td>Glu</td><td>Asp 420</td><td>Tyr</td><td>Arg</td><td>Pro</td><td>Pro</td>

<td>Pro</td><td>Cheese</td><td>phe 435</td><td>Glu</td><td>Asp</td><td>Underworld</td><td>lys</td><td>lys 440</td>

<td>Pro</td><td>Thr 450</td><td>How much</td><td>Pro</td><td>own</td><td>Arg</td><td>Leu 455</td><td>ala</td>

<td>ala 465</td><td>Gin</td><td>Underworld</td><td>Underworld</td><td>Arg</td><td>Glu 470</td><td>Cys</td><td>Trj?</td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Arg</td><td>How much 485</td><td>lys</td><td>lys</td><td>Thr</td>

<td>Glu</td><td>lys</td><td>Pro</td><td>lys</td><td>val</td><td>How much</td><td>Gin</td><td></td>

265 270

Glu His Gly Ser Leu Tyr Asp Phe, 285

His Leu Ala Leu Arg Leu Ala Val 300

Leu His Val Glu Ile Phe Gly Thr 315 320

Arg Asp Phe Lys Ser Arg Asn Val 330 335

Cys Ile Ala Asp Leu Gly Leu Ala 345 350

Tyr Leu Asp Ile Gly Asn Asn Pro 365

Ala Pro Glu Val Leu Asp Glu Gin 380

Tyr Lys Trp Thr Asp Ile Trp Ala 395 400

Ala Arg Arg Thr Ile Val Asn Gly

A 1 Π A 1 R

Phe Tyr Asp Val Val Pro Asn Asp 425 430

Val Val Cys Val Asp Gin Gin Thr 445

Ala Asp Pro Val Leu Ser Gly Leu 460

Tyr Pro Asn Pro Cheese Ala Arg Leu 475 480

Leu Gin Lys Ile Cheese Asn Ser Pro 490 495

500 &lt;210&gt; 94 &lt;211&gt; 1512 &lt;212&gt; DNA &lt;213&gt; Monkey, Crab-eating macaque &lt;400&gt; 94

151

<td>atgaccttgg gctccccgag gagaggcctt ctgatgctgc tgatggcctt ggtgacccag</td><td> 60</td>

<td>ggtgaccccg tgaagccctc tcggggcccg ctggtgacct gcacatgtga gagcccacat</td><td> 120</td>

<td>tgcagggggc ctacctgcca gggggcctgg tgcacagtag tgctggtgcg ggaggagggg</td><td> 180</td>

<td>aggcaccccc aggaacatcg gggctgcggg aacttgcaca gggagctctg cagggggcgc</td><td> 240</td>

<td>cccaccgagt tcgtcaacca ctactgctgt gacagccacc tctgcaaccg caacgtgtcc</td><td> 300</td>

<td>ctggtgctgg aggccaccca aactccttcg gagcagccgg gaacagacag ccagctggcc</td><td> 360</td>

<td>ctgatcctgg gccccgtgct ggccttgctg gccctggtgg ccctgggtgt cgtgggcctg</td><td> 420</td>

<td>tggcatgtcc gacggaggca ggagaagcag cggggcctgc acagcgagct gggagagtćc</td><td> 480</td>

<td>agtctcatcc tgaaagcatc tgagcagggc gacagcatgt tgggggacct cctggacagt</td><td>54 (P</td>

<td>gactgcacca cagggagtgg ctcggggctc cccttcctgg tgcagaggac agtggcacgg</td><td> 600</td>

<td>caggttgcct tggtggagtg tgtgggaaaa ggccgctatg gcgaagtgtg gcggggcttg</td><td> 660</td>

<td>tggcacggtg agagtgtggc cgtcaagatc ttctcctcga gggacgaaca gtcctggttc</td><td> 720</td>

<td>cgggagactg agatctacaa cacagtgttg ctcagacacg acaacatcct aggcttcatc</td><td> 780</td>

<td>gcctcagaca tgacctcccg caactcgagc acgcagctgt ggctcatcac gcattaccac</td><td> 840</td>

<td>gagcacggct ccctctacga ctttctgcag agacagacgc tggagccgca tttggctctg</td><td> 900</td>

<td>aggctagctg tgtccgcagc ctgtggcctg gcacacctgc acgtggagat cttcggtaca</td><td> 960</td>

<td>cagggcaaac cggccattgc ccaccgtgac ttcaagagcc gcaacgtgct ggtcaagagc</td><td> 1020</td>

<td>aacctgcagt gttgcattgc tgacctgggc ctggctgtga tgcactcaca gggcagcgat</td><td> 1080</td>

<td>tacctggaca tcggcaacaa cccgag agta ggcaccaaga ggtacatggc acccgaggtg</td><td> 1140</td>

<td>ctggatgagc agatccgcac ggactgcttt gagtcctata agtggactga catctgggcc</td><td> 1200</td>

<td>tttggcctgg tgctgtggga gatcgcccgc cggaccatcg tgaacggcat cgtggaggac</td><td> 1260</td>

<td>tatagaccac ccttctatga tgtggtgccc aatgacccca gctttgagga catgaagaag</td><td> 1320</td>

<td>gtggtgtgtg tggatcagca gacccccacc atccctaacc ggctggctgc agacccggtc</td><td> 1380</td>

<td>ctctcaggcc tagctcagat gatgcgggag tgctggtacc caaacccctc tgcccgactc</td><td> 1440</td>

<td>actgcgctgc ggatcaagaa gacactacag aaaattagca acagtccaga gaagcccaaa</td><td> 1500</td>

<td>gtgattcagt ag</td><td> 1512</td>

&lt;210&gt; 95 &lt;211&gt; 1332 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 95 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

152

<td>acctgcactg tctctggtgg ctccatgagc agtggtgaat actactggaa ctggatccgc</td><td> 120</td>

<td>cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagtacctac</td><td> 180</td>

<td>tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc</td><td> 240</td>

<td>tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag</td><td> 300</td>

<td>tcagtggctg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcctcc</td><td> 360</td>

<td>accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca</td><td> 420</td>

<td>gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac</td><td> 480</td>

<td>tcaggcgctc tgaccagcgg cgtgcacacc ttcccagctg tcctacagtc ctcaggactc</td><td> 540</td>

<td>tactccctca gcagcgtggt gaccgtgccc tccagcaact tcggcaccca gacctacacc</td><td> 600</td>

<td>tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agacagttga gcgcaaatgt</td><td> 660</td>

<td>tgtgtcgagt gcccaccgtg cjscagcacca cctgtggcag gaccgtcagt cttcctcttc</td><td> 720</td>

<td>cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg</td><td> 780</td>

<td>gtggacgtga gccacgaaga ccccgaggtc cagttcaact ggtacgtgga cggcgtggag</td><td> 840</td>

<td>gtgcataatg ccaagacaaa gccacgggag gagcagttca acagcacgtt ccgtgtggtc</td><td> 900</td>

<td>agcgtcctca ccgttgtgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc</td><td> 960</td>

<td>tccaacaaag gcctcccagc ccccatcgag aaaaccatct ccaaaaccaa agggcagccc</td><td> 1020</td>

<td>cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc</td><td> 1080</td>

<td>agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc</td><td> 1140</td>

<td>aatgggcagc cggagaacaa ctacaagacc acacctccca tgctggactc cgacggctcc</td><td> 1200</td>

<td>ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc</td><td> 1260</td>

<td>tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg</td><td> 1320</td>

<td>tctccgggta aa</td><td> 1332</td>

&lt;210&gt; 96 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; compatible primer used for ECD cloning with ALK-1 &lt;400&gt; 96 acggcccagc cggccgaccc tgtgaagccg tct 33 &lt;210&gt; 97 &lt;211&gt; 47 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Inverse primer used for ECD cloning with ALK-1 &lt;400&gt; 97 actaagcttt taatgatgat gatgatgatg ctggccatct gttcccg 47

153 &lt;210&gt; 98 &lt;211&gt; 103 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="2"> &lt;400&gt; 98</td><td colspan="2" rowspan="2">Lys Pro Cheese Arg Gly Pro Leu Val</td><td rowspan="3">Thr Cys</td><td rowspan="3">Thr</td><td rowspan="3">Cys 15</td><td rowspan="3">Glu</td>

<td rowspan="2">Asp 1</td><td rowspan="2">Pro Val</td>

<td> 5</td><td> 10</td>

<td>Cheese</td><td>Pro His</td><td>Cys Lys Gly Pro Thr Cys</td><td>Arg Gly</td><td>Ala Trp</td><td>Cys</td><td>Thr</td><td>val</td>

<td></td><td></td><td> 20 25</td><td></td><td></td><td> 30</td><td></td><td></td>

<td>val</td><td>Leu Val</td><td>Arg Glu Glu Gly Arg His</td><td>Pro Gin</td><td>Glu His</td><td>Arg</td><td>Gly</td><td>Cys</td>

<td></td><td> 35</td><td> 40</td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gly</td><td>Asn Leu</td><td>His Arg Glu Leu Cys Arg</td><td>Gly Arg</td><td>Pro Thr</td><td>Glu</td><td>phe</td><td>val</td>

<td></td><td> 50</td><td> 55</td><td></td><td> 60</td><td></td><td></td><td></td>

<td>own</td><td>His Tyr</td><td>Cys Cys Asp Ser His Leu</td><td>Cys Asn</td><td>His Asn</td><td>val</td><td>Cheese</td><td>Leu</td>

<td> 65</td><td></td><td> 70</td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>val</td><td>Leu Glu</td><td>Ala Thr Gin Pro Pro Ser</td><td>Glu Gin</td><td>Pro Gly</td><td>Thr</td><td>Asp</td><td>Gly</td>

<td></td><td></td><td> 85</td><td> 90</td><td></td><td></td><td> 95</td><td></td>

<td>Gin</td><td>His His</td><td>His His His His</td><td></td><td></td><td></td><td></td><td></td>

100 &lt;210&gt; 99 &lt;211&gt; 387 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 99

<td>atggagacag</td><td>acacactcct</td><td>gctatgggta</td><td>ctgctgctct</td><td>gggttccagg</td><td>ttccactggt</td><td> 60</td>

<td>gacgcggccc</td><td>agccggccga</td><td>ccctgtgaag</td><td>ccgtctcggg</td><td>gcccgctggt</td><td>gacctgcacg</td><td> 120</td>

<td>tgtgagagcc</td><td>cacattgcaa</td><td>ggggcctacc</td><td>tgccgggggg</td><td>cctggtgcac</td><td>agtagtgctg</td><td> 180</td>

<td>gtgcgggagg</td><td>aggggaggca.</td><td>cccccaggaa</td><td>catcggggct</td><td>gcgggaactt</td><td>gcacagggag</td><td> 240</td>

<td>ctctgcaggg</td><td>ggcgccccac</td><td>cgagttcgtc</td><td>aaccactact</td><td>gctgcgacag</td><td>ccacctctgc</td><td> 300</td>

<td>aaccacaacg</td><td>tgtccctggt</td><td>gctggaggcc</td><td>acccaacctc</td><td>cttcggagca</td><td>gccgggaaca</td><td> 360</td>

<td>gatggccagc</td><td>atcatcatca</td><td>tcatcat</td><td></td><td></td><td></td><td> 387</td>

&lt;210&gt; 100 &lt;211&gt; 444 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 100

154

<img file="PL2447283T3_D0020.tif" />

155

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

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

Cheese Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 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 Cheese Asp Ile Ala Val Glu Trp Glu Cheese Asn Gly Gin Pro 370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Cheese 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 &lt;210&gt; 101 &lt;211&gt; 645 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 101

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagacacc</td><td> 60</td>

<td>ctctcctgta</td><td>gggccagtca</td><td>gagtgtcagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>ggtacatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcaccc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcag</td><td>cagtatggta</td><td>gctcgccgat</td><td>caccttcggc</td><td> 300</td>

156

<td>caagggacac</td><td>gactggagat</td><td>taaacgaact</td><td>gtggctgcac</td><td>catctgtctt</td><td>catcttcccg</td><td> 360</td>

<td>ccatctgatg</td><td>agcagttgaa</td><td>atctggaact</td><td>gcctctgttg</td><td>tgtgcctgct</td><td>gaataacttc</td><td> 420</td>

<td>tatcccagag</td><td>aggccaaagt</td><td>acagtggaag</td><td>gtggataacg</td><td>ccctccaatc</td><td>gggtaactcc</td><td> 480</td>

<td>caggagagtg</td><td>tcacagagca</td><td>ggacagcaag</td><td>gacagcacct</td><td>acagcctcag</td><td>cagcaccctg</td><td> 540</td>

<td>acgctgagca</td><td>aagcagacta</td><td>cgagaaacac</td><td>aaagtctacg</td><td>cctgcgaagt</td><td>cacccatcag</td><td> 600</td>

<td>ggcctgagct</td><td>cgcccgtcac</td><td>aaagagcttc</td><td>aacaggggag</td><td>agtgt</td><td></td><td> 645</td>

&lt;210&gt; 102 &lt;211&gt; 215 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="3"> &lt;400&gt; 102</td><td rowspan="3">Leu</td><td colspan="11" rowspan="2">Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro</td><td rowspan="3">Gly</td>

<td rowspan="2">Glu 1</td><td rowspan="2">How much</td><td rowspan="2">VaJ |</td>

<td> 5</td><td colspan="6"> 10</td><td colspan="4"> 15</td>

<td>Glu</td><td>Arg</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>Thr</td><td>val</td><td>ala</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>ala</td><td>Pro</td><td>Cheese</td><td>val</td><td>phe</td><td>How much</td><td>phe</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>Glu</td><td>Gin</td><td>Leu</td><td>lys</td><td>Cheese</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>ala</td><td>Cheese</td><td>val</td><td>val</td><td>Cys</td><td>Leu</td><td>Leu</td><td>own</td><td>own</td><td>phe</td><td>Tyr</td><td>Pro</td><td>Arg</td><td>Glu</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>ala</td><td>lys</td><td>val</td><td>Gin</td><td>Trp</td><td>lys</td><td>val</td><td>Asp</td><td>own</td><td>ala</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>own</td><td>Cheese</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Gin</td><td>Glu</td><td>Cheese</td><td>val</td><td>Thr</td><td>Glu</td><td>Gin</td><td>Asp</td><td>Cheese</td><td>lys</td><td>Asp</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Cheese</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Cheese</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>lys</td><td>ala</td><td>Asp</td><td>Tyr</td><td>Glu</td><td>lys</td><td>His</td><td>lys</td><td>val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Tyr</td><td>ala</td><td>Cys</td><td>Glu</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>val</td><td>Thr</td><td>lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Cheese</td><td>phe</td><td>own</td><td>Arg</td><td>Gly</td><td>Glu</td><td>Cys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

157 &lt;210&gt; 103 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 103

<td>caggtgcagc</td><td>tgcaggagtc gggcccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg ctccatgagc</td><td>agtggtgaat</td><td>actactgg &lt;ia</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagtacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag tcgagttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagag</td><td> 300</td>

<td>tcagtggctg</td><td>ggtttgacta ctggggccag</td><td>ggaaccctgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 104 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 104

<td colspan="10">Gin Val Gin Leu Gin Glu Cheese Gly Pro Gly</td><td colspan="2" rowspan="2">Leu Val</td><td rowspan="2">lys</td><td rowspan="2">Pro</td><td rowspan="2">Cheese 15</td><td rowspan="2">Gin</td>

<td colspan="3"> 1</td><td colspan="2"> 5</td><td colspan="5"> 10</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Thr</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Underworld</td><td>Cheese</td><td>Cheese</td><td>Gly</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Glu</td><td>Tyr</td><td>Tyr</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>His</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Trp</td><td>How much</td><td>Gly</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>own</td><td>Pro</td><td>Cheese</td>

<td></td><td> 50</td><td> |</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td> |</td><td></td><td></td>

<td>Leu</td><td>lys</td><td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>How much</td><td>Cheese</td><td>val</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td>Leu</td><td>lys</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cys</td><td>ala</td><td>Arg</td><td>Glu</td><td>Cheese</td><td>val</td><td>ala</td><td>Gly</td><td>phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Leu Val Thr Val Ser Ser 115 &lt;210&gt; 105 &lt;211&gt; 58 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Multiplication starter used for full length cloning 1.12.1 &lt;400&gt; 105

158 tcttcaagct tgatatctct agaagccgcc accatgaaac acctgtggtt cttcctcc 58 &lt;210&gt; 106 &lt;211&gt; 46 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Multiplication starter used for full length cloning 1.12.1 &lt;400&gt; 106 ttctctgatc agaattccta ctatttaccc ggagacaggg agaggc 46 &lt;210&gt; 107 &lt;211&gt; 48 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Multiplication starter used for full length cloning 1.12.1 &lt;400&gt; 107 tcttcaagct tcccgggagc cgccaccatg gaaaccccag cgcagctt 48 &lt;210&gt; 108 &lt;211&gt; 49 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Multiplication starter used for full length cloning 1.12.1 &lt;400&gt; 108 ttctttgatc agaattctca ctaacactct cccctgttga agctctttg 49 &lt;210&gt; 109 &lt;210&gt; 109 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Mutagenic oligonucleotide &lt;400&gt; 109 ctccagggga aagagccacc ctctcctgta gg 32 &lt;210&gt; 110 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213&gt; Artificial

159 &lt;220&gt;

&lt;223&gt; Mutagenic oligonucleotide &lt;400&gt; 110 cctacaggag agggtggctc tttcccctgg ag 32 &lt;210&gt; 111 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Mutagenic oligonucleotide &lt;400&gt; 111 ggtggctcca tcagcagtgg tgaatactac 30 &lt;210&gt; 112 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Mutagenic oligonucleotide &lt;400&gt; 112 gtagtattca ccactgctga tggagccacc 30 &lt;210&gt; 113 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 113

Glu Ile Val Leu Thr Gin Ser Pro Gly 1 5 &lt;210&gt; 114 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 114

Gin Val Gin Leu Gin Glu Cheese Gly

5 &lt;210&gt; 115 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

160 &lt;223&gt; Compatible primer &lt;400&gt; 115 agcgggccca gagggaccat g 21 &lt;210&gt; 116 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Reverse primer &lt;400&gt; 116 cagaaaggaa tcaggtgctc ctgggcta 28 &lt;210&gt; 117 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Compatible starter &lt;400&gt; 117 gattatggcc ttgggctccc ccaggaaa 28 &lt;210&gt; 118 &lt;211&gt; 37 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Reverse primer &lt;400&gt; 118 gggctattga atcactttag gcttctctgg actgttg &lt;210&gt; 119 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; TaqMan probe (ID1 probe) &lt;400&gt; 119 ccagcacgtc atcgactaca tcaggga 27 &lt;210&gt; 120 &lt;211&gt; 22 &lt;212&gt; DNA

161 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Starter Taqman PCR (ID1-F) &lt;400&gt; 120 aaggtgagca aggtggagat tc 22 &lt;210&gt; 121 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Starter Taqman PCR (ID1-R) &lt;400&gt; 121 ttccgagttc agctccaact g 21 &lt;210&gt; 122 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 122

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

Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 &lt;210&gt; 123 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 123

Gly Gly Cheese Ile Ser Cheese Gly Gly Tyr Tyr Trp Ser 15 10 &lt;210&gt; 124 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 124

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

Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 &lt;210&gt; 125 &lt;211&gt; 12

162 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 125

Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr Trp Ser 15 10 &lt;210&gt; 126 &lt;211&gt; 325 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 126

<td>gaaattgtgt</td><td>tgacgcagtc</td><td>tccaggcacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagacacc</td><td> 60</td>

<td>ctctcctgta</td><td>gggccagtca</td><td>gagtgtcagc</td><td>agcagctact</td><td>tagcctggta</td><td>ccagcagaaa</td><td> 120</td>

<td>cctggccagg</td><td>ctcccaggct</td><td>cctcatctat</td><td>ggtacatcca</td><td>gcagggccac</td><td>tggcatccca</td><td> 180</td>

<td>gacaggttca</td><td>gtggcagtgg</td><td>gtctgggaca</td><td>gacttcaccc</td><td>tcaccatcag</td><td>cagactggag</td><td> 240</td>

<td>cctgaagatt</td><td>ttgcagtgta</td><td>ttactgtcag</td><td>cagtatggta</td><td>gctcgccgat</td><td>caccttcggc</td><td> 300</td>

<td>caagggacac</td><td>gactggagat</td><td>taaac</td><td></td><td></td><td></td><td> 325</td>

&lt;210&gt; 127 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human

<td colspan="4"> &lt;400&gt; 127</td><td rowspan="2">Thr 5</td><td colspan="4" rowspan="2">Gin Ser Pro Gly</td><td rowspan="2">Thr 10</td><td rowspan="2">Leu</td><td colspan="2" rowspan="2">Leu cheese</td><td rowspan="2">Cheese</td><td rowspan="2">Pro 15</td><td rowspan="2">Gly</td>

<td>Glu 1</td><td>How much</td><td>val</td><td>Leu</td>

<td>Glu</td><td>Arg</td><td>Asp</td><td>Thr twenty</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td>Cheese thirty</td><td>Cheese</td><td>Cheese</td>

<td>Tyr</td><td>Leu</td><td>ala 35</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys 40</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro 45</td><td>Arg</td><td>Leu</td><td>Leu</td>

<td>How much</td><td>Tyr 50</td><td>Gly</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Arg 55</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro 60</td><td>Asp</td><td>Arg</td><td>phe</td><td>Cheese</td>

<td>Gly 65</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr 70</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr 75</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Glu 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala 85</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin 90</td><td>Gin</td><td>Tyr</td><td>Gly</td><td>Cheese</td><td>Cheese 95</td><td>Pro</td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td>

100 105 &lt;210&gt; 128 &lt;211&gt; 1332 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 128

163 caggtgcagc tgcaggagtc gggtccagga acctgcactg tctctggtgg ctccatgagc cagcacccag ggaagggcct ggagtggatt tacaacccgt ccctcaagag tcgagttacc tccctgaagc tgagctctgt gactgccgcg tcagtggctg ggtttgacta ctggggccag accaagggcc catcggtctt ccccctggcg gcggccctgg gctgcctggt caaggactac tcaggcgctc tgaccagcgg cgtgcacacc tactccctca gcagcgtggt gaccgtgccc tgcaacgtag atcacaagcc cagcaacacc tgtgtcgagt gcccaccgtg cccagcacca cccccaaaac ccaaggacac cctcatgatc gtggacgtga gccacgaaga ccccgaggtc gtgcataatg ccaagacaaa gccacgggag agcgtcctca ccgttgtgca ccaggactgg tccaacaaag gcctcccagc ccccatcgag cgagaaccac aggtgtacac cctgccccca agcctgacct gcctggtcaa aggcttctac aatgggcagc cggagaacaa ctacaagacc ttcttcctct acagcaagct caccgtggac tcatgctccg tgatgcatga ggctctgcac tctccgggta aa &lt;210&gt; 129 &lt;211&gt; 355 &lt;212&gt; DNA &lt;213&gt; Human ctggtgaagc agtggtgaat gggtacatct atatcagtag gacacggccg ggaaccctgg ccctgctcca ttccccgaac ttcccagctg tccagcaact aaggtggaca cctgtggcag tcccggaccc cagttcaact gagcagttca ctgaacggca aaaaccatct tcccgggagg cccagcgaca acacctccca aagagcaggt aaccactaca cttcacagac actactggaa attacagtgg acacgtctaa tgtattactg tcaccgtctc ggagcacctc cggtgacggt tcctacagtc tcggcaccca agacagttga gaccgtcagt ctgaggtcac ggtacgtgga acagcacgtt aggagtacaa ccaaaaccaa agatgaccaa tcgccgtgga tgctggactc ggcagcaggg cgcagaagag cctgtccctc ctggatccgc gagtacctac gaaccagttc tgcgagagag ctcagcctcc cgagagcaca gtcgtggaac ctcaggactc gacctacacc gcgcaaatgt cttcctcttc gtgcgtggtg cggcgtggag ccgtgtggtc gtgcaaggtc agggcagccc gaaccaggtc gtgggagagc cgacggctcc gaacgtcttc cctctccctg

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1332 &lt;400&gt; 129

<td>caggtgcagc</td><td>tgcaggagtc</td><td>gggtccagga</td><td>ctggtgaagc</td><td>cttcacagac</td><td>cctgtccctc</td><td> 60</td>

<td>acctgcactg</td><td>tctctggtgg</td><td>ctccatgagc</td><td>agtggtgaat</td><td>actactggaa</td><td>ctggatccgc</td><td> 120</td>

<td>cagcacccag</td><td>ggaagggcct</td><td>ggagtggatt</td><td>gggtacatct</td><td>attacagtgg</td><td>gagtacctac</td><td> 180</td>

<td>tacaacccgt</td><td>ccctcaagag</td><td>tcgagttacc</td><td>atatcagtag</td><td>acacgtctaa</td><td>gaaccagttc</td><td> 240</td>

<td>tccctgaagc</td><td>tgagctctgt</td><td>gactgccgcg</td><td>gacacggccg</td><td>tgtattactg</td><td>tgcgagagag</td><td> 300</td>

<td>tcagtggctg</td><td>ggtttgacta</td><td>ctggggccag</td><td>ggaaccctgg</td><td>tcaccgtctc</td><td>ctcag</td><td> 355</td>

&lt;210&gt; 130 &lt;211&gt; 503 &lt;212&gt; PRT &lt;213&gt; Human

164

<td colspan="3"> &lt;400&gt; 130</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Underworld 1</td><td>Thr</td><td>Leu</td><td>Gly</td><td>Cheese 5</td><td>Pro</td><td>Arg</td><td>lys</td><td>Gly</td><td>Leu 10</td><td>Leu</td><td>Underworld</td><td>Leu</td><td>Leu</td><td>Underworld 15</td><td>ala</td>

<td>Leu</td><td>val</td><td>Thr</td><td>Gin twenty</td><td>Gly</td><td>Asp</td><td>Pro</td><td>val</td><td>lys 25</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Gly</td><td>Pro thirty</td><td>Leu</td><td>val</td>

<td>Thr</td><td>Cys</td><td>Thr 35</td><td>Cys</td><td>Glu</td><td>Cheese</td><td>Pro</td><td>His 40</td><td>Cys</td><td>lys</td><td>Gly</td><td>Pro</td><td>Thr 45</td><td>Cys</td><td>Arg</td><td>Gly</td>

<td>ala</td><td>Trp 50</td><td>Cys</td><td>Thr</td><td>val</td><td>val</td><td>Leu 55</td><td>val</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gly 60</td><td>Arg</td><td>His</td><td>Pro</td><td>Gin</td>

<td>Glu 65</td><td>His</td><td>Arg</td><td>Gly</td><td>Cys</td><td>Gly 70</td><td>own</td><td>Leu</td><td>His</td><td>Arg</td><td>Glu 75</td><td>Leu</td><td>Cys</td><td>Arg</td><td>Gly</td><td>Arg 80</td>

<td>Pro</td><td>Thr</td><td>Glu</td><td>phe</td><td>val 85</td><td>own</td><td>His</td><td>Tyr</td><td>Cys</td><td>Cys 90</td><td>Asp</td><td>Cheese</td><td>His</td><td>Leu</td><td>Cys 95</td><td>own</td>

<td>His</td><td>own</td><td>val</td><td>Cheese 100</td><td>Leu</td><td>val</td><td>Leu</td><td>Glu</td><td>ala 105</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Cheese 110</td><td>Glu</td><td>Gin</td>

<td>Pro</td><td>Gly</td><td>Thr 115</td><td>Asp</td><td>Gly</td><td>Gin</td><td>Leu</td><td>ala 120</td><td>Leu</td><td>How much</td><td>Leu</td><td>Gly</td><td>Pro 125</td><td>val</td><td>Leu</td><td>ala</td>

<td>Leu</td><td>Leu 130</td><td>ala</td><td>Leu</td><td>val</td><td>ala</td><td>Leu 135</td><td>Gly</td><td>val</td><td>Leu</td><td>Gly</td><td>Leu 140</td><td>Trp</td><td>His</td><td>val</td><td>Arg</td>

<td>Arg 145</td><td>Arg</td><td>Gin</td><td>Glu</td><td>lys</td><td>Gin 150</td><td>Arg</td><td>Gly</td><td>Leu</td><td>His</td><td>Cheese 155</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Cheese 160</td>

<td>Cheese</td><td>Leu</td><td>How much</td><td>Leu</td><td>lys 165</td><td>ala</td><td>Cheese</td><td>Glu</td><td>Gin</td><td>Gly 170</td><td>Asp</td><td>Thr</td><td>Underworld</td><td>Leu</td><td>Gly 175</td><td>Asp</td>

<td>Leu</td><td>Leu</td><td>Asp</td><td>Cheese 180</td><td>Asp</td><td>Cys</td><td>Thr</td><td>Thr</td><td>Gly 185</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Leu 190</td><td>Pro</td><td>phe</td>

<td>Leu</td><td>val</td><td>Gin 195</td><td>Arg</td><td>Thr</td><td>val</td><td>ala</td><td>Arg 200</td><td>Gin</td><td>val</td><td>ala</td><td>Leu</td><td>val they in</td><td>Glu</td><td>Cys</td><td>val</td>

<td>Gly</td><td>lys 210</td><td>Gly</td><td>Arg</td><td>Tyr</td><td>Gly</td><td>Glu 215</td><td>val</td><td>Trp</td><td>Arg</td><td>Gly</td><td>Leu 220</td><td>Trp</td><td>His</td><td>Gly</td><td>Glu</td>

<td>Cheese 225</td><td>val</td><td>ala</td><td>val</td><td>lys</td><td>How much 230</td><td>phe</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>Asp 235</td><td>Glu</td><td>Gin</td><td>Cheese</td><td>Trp</td><td>phe 240</td>

<td>Arg</td><td>Glu</td><td>Thr</td><td>Glu</td><td>How much 245</td><td>Tyr</td><td>own</td><td>Thr</td><td>val</td><td>Leu 250</td><td>Leu</td><td>Arg</td><td>His</td><td>Asp</td><td>own 255</td><td>How much</td>

165

Leu Gly Phe Ile Ala Ser Asp Met Thr Ser Arg Asn Ser Ser Thr Gin 260 265 270

Leu Trp Leu Ile 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

Cheese Ala Ala Cys Gly Leu Ala His Leu His Val Glu Ile Phe Gly Thr 305 310 315 320

Gin Gly Lys Pro Ala Ile Ala His Arg Asp Phe Lys Ser Arg Asn Val 325 330 335

Leu Val Lys Cheese Asn Leu Gin Cys Cys Ile Ala Asp Leu Gly Leu Ala 340 345 350

Val Met His Ser Gin Gly Ser Asp Tyr Leu Asp Ile 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

Ile Arg Thr Asp Cys Phe Glu Ser Tyr Lys Trp Thr Asp Ile Trp Ala 385 390 395 400

Phe Gly Leu Val Leu Trp Glu Ile Ala Arg Arg Thr Ile Val Asn Gly 405 410 415

Ile 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 Ile 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 Cheese Ala Arg Leu 465 470 475 480

Thr Ala Leu Arg Ile Lys Lys Thr Leu Gin Lys Ile Ser Asn Ser Pro 485 490 495

Glu Lys Pro Lys Val Ile Gin 500 &lt;210&gt; 131 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 131

166

<img file="PL2447283T3_D0021.tif" />

&lt;210&gt; 132 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 132

<img file="PL2447283T3_D0022.tif" />

&lt;210&gt; 133 &lt;211&gt; 96 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 133

167

<img file="PL2447283T3_D0023.tif" />

&lt;210&gt; 134 &lt;211&gt; 99 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 134

<img file="PL2447283T3_D0024.tif" />

&lt;210&gt; 135 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 135

168

<td>Glu</td><td>How much</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td colspan="2">Cheese</td><td>Pro</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>val</td><td></td><td>Cheese</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td colspan="2"> 30</td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td colspan="2">Arg</td><td>Leu</td><td>Leu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>Tyr</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>Asp</td><td colspan="2">Arg</td><td>phe</td><td>Cheese</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td colspan="2">Arg</td><td>Leu</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>His</td><td>phe</td><td>Gly</td><td colspan="2">Cheese</td><td>Cheese</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>How much</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 136 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 136

Gly Tyr Tyr Trp Cheese I

5