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

Patent holder (s):

Amgen Fremont Inc. in Fremont, California, United States of America (US).

Pfizer Inc. in New York, New York, United States of America (US).

Inventor (s):

Shannon Marie Karlicek of San Diego, California (US).

Sirid-Aimée Kellermann at Menlo Park, California (US).

James Arthur Thomson of San Diego, California (US).

Jianying Wang of San Diego, California (US). Grant Raymond Wickman of San Diego, California (US).

Jingchuan Zhang of Boulder, Colorado (US). Michael Aidan North in Rancho Santa Fe, California (US).

Karin Kristina Amundson of San Diego, California (US).

Vahe Bedian of East Lyme, Connecticut (US).

Shelley Sims Belouski of Camarillo, California (US).

Dana Dan Hu-Low of Encinitas, California (US).

Xin Jiang of San Diego, California (US).

© Authorized representative:

Mr. Drs. CJJ van Loon cs at 2508 DH The Hague.

© Human monoclonal antibodies against activin receptor-like kinase-1.

NL C 1032452

The content of this patent differs from the original filed description with claim (s) and possible drawing (s). The documents originally filed can be viewed at the Netherlands Patent Office. The Netherlands Patent Office is the Office for Industrial Property, an agency of the Ministry of Economic Affairs

Title: human monoclonal antibodies to activin receptor-like kinase-1

This application claims priority under 35 USC § 119 (e) of

U.S. Provisional Application 60 / 715,292, filed September 7, 2005, which is incorporated by reference herein in its entirety.

Field of the Invention

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

Background of the Invention

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

In general, TGF-beta superfamily ligands exert their biological activities through binding to heteromeric receptor complexes of two types (I and II) of serine / threonine kinases. Type II receptors are constitutively active kinases that phosphorylate type I receptor upon ligand binding. Activated type I kinases, in turn, phosphorylate downstream signaling molecules, including the various Smads, that translocate to the nucleus and lead to a transcriptional response. Heldin et al. Nature, 1997, vol. 390, pp. 465-471. In the case of ALK1, we have demonstrated that Smadl is specifically phosphorylated and translocated to the nucleus, where it directly regulates the expression of the Smadl responsive genes Id1 and EphB2.

ALK-1 is highly and selectively expressed in endothelial cells and other highly vascularized tissues, such as placenta or brain. We have shown by Affymetrix profiling and real-time RT-PCR that the expression of ALK-1 in endothelial cells greatly exceeds the expression of its co-receptors activin type II and endogline, its ligand TGF-beta-1 or ALK-5. Mutations in ALK-1 have been associated with hereditary haemorrhagic telangiectasia (HHT), suggesting a critical role for ALK-1 in the control of blood vessel development or repair. Abdalla et al. J. Med. Genet., 2003, vol. 40, pp. 494-502; Sadick et al. Hematological The Hematology J., 2005, vol. 90, 818-828. Furthermore, two independent studies of ALK-1 knockout mice provide conclusive in vivo evidence for ALK-1 function during angiogenesis. Oh et al. Proc Natl Acad Sci USA, 2000, vol. 97, pp. 2626-2631; Umess et al. Nature Genetics, 2000, vol. 26, pp. 328-331.

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

In addition to a role in solid tumor growth and metastasis, there are other conditions with an angiogenic component worth noting, for example, arthritis, psoriasis, neovascular age-related macular degeneration and diabetic retinopathy. Bonnet et al. "Osteoarthritis, Angiogenesis and Inflammation," Rheumatology, 2005, vol. 44, pp. 7-16; Creamer et al. "Angiogenesis in Psoriasis," Angiogenesis, 2002, vol. 5, pp. 231-236; Clavel et al. "Recent data on the role for angiogenesis in rheumatoid arthritis," Joint Bone Spine, 2003, vol. 70, pp. 321-326; Anandarajah et al. "Pathogenesis of psoriatic arthritis," Curr. On in. Rheumatol., 2004, vol. 16, pp. 338-343; Ng et al. "Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration," Can. J. Ophthalmol., 2005, vol. 40, pp. 352-368;

Witmer et al. "Vascular endothelial growth factors and angiogenesis in eye disease," Progress in Retinal & Eye Research, 2003, vol. 22, pp. 1-29; Adamis et al. "Angiogenesis and ophthalmic disease," Angiogenesis, 1999, vol. 3, pp. 9-14.

Anti-angiogenic therapies are expected to be chronic in nature. Accordingly, targets with highly selective endothelial function, such as ALK-1, are preferred to reduce attrition due to side effects. Furthermore, given the remarkable divergence of the ALK1 ECD from ECDs of other members of the ALK family, mAb raised against the human ALK-1 ECD is expected to be selectively targeted against ALK-1. Based on these considerations, a monoclonal antibody to the ALK-1 extracellular domain, which can inhibit dimerization with the type II receptor and thus block Smadl phosphorylation and the downstream transcriptional response, is highly desirable.

R&D Systems, Ine. makes and markets a monoclonal anti-human ALK-1 antibody (Cat. # MAB370) produced by a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse treated with purified NSO-derived recombinant human ALK-1 extracellular domain was immunized. We have shown that this antibody neither neutralizes the interaction between ALK-1 and TGF-beta-1, nor abolishes Smadl phosphorylation. Rabbit antisera have been generated against a synthetic peptide corresponding to part of the intracellular juxtamembrane region of ALK-1 (amino acid residues 145-166), linked to keyhole limpet haemocyanin (KLH) (US Patent 6,692,925) and against the entire ALK-1 extracellular domain except the leader sequence (Lux et al., J. Biol. Chem., 1999, vol. 274, pp. 9984-9992). Abdalla et al (Human Mol. Gen., 2000, vol. 9, pp. 1227-1237) report the formation of a polyclonal antibody to ALK-1 using a recombinant vaccinia virus construct. R&D Systems, Ine. makes and markets a polyclonal anti-human ALK-1 antibody (Cat. # AF370) produced in goats immunized with purified NSO-derived recombinant human ALK-1 extracellular domain.

To date, no fully human monoclonal antibodies against the ECD of ALK-1 have been reported and no one has demonstrated the efficacy of a monoclonal antibody against the ECD of ALK-1 in disabling the ALK-1 / TGF-beta-1 / Smadl signal route.

Summary of the Invention

The invention relates to isolated neutralizing anti-ALK-1 monoclonal antibodies or antigen-binding pieces thereof that bind to primate ALK-1, preferably the ECD of primate ALK1, more preferably the ECD of human ALK-1. In a preferred embodiment, the neutralizing antibodies are fully human monoclonal antibodies or antigen-binding stretches thereof.

In another aspect, the present invention is an anti-ALK-1 antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof disables the ALK-1 / TGF-beta-1 / Smadl signal pathway.

In a preferred embodiment, the antibodies are fully human monoclonal antibodies or antigen-binding stretches thereof.

In another aspect, the present invention 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 stimulated angiogenesis. In a preferred embodiment, the antibodies are fully human monoclonal antibodies or antigen-binding stretches thereof.

In another aspect, the present invention 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.

In another aspect, the present invention is a well-tolerated, injectable, full-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 is.

In another aspect, the present invention is an anti-ALK-1 antibody or antigen-binding portion thereof, which inhibits antibody or antigen-binding portion thereof up-regulation of a specific downstream target gene of ALK-1, Id1. In a preferred embodiment, the antibodies are fully human monoclonal antibodies or antigen-binding stretches thereof.

In another aspect, the present invention is an anti-ALK-1 monoclonal antibody or antigen-binding portion thereof, wherein its antibody or antigen-binding portion is described in terms of at least one of several functional properties as described below.

For example, in one embodiment, the antibody or antigen-binding portion thereof binds to the extracellular domain of primates ALK-1 with t

an avidity value of 1 μΜ or less, such as enjoyment by surface plasmon resonance. In a further embodiment, the antibody or piece binds to the extracellular domain of primates ALK-1 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, as measured by surface plasmon resonance. In certain embodiments, the avidity value is 0.1 µM to 1 µΜ. In other embodiments, the avidity value is 1 µM to 100 nM. In other embodiments, the avidity value is 1 µM to 5 nM. In other embodiments, the avidity value is 1 µM to 500 µM. In other embodiments, the avidity value is 1 µM to 100 µM. In other embodiments, the avidity is 1 pM to 10 pM.

In another embodiment, the antibody or antigen-binding portion thereof binds to the extracellular domain of human ALK-1 with an avidity value of 100 nM or less, as measured by surface plasmon resonance. In a further embodiment, the antibody or piece binds to the extracellular domain of human ALK-1 with 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, less than 10 pM, or less than 1 pM, as measured by surface plasmon resonance. In certain embodiments, the avidity value is 1 µM to 100 nM. In other embodiments, the avidity value is 1 µM to 5 nM. In other embodiments, the avidity value is 1 µM to 500 µM. In other embodiments, the avidity value is 1 µM to 100 µM. In other embodiments, the avidity is 1 pM to 10 pM.

In another embodiment, the antibody or piece thereof has an off value (k_YOUIT) for 5 x 10 'human ALK-1³ s¹ or lower as measured by surface plasmon resonance. For example, in certain embodiments, the antibody or piece has a calf for human ALK-1 of less than 10 '³ s¹, less than 5 x 10^-4 s¹, lower than 10⁴ s¹, less than 5 x 10 '⁵ s¹, lower than 10 ' ⁵ s¹, or less than 5 x 10 ⁶ s¹. In other embodiments, the calf is 10⁶ s¹ to 10-⁴ s¹. In other embodiments, the k_youit 10-⁶ s¹ up to 5 x 10 ⁵ s¹.

In another embodiment, the antibody or piece thereof binds to primates ALK-1 with a Kd of 1000 nM or less. In a further embodiment, the antibody or piece binds to human ALK-1 with a Kd of less than 500 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, or less than 1 nM, such as measured by surface plasmon resonance. In certain embodiments, the Kd is 1 pM to 100 nM. In other embodiments, the Kd is from 100 nM to 10 nM. In other embodiments, Kd is 50 nM to 0.1 nM. Such Kd values ​​can be measured by any technique known to those skilled in the art, such as by ELISAs, RIAs, flow cytometry, or surface plasmon resonance, such as BIACORE ™.

In another embodiment, the antibody or piece thereof has a greater binding affinity for primate ALK-1 (Kd (P)) than for rodent ALK-1 (Kd (R)). In one embodiment, the antibodies or antigen-binding portions thereof of the present invention have a Kd (R) / Kd (P) greater than or equal to 1.5. In a further embodiment, the antibodies or antigen-binding portions thereof of the present invention have a Kd (R) / Kd (P) greater than or equal to 2, greater than or equal to 3, greater than or equal to 5, greater is greater than or equal to 10, greater 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 primate ALK-1 and for rodent ALK-1 can be measured by any technique known to those skilled in the art, such as by flow cytometry, ELISA, RIA, or surface plasmon resonance, such as BIACORE ™.

In another embodiment, the anti-ALK-1 antibody or piece thereof has an IC50 of 500 nM or less, as measured by their ability to inhibit up-regulation of a specific downstream target gene of ALK-1, Id1. In a further embodiment, said IC50 is less than 300 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In certain embodiments, the IC50 is 1 nM to 500 nM. In other embodiments, the IC50 is from 5 nM to 250 nM. In other embodiments, the IC50 is 10 nM to 100 nM.

In another embodiment, the anti-ALK-1 antibody or piece thereof has an IC50 of 250 nM or less, as measured by their ability to inhibit Smadl phosphorylation determined by Western Blotting using Odyssey Infrared Imaging System. In a further embodiment, said IC50 is less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, or less than 1 nM. In certain embodiments, the IC50 is 1 nM to 250 nM. In other embodiments, the IC50 is 5 nM to 200 nM. In other embodiments, the IC50 is 10 nM to 100 nM.

In another embodiment, the anti-ALK-1 antibody or piece of it inhibits human vessel angiogenesis in an SC ID mouse with a graft of human foreskin tissue implanted intradermally with human melanoma M24 with tumor cells, as determined by IHC analysis of human CD-31 signal assay by at least 40% compared to a control sample. In a further embodiment, the anti-ALK-1 antibody or piece thereof inhibits angiogenesis of human vessels in a SCID mouse with a graft of human foreskin tissue implanted intradermally with human melanoma M24 with tumor cells by at least 30%, at least 40%, at least 50%, or at least 60% compared to a control sample.

In another embodiment, the anti-ALK-1 antibody or stretch thereof has an EC50 of 500 nM or less, as measured by their ability to inhibit human vessel angiogenesis in an SCID mouse with a graft of human foreskin tissue, which contains intradermal human melanoma M24 with tumor cells have been implanted. In a further embodiment, said EC50 is less than 400 nM, less than 300 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, or less than 5 nM. In certain embodiments, the EC50 is 5 nM to 500 nM. In other embodiments, the IC50 is 25 nM to 300 nM. In other embodiments, the IC50 is from 50 nM to 150 nM.

In another embodiment, the anti-ALK-1 antibody or piece thereof inhibits angiogenesis of human vessels in a SCID mouse with a graft of human foreskin tissue implanted intradermally with a mixture of collagen plus human macrovascular endothelial cells, as determined by IHC analysis of human CD-31 signal assay by at least 25% compared to a control sample. In a further embodiment, the anti-ALK-1 antibody or piece thereof inhibits angiogenesis of human vessels in a SCID mouse with a graft of human foreskin tissue implanted intradermally with at least 50% collagen compared to a control sample. In a further embodiment, the anti-ALK-1 antibody or stretch thereof inhibits by at least 75%, by at least 80%, by at least 85%, by at least 90% or at least 95% compared to the control.

In another embodiment, the anti-ALK-1 antibody or piece 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.

In another embodiment, the anti-ALK-1 antibody or portion thereof 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 (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 another embodiment, the anti-ALK-1 antibody or stretch thereof binds to the same epitope of ALK-1 as an antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1 (M29IZD19A); 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 another embodiment, the anti-ALK-1 antibody or piece thereof binds to ALK-1 with substantially the same Kd as 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.

In another embodiment, the anti-ALK-1 antibody or piece thereof binds to ALK-1 with substantially the same calf as 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.

A further aspect of the present invention is an antibody or antigen-binding portion thereof having at least one of the previously described functional properties, and comprising a Vh domain that is at least 90% identical in amino acid sequence to one of SEQ ID NOs: 6; 10; 14; 18; 22;

26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104. In one embodiment, said Vh domain is at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% identical in amino acid sequence to any of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104.

In a further embodiment, the antibody or piece thereof has at least one of the previously described functional properties, and comprises a Vh domain comprising one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or different from one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82;

86; 90; or 104 in that it has at least one conservative amino acid substitution. For example, the Vh domain can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9,

10,11, 12, 13, 14 or 15 conversative amino acid substitutions of any of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104. In a further embodiment, any of these conservative amino acid substitutions may occur in the CDR1, CDR2, and / or CDR3 regions.

A further aspect of the present invention is an antibody or antigen-binding portion thereof having at least one of the previously described functional properties, and comprising a V1 domain that is at least 90% identical in amino acid sequence to one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127. In one embodiment, said V1 domain is at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% identical in amino acid sequence to any of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127.

In a further embodiment, the antibody or piece thereof has at least one of the previously described functional properties, and comprises a V1 domain comprising one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or is 127, or is different from one of SEQ ID Nos: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127 in that it has at least one conservative amino acid substitution. For example, the V1 domain can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9,

10,11,12,13,14 or 15 conservative amino acid substitutions of one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127. In a further embodiment, any of these conservative amino acid substitutions may occur in the CDR1, CDR2, and / or CDR3 regions.

Another aspect of the present invention is an antibody or antigen-binding portion thereof having at least one of the previously described functional properties, wherein the V1 and Vh domains are each at least 90% identical in amino acid sequence to the V1 and Vh domains, respectively, of one 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, the V1 and Vh domains are each at least 91%, 93%, 95%, 97%, 99%, or 100% identical in amino acid sequences to the V1 and Vh domains of one of the monoclonal antibodies 1.11.1, 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.

In another aspect of the present invention, a monoclonal antibody or antigen-binding portion thereof selected from the group consisting of: a) an antibody or portion thereof comprising a Vh domain as shown in SEQ ID NO: 6, and a V1 domain as shown in SEQ ID NO: 8; b) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 10, and a V1 domain as shown in SEQ ID NO: 12; c) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 14 and a V1 domain as shown in SEQ ID NO: 16; d) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 18, and a V1 domain as shown in SEQ ID NO: 20; e) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 22 and a V1 domain as shown in SEQ ID NO: 24; f) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 26 and a V1 domain as shown in SEQ ID NO: 28; g) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 30 and a V1 domain as shown in SEQ ID NO: 32; h) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 34 and a V1 domain as shown in SEQ ID NO: 36; i) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 38 and a V1 domain as shown in SEQ ID NO: 40; j) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 42 and a V1 domain as shown in SEQ ID NO: 44; k) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 46 and a V1 domain as shown in SEQ ID NO: 48; 1) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 50 and a V1 domain as shown in SEQ ID NO: 52; m) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 54 and a V1 domain as shown in SEQ ID NO: 56; n) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 58 and a V1 domain as shown in SEQ ID NO: 60; o) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 62 and a V1 domain as shown in SEQ ID NO: 64; p) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 66 and a V1 domain as shown in SEQ ID NO: 68; q) an antibody or antigen-binding portion thereof comprising a Vh domain as shown in SEQ ID NO: 70 and a V1 domain as shown in SEQ ID NO: 72; r) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 74 and a V1 domain as shown in SEQ ID NO: 76; s) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 78 and a V1 domain as shown in SEQ ID NO: 80; t) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 82 and a V1 domain as shown in SEQ ID NO: 84; u) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 86 and a V1 domain as shown in SEQ ID NO: 88; v) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 90 and a

V1 domain as shown in SEQ ID NO: 92; w) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 104 and a V1 domain as shown in SEQ ID NO: 127; x) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 6 and a V1 domain as shown in SEQ ID NO: 127; and y) an antibody or piece thereof comprising a Vh domain as shown in SEQ ID NO: 104 and a V1 domain as shown in SEQ ID NO: 8.

In a further embodiment, for each of the antibodies or pieces thereof as described above in groups a) to v), the Vh and / or V1 domains may differ from the specific SEQ ID NOs mentioned therein by at least one conservative amino acid substitution. For example, the Vh and / or V1 domains may differ from said SEQ ID NO by 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions. In a further embodiment, any of these conservative amino acid substitutions can occur in the CDR1, CDR2, and / or CDR3 regions.

In another embodiment, the present invention provides a monoclonal antibody or antigen-binding portion thereof having at least one of the previously described functional properties, wherein the Vh domain is independently selected from one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence different from any of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, by at least one conservative amino acid substitution, and the V1 domain is independently selected from one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or a sequence different from any of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, by at least one conservative amino acid substitution. For example, the Vh and V1 domains can each differ from SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, and 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or

127, respectively, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.

In a further embodiment, the present invention provides a monoclonal antibody or antigen-binding piece thereof having at least one of the previously described functional properties, wherein said antibody or piece comprises Vh CDR1, CDR2 and CDR3 sequences independently selected from the heavy chain CDR1, CDR2 , or CDR3 sequences, respectively, found in one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or a sequence different from any of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, by at least one conservative amino acid substitution. For example, the Vh CDR1, CDR2 and CDR3 may differ from the CDR1, CDR2 and CDR3, respectively, from one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.

In a further embodiment, the present invention provides a monoclonal antibody or antigen-binding piece thereof having at least one of the previously described functional properties, wherein said antibody or piece comprises V1 CDR1, CDR2 and CDR3 sequences independently selected from the light chain CDR1, CDR2 , or CDR3 sequences, respectively, found in one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or a sequence different from any of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, by at least one conservative amino acid substitution. For example, the V1 CDR1, CDR2 and CDR3 may be different from the CDR1, CDR2 and CDR3, respectively, from one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.

The present invention further provides a monoclonal antibody or antigen binding stretch thereof having at least one of the previously described functional properties, wherein said antibody or antigen binding stretch comprises the Vh and V1 CDR1, the Vh and V1 CDR2, and the Vh and V1 CDR3 as found in 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;

I. 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.

The present invention further provides a monoclonal antibody or antigen-binding piece thereof having at least one of the previously described functional properties, wherein said antibody or antigen-binding piece comprises a heavy chain comprising a human Vh 4-31, Vh 3II, Vh 3 -15, Vh 3-33, Vh 4-61 or Vh 4-59 gene are used. In some embodiments, the heavy chain uses a human Vh 3-33 gene, a human D 6-19 gene and a human Jh 3B gene; a human Vh 4-31 gene, a human D 6-19 gene and a human Jh 4B gene; a human Vh 4-61 gene, a human D 6-19 gene and a human Jh 4B gene; a human Vh 431 gene, a human D 3-3 gene and a human Jh 3B gene; a human Vh 4-31 gene and a human Jh 3B gene; a human Vh 4-59 gene, a human D 6-19 gene and a human Jh 4B gene; a human Vh 3-11 gene, a human D 3-22 gene and a human Jh 6B gene; a human Vh 3-15 gene, a human D 3-22 gene and a human Jh 4B gene; a human Vh 4-31 gene, a human D 5-12 gene and a human Jh 6B gene; a human Vh 431 gene, a human D 4-23 gene and a human Jh 4B gene; a human Vh 4-31 gene, a human D 2-2 gene and a human Jh 5B gene; a human Vh 4-31 gene and a human Jh 6B gene; human Vh 3-15 gene, a human D 1-1 gene and a human Jh 4B gene; a human Vh 3-11 gene, a human D 6-19 gene and a human Jh 6B gene; a human Vh 3-11 gene, a human D 3-10 gene and a human Jh 6B gene; or a human Vh 3-11 gene, a human D 6-6 gene and a human Jh 6B gene.

The present invention further provides a monoclonal antibody or antigen-binding piece thereof having at least one of the previously described functional properties, wherein said antibody or antigen-binding piece comprises a light chain comprising a human V_K A27, V_K A2, V_K Al, V_K A3, V_K B3, V_k B2, V_k LI or V_K L2 gene used. In some embodiments, the light chain uses a human Vk LI gene and a human Jk 4 gene; a human Vk A27 gene and a human Jk 5 gene or a human Jk 4 gene; a human Vk B3 gene and a human Jk 1 gene; a human Vk L2 gene and a human Jk 3 gene; a human Vk A2 gene and a human Jk 1 gene; a human Vk A3 gene and a human Jk 4 gene; a human Vk Al gene and a human Jk 1 gene; a human Vk B2 gene and a human Jk 4 gene; or a human Vk A2 gene and a human Jk 1 gene.

The present invention further provides a monoclonal antibody or antigen binding piece thereof having at least one of the previously described functional properties, wherein said antibody or antigen binding piece comprises one or more heavy chain and / or light chain FR1, FR2, FR3 or FR4 amino acid sequence as found in 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.

The present invention further provides a monoclonal antibody comprising the amino acid sequences shown 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.

In a further embodiment of the present invention, one of the previously described antibodies is or is an IgG, an IgM, an IgE, an IgA, or an IgD molecule. For example, the antibody can be an IgGi or IgGfe.

Another embodiment provides one of the antibodies or antigen-binding stretches described above, comprising a Fab fragment, an F (ab ') 2 fragment, a Fv fragment, a single chain Fv fragment, a single chain Vh fragment, a single chain V1 fragment, a humanized antibody, a chimeric antibody or a bispecific antibody.

In a further embodiment, a derivatized antibody or antigen binding stretch comprising one of the antibodies or stretches thereof as previously described and at least one additional molecular entity. For example, the at least one additional molecular entity may be a different antibody (e.g. a bispecific antibody or a diabody), a detection agent, a label, a cytotoxic agent, a pharmaceutical, and / or a protein or peptide that associates the antibody or antibody piece with another molecule (such as a streptavidin core region or a polyhistidine tag). For example, useful detection agents capable of derivatizing an antibody or antigen-binding piece of the invention include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrine, lanthanide phosphors, and the like. An antibody can also be labeled with enzymes useful for detection, such as horseradish peroxidase, 6-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. In a further embodiment, the antibodies or pieces thereof of the present invention may also be labeled with biotin, or with a predetermined polypeptide epitope recognized by a secondary reporter (e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In yet another embodiment of the present invention, any of the antibodies or pieces thereof can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group.

In some embodiments, the anti-ALK-1 antibodies or antigen binding stretches described herein are linked to a solid support.

In some embodiments, the C-terminal heavy chain lysine is cleaved from one of the anti-ALK-1 antibodies of the invention.

In various embodiments of the invention, the heavy and light chains of the anti-ALK-1 antibodies may optionally comprise a signal sequence.

The present invention also provides a pharmaceutical composition comprising one of its antibodies or antigen-binding stretches as described above and a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of its antibodies or antigen-binding stretches as described herein. In one particular embodiment, an isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1, which encodes a heavy chain sequence. In another particular embodiment, an isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 3, which encodes a light chain sequence.

In another particular embodiment, an isolated nucleic acid molecule comprises a polynucleotide comprising an open reading frame of the cDNA sequence of a clone deposited under an ATCC

Accession number PTA-6864. In another particular embodiment, an isolated nucleic acid molecule comprises a polynucleotide comprising an open reading frame of the clone cDNA sequence deposited under an ATCC Accession Number PTA-6865.

In another particular embodiment, an isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 95 or 128, both of which encode a heavy chain. In another particular embodiment, an isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 101, which encodes a light chain sequence.

The invention 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.

Another embodiment provides a host cell comprising any of the vectors described herein or comprising any of the nucleic acid molecules described herein. The present invention also provides an isolated cell line that produces one of the antibodies or antigen binding stretches as described herein or which produces the heavy chain or light chain of any of said antibodies or said antigen binding stretches.

In another embodiment, the present invention relates to a method of producing an anti-ALK-1 antibody or antigen-binding portion thereof, comprising culturing one of the host cells or cell lines described herein under suitable conditions and recovering said antibody or antigen binding piece.

The present invention also relates to a non-human transgenic animal or transgenic plant comprising any of the nucleic acids described herein, wherein the non-human transgenic animal or transgenic plant expresses said nucleic acid.

The present invention further provides a method of isolating an antibody or antigen-binding portion thereof that binds to ALK-1, comprising the step of isolating the antibody from the non-human transgenic animal or transgenic plant as described herein.

In another embodiment, the invention relates to a hybridoma deposited under an ATCC Accession Number of PTA-6808.

The present invention also provides a method for determining whether a substance inhibits upregulation of a specific downstream target gene of ALK-1, Id1, the method comprising contacting a first sample of cells expressing Id1 with the substance and determining whether Id1 expression is inhibited, whereby a reduced level of Id1 expression in the first sample of cells contacted with the substance compared to a control sample of cells, it is indicative that said substance inhibits Id1 expression. The present invention further provides the method, wherein the substance is an antibody that binds to the extracellular domain of ALK-1.

The present invention also provides a method of treating abnormal cell growth in a mammal in need thereof, comprising the step of administering to said mammal any of its antibodies or antigen-binding stretches, or any of the pharmaceutical compositions, as herein described. The present invention further provides a method of treating abnormal cell growth in a mammal in need thereof with 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 suitable conditions that allow expression of said nucleic acid molecules. In another embodiment, the method of treating abnormal cell growth further comprises administering an amount of one or more substances selected from anti-tumor agents, anti-angiogenesis agents, signal transduction inhibitors, and anti-proliferative agents, which act together in the treating said abnormal cell growth. In particular embodiments, said abnormal cell growth is cancerous.

The present invention also provides an isolated Cynomolgus monkey ALK-1 protein having an amino acid sequence of SEQ ID NO: 93. The present invention further provides an isolated nucleic acid molecule encoding a protein having an amino acid sequence of SEQ ID NO: 93. The present invention further provides an isolated nucleic acid molecule of SEQ ID NO: 94.

Brief description of the figure

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

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

Figure 3 shows Kd determination of the recombinant 1.12.1 antibody binding to cell surface ALK-1. (a) human, (b) Cyno.

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

1.12.1 (M29I / D19A) indicates the mAb 1.12.1 variant which was 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) indicates the mAb 1.12.1 variant which was expressed recombinant mAb containing a specific single amino acid mutation in which the methionine at position 29 in the heavy chain was replaced by isoleucine.

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

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

1.12.1 refers to the mAb 1.12.1 variant isolated from the hybridoma.

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

1.12.1 (M29I / D19A) indicates the mAb 1.12.1 variant which was 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) indicates the mAb 1.12.1 variant which was expressed recombinant mAb containing a specific single amino acid mutation in which the methionine at position 29 in the heavy chain was replaced by isoleucine.

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

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

1.12.1 refers to the mAb 1.12.1 variant isolated from the hybridoma.

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

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

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

1.12.1 (M29I / D19A) indicates the mAb 1.12.1 variant which was 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.KM29I / D19A) denotes the Fab fragment of mAb 1.12.1 (M29I / D19A) prepared by digestion of 1.12.1 (M29I / D19A) IgG1 using papain.

Figure 6 shows ALK-1 internment, (a) Monitor for cell surface residual neutralizing antibody. (b) Monitor leftover cell surface receptor ALK-1.

Figure 7A shows alignment of variable domain sequences for antiALK-1 antibodies of the invention with germline sequences. Mutations compared to germline are shown in bold. CDR sequences are underlined. Figure 7B shows alignment of the predicted amino acid sequences of light chain variable domains for anti-ALK-1 antibodies 1.12.1,1.14.1,

1,162.1, 1.31.1, 4.62.1 and 4.72.1 with the human germline A27 Vk sequence. Figures 7C and 7D show alignment of the predicted amino acid sequences of heavy 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 the human germline 4-31 Vh sequence.

Figure 8 shows an example of the histological (H&E staining) analysis of a section of the graft from human skin post surgery.

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

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

Figure 10 shows an immunofluorescent image of human (red) and mouse (green) vessels of the M24 with tumor in the human foreskin SCID chimera mouse.

Figure 11 shows the IHC image of human vessels (brown) of the M24 with tumor in the human foreskin SCID chimera mouse.

Figure 12 shows the representative immunofluorescent images of human (red) and mouse (green) vessels of the control and the 1.12.1 (M29I / D19A) antibody treated (10 mg / kg) M24 with tumors in the human foreskin SCID chimera mouse.

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

Figure 14 shows the SCID mouse plasma concentration of the 1.12.1 (M29I / D19A) antibody.

Figure 15 shows the estimated EC50 for the 1.12.1 (M29I / D19A) antibody in the M24 foreskin SCID chimera model. The control value at 100% was given an artificial serum concentration of 0.1 nM for graphic purposes. This does not lead to any other apparent EC50.

Detailed Description of the Invention

Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings commonly understood to those skilled in the art. Furthermore, unless otherwise required by the context, singular terms will include the plural and plural terms will include the singular. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are as known and commonly known in the art used.

The methods and techniques of the present invention are generally conducted according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the description unless otherwise indicated. See, e.g., Sambrook J. & Russell D .. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 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), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as usually performed in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry as described herein are as known and commonly used in the art.

The following terms, unless otherwise indicated, will be deemed to have the following meanings:

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

As used herein, the acronym "mAb" indicates a monoclonal antibody.

As used herein, an antibody referred to in number is a monoclonal antibody (mAb) obtained from the hybridoma of the same number. For example, monoclonal antibody

1.12.1 obtained from hybridoma 1.12.1.

1.12.1 refers to the mAb 1.12.1 variant isolated from the hybridoma.

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

1.12.1 (M29I / D19A) indicates the mAb 1.12.1 variant which was 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) indicates the mAb 1.12.1 variant which was expressed recombinant mAb containing a specific single amino acid mutation in which the methionine at position 29 in the heavy chain was replaced by isoleucine.

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

As used herein, unless otherwise indicated, abnormal cell growth indicates cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition).

As used herein, the term adjacent is used to denote nucleotide sequences directly linked together without nucleotides in between. For example, the pentanucleotide 5'-AAAAA-3 'is adjacent to the trinucleotide 5'-TTT-3' when the two are so joined: 5'-AAAAATTT-3 'or 5'-TTTAAAAA-3', but not when the two are thus connected: 5'-AAAAACTTT-3 '.

The term agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, alleviating a disease, disorder, or condition indicates decreasing the severity of the symptoms of the disease, disorder, or condition. This includes, but is not limited to, influencing the size, growth and / or mass of a tumor, the size or progression of metastasis, and the like, in a patient compared to the same parameters in the patient before or in the absence of the method of treatment.

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

As used herein, the term "Smad" refers to Smad domain proteins, which are found in a species range from nematodes to humans. These highly conserved proteins contain an N-terminal MH1 domain that contacts DNA, and are separated from the C-terminal MH2 domain by a short left molecule region, the latter domain strikingly similar to forkhead-associated (FHA) domains. FHA and Smad (MH2) domains share a common structure consisting of a sandwich of eleven beta strands in two plates with Greek key topology. Smad proteins mediate signaling by the TGF-beta / activin / BMP-2/4 cytokines of receptor Ser / Thr protein kinases at the cell surface to the nucleus. Smad proteins are classified into three functional classes: the receptor-regulated Smads (R-Smads), including Smadl, -2, -3, -5, and -8, each of which is involved in a ligand-specific signaling pathway; the co-mediator Smads (co-Smads), including Smad4, which interacts with R-Smads to participate in signaling; and the inhibitory Smads (I-Smads), including Smad-6 and -7, which block the activation of R-Smads and Co-Smads, thereby negatively regulating signaling pathways.

As used herein, the term "TGF-beta" refers to the transforming growth factors-beta, which are a family of multi-functional cytokines (TGF-beta 1-5) that regulate cell growth and differentiation.

Transformation growth factor (TGF) is one of many characterized growth factors that occur in nature. He plays critical roles in “SCID” mice with severe combined immunodeficiency. Many cells synthesize TGF-beta and essentially all have specific receptors for this peptide. TGF-beta regulates the actions of many other peptide growth factors and determines the positive or negative direction of their effects. TGF-beta is a tumor suppression cytokine with growth inhibitory effects in epithelial cells. TGF-β can also act as a tumor promoter by generating an epithelial-to-mesenchymal junction. TGF-β inactivates several proteins involved in cell cycle progression and therefore exerts its growth inhibitory effects on epithelial cells by stopping them in the G1 phase of the cell cycle. The protein functions as a disulfide-linked homodimer. Its sequence is characterized by the presence of several C-terminal cysteine ​​residues, which form interlocking disulfide bridges arranged in a knot-like topology. A similar cystine knot setup has been observed in the structures of some enzyme inhibitors and neurotoxins that bind to Ca²⁺channels with voltage port, although the exact topology differs. TGF-beta genes are differentially expressed, suggesting that the different TGF-beta species may have distinct physiological roles in vivo.

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

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

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

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

Full Name Three-Letter Code One-Letter Code

<td>Asparagine hour</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 H</td><td></td>

<td>Tyrosine</td><td>Tyr</td><td>Y.</td>

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

<td>Asparagine</td><td>Asn</td><td>N</td>

<td>Glutamine</td><td>Gin</td><td>Q</td>

<td>Serine</td><td>Ser</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>a</td>

<td>Valine</td><td>Val</td><td>V</td>

<td>Leucine</td><td>Leu</td><td>L</td>

<td>Isoleucine</td><td>Ile</td><td>I</td>

<td>Methionine</td><td>With</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>W.</td>

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology — A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated by reference herein.

A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue with a side chain R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percent sequence identity or similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for carrying out this adjustment are known to the skilled person. See, e.g., Pearson, Methods Mol. Biol. 243: 307-31 (1994).

Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine tyrosine, lysine-arginine, alanine valaline, glutamate aspartate, and asparagine glutamine.

Alternatively, a conservative replacement is any positive value change in the PAM250 log probability matrix described in Gonnet et al., Science 256: 1443-45 (1992), incorporated herein by reference. A moderately conservative replacement is any change with a non-negative value in the PAM250 log probability matrix.

In certain embodiments, amino acid substitutions for an antiALK-1 antibody or antigen-binding portion thereof are those that: (1) decrease sensitivity to proteolysis, (2) decrease sensitivity to oxidation, (3) alter binding affinity for the formation of protein complexes, and ( 4) confer or modify other physicochemical or functional properties of such analogs, but still maintain specific binding to ALK-1. Analogs can include various substitutions in the normally occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, can be made in the normally occurring sequence, for example, in the stretch of the polypeptide outside the domain (s) that form intermolecular contacts. Amino acid substitutions can also be made in the domain (s) that form intermolecular contacts that can enhance the activity of the polypeptide. A conservative amino acid substitution should not substantially alter the structural characteristics of the mother sequence; e.g. a replacement amino acid should not alter the anti-parallel β plate that forms the immunoglobulin binding domain that exists in the mother sequence, or disrupt other types of secondary structure that characterizes the mother sequence. In general, glycine and proline should not be used in an anti-parallel β plate. Examples of art recognized 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, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354: 105 (1991), incorporated herein by reference.

Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using degrees of tolerance assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. E.g. contains GCG programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See eg GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percentage sequence use of the areas of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132 : 185-219 (2000)). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997); incorporated herein by reference.

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a wide variety of organisms, it is preferable to compare amino acid sequences. The term analog as used herein refers to polypeptides comprising a segment of at least 25 amino acids that has substantial similarity to a stretch of a derived naturally occurring amino acid sequence and which has at least one of the properties of the naturally occurring polypeptide. Polypeptide analogs typically include a conservative amino acid substitution (or addition or deletion) over the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, preferably at least 50 amino acids in length or longer, and can often be as long as a naturally occurring full length polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide 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), which are incorporated by reference herein. Such compounds are often developed using computerized molecular modeling.

Peptide mimetics structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. In general, peptidomimetics structurally resemble a polypeptide paradigm (i.e. a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have optionally replaced one or more peptide linkages with a linkage selected from the group consisting of: —CH2NH—, —CH₂S — CH 2 —CH 2 — CH — CH — (cis and trans) —COCH 2 — CH (OH) CH 2 — and CH 2 SO — by methods well known in the art. Systematic replacement of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to generate more stable peptides. In addition, limited (constrained) peptides, comprising a consensus sequence or a substantially identical consensus sequence variation, can be generated by methods known in the art (Rizo and GieraschAnn. Rev. Biochem. 61: 387 (1992), incorporated herein by reference). ; e.g. by adding internal cysteine ​​residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

An intact antibody or "immunoglobulin" (Ig) comprises at least two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. There are only two types of light chain: λ and k. 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 isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated by reference in its entirety for all purposes). In a preferred embodiment, the antibody is an IgG and is of an IgG1, IgG2, IgG3 or IgG4 subtype. In a more preferred embodiment, the anti-ALK-1 antibody is of subclass IgG2.

Each heavy chain includes a heavy chain variable domain (Vh) and a heavy chain constant region (Ch). The heavy chain constant region includes three domains, CHI, CH2 and CH3. Each light chain includes a light chain variable domain (V1) and a light chain constant region. The light chain constant region includes one domain, Cl · In light and heavy chains, the variable and constant regions are linked by a J region of about 12 or more amino acids, the heavy chain also having a D region of about 3 or more amino acids includes. The Vh and V1 regions can be further divided into areas of hypervariability, called "complementarity determining regions" (CDR), with more conserved regions in between, called "framework regions" (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Amino acids are assigned to each domain according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and

1991)), or Chothia &amp; Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989).

The variable domains of each heavy / light chain pair (Vh and V1) form the antibody binding site that interacts with an antigen. Thus, for example, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The constant regions of the antibodies can promote the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g. effector cells) and the first component (Clq) of the classical complement system.

Antibodies must have sufficient diversity in antigen binding to recognize every possible pathogen (many V regions) while retaining the biological effectiveness of their C regions (few C regions). Ig genes are randomly spliced ​​from gene segments allowing many V regions to be used with only a few C regions. Gene segments encoding Ig H, kappa and lambda chains have been found on three different chromosomes. During B cell development, recombinase enzymes remove introns and some exons from the DNA and splice segments into functional Ig genes.

Ig gene segments in mammals are arranged in groups of “variable” (V), “diversity” (D), “joining (joining)” (J), and “constant” (C) exons. V kappa (Vx) segments each encode the first two CDR and three FR of the kappa chain V region, plus some residues of CDR3. J kappa (Jx) segments each encode the remainder of CDR3 and the fourth FR. C kappa (Cx) encodes the complete C region of the kappa light chain. DNA encoding human kappa chain includes about 40 functional V kappa (Vx) segments, five J kappa (Jx) segments, and one C kappa (Cx) gene segment, as well as some gene segments containing stop codons ("pseudogenes"). Human lambda (λ) chain DNA contains approximately 30 functional V lambda (VX) segments and four functional sets of J lambda (J X) and C lambda (CX) segments. A given J lambda (JX) always pairs with its corresponding C lambda (CX), unlike J kappa (Jk) which all pair with the same C kappa (Ck). Human H chain DNA includes approximately 50 functional Vh segments, 30 Dh segments, and six Jh segments. The first two CDR and three FR of the heavy chain variable domain are encoded by Vh.

CDR3 is encoded by some nucleotides of Vh, all of Dh, and part of Jh, while FR4 is encoded by the rest of the Jh gene segment. There are also individual gene segments in the DNA for each heavy chain domain and membrane region of each isotype, arranged in the order in which they are expressed by B cells.

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

The term "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody, which, due to its origin or source of origin: (1), is not associated with naturally associated components that it contains in its native state, (2) is free from other proteins from the same species, (3) is expressed by a cell from another species, or (4) does not exist in nature. Thus, a polypeptide, which has been chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived, will be "isolated" from its naturally associated components. A protein can also be made substantially free of naturally associated components by isolation using techniques of protein purification well known in the art.

Examples of isolated antibodies include, but are not limited to, an anti-ALK-1 antibody affinity purified using ALK-1, and an anti-ALK-1 antibody synthesized by a cell line in vitro.

A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60 to

75% of a sample shows a single polypeptide species. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein may typically comprise about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more usually about 95%, and may preferably be greater than 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band after staining the gel with a dye well known in the art. For certain purposes, higher resolution can be provided using HPLC or other purifying agents well known in the art.

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

The term "analog" or "polypeptide analog" as used herein refers to a polypeptide comprising a segment of substantial similarity to some reference amino acid sequence and substantially the same function or activity as the reference amino acid sequence. Polypeptide analogs typically include a conservative amino acid substitution (or insertion or deletion) from the reference sequence. Analogs can be at least 20 or 25 amino acids in length, or can be at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids in length or longer, and can often be as long as the full-length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analog antibodies with 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 germline replacements amino acid sequence. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art by following the directions herein.

The term antigen-binding piece of an antibody (or simply antibody piece), as used herein, refers to one or more fragments of an antibody that retain the ability of specific binding to an antigen (e.g. ALK-1 or ECD of ALK- 1). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments encompassed by the term antigen binding stretch of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V1, Vh, Cl and ChI domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the Vh and ChI domains;

(iv) an Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a Vh domain ; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V1 and Vh, are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker molecule that allows them to be made as a single protein chain in which the V1 and Vh regions pair to form monovalent molecules (known as single chain Fv (scFv)); see, e.g., Bird et al. Science 242: 423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be included in the term antigen binding stretch of an antibody. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies expressing Vh and Vl domains on a single polypeptide chain, but using a linker molecule that is too short to allow pairing between the two domains on the same chain, forcing the domains to mate with create complementary domains from another chain and two antigen binding sites (see, e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al. Structure 2: 1121-1123 (1994)).

Still further, an antibody or antigen-binding piece thereof may form part of larger immunoadhesion molecules formed by covalent or noncovalent association of the antibody or antibody piece with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. Human. Antibodies and Hybridomas 6: 93-101 (1995)) and use of a cysteine ​​residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. Mol. Immunol. 31: 10471058 (1994)). Other examples include where one or more CDRs from an antibody are included in a molecule, covalent or noncovalent, to make it an immunoadhesin that specifically binds to an antigen of interest, such as ALK-1 or ECD of ALK-1. In such embodiments, the CDR (s) may be included as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be included noncovalently.

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

As used herein, the term "human antibody" refers to any antibody in which the variable and constant domain sequences are human sequences. The term includes antibodies with sequences derived from human genes, but which have been altered, e.g., to reduce potential immunogenicity, increase affinity, eliminate cysteines that could cause unwanted folding, etc. The term also includes antibodies produced recombinantly in non-human cells, which could give glycosylation that is not typical of human cells. These antibodies can be prepared in various ways, as described below.

As used herein, the term "neutralizing antibody," "an inhibitory antibody" or antagonist antibody indicates an antibody that inhibits the ALK-1 / TGF-beta-1 / Smadl signaling pathway. In a preferred embodiment, the antibody inhibits the ALK-1 / TGF-beta-1 / Smadl signaling pathway by at least about 20%, preferably 40%, more preferably 60%, even more preferably 80%, or even more preferably 85 %.

For example, the neutralizing or inhibiting potential of human anti-ALK-1 antibodies can be determined by their ability to inhibit up-regulation of a specific downstream target gene of ALK-1, Id1, as presented in Example 12; to inhibit Smadl phosphorylation determined by Western Blotting using Odyssey Infrared Imaging System from LI-COR Biosciences, as presented in Example

13.

The term "chimeric antibody" as used herein denotes an antibody comprising regions from two or more different antibodies. For example, one or more of the CDRs of a chimeric body may be from a human anti-ALK-1 body. In another example, all CDRs can be from human anti-ALK-1 antibodies. In another example, the CDRs from more than one human anti-ALK-1 antibody can be combined into a chimeric antibody. E.g. a chimeric antibody may be a light chain CDR1 from a first human anti-ALK-1 antibody, a light chain CDR2 from a second human anti-ALK-1 antibody, and a third human anti-light chain CDR3. ALK-1 antibody, and heavy chain CDRs may be derived from one or more other anti-ALK-1 antibodies. Furthermore, the framework regions may be from one of the anti-ALK-1 antibodies from which one or more of the CDRs have been taken or from one or more different human antibodies. In addition, as discussed earlier herein, chimeric antibody includes an antibody comprising a stretch derived from the germline sequences of more than one species.

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

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

The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentrations in a biosensor matrix, for example using the BIACORE ™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). for further descriptions, see Jonsson et al., Ann. Biol. Clin. 51: 19-26 (1993); Jonsson et al., Biotechniques 11: 620-627 (1991); Jonsson et al., J. Mol. Recognit. 8: 125131 (1995); and Johnsson et al., Anal. Biochem. 198: 268-277 (1991).

The term "affinity" indicates a measure of the attraction between an antigen and an antibody. The intrinsic appeal of the antibody to the antigen is typically expressed as the binding affinity equilibrium constant (Kd) of a given antibody-antigen interaction. An antibody is said to bind specifically to an antigen when the Kd is &lt;1 mM, preferably &lt;100 nM. a Kd binding affinity constant can be measured by surface plasmon resonance, for example using the BIACORE ™ system as discussed in Examples 7 and 8.

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

The term "avidity" refers to the functional combination strength of an antibody with its antigen based on both affinity and valence of the antibody. As used herein, this term describes the increased affinity that arises from multiple antigen binding sites on an immunoglobulin.

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

The term "epitope" encompasses any protein determinant with the ability of specific binding to an immunoglobulin or T cell receptor or other interaction with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structure characteristics, as well as specific charge characteristics. An epitope can be "linear" or "conformational". In a linear epitope, all points of interaction between the protein and the molecule interacting (such as an antibody) occur linearly along the protein's primary amino acid sequence. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from each other. Once a desired epitope on an antigen has been determined, it is possible to generate antibodies against that epitope, e.g. using the techniques described in the present invention. Alternatively, in the process of their discovery, the generation and characterization of antibodies may reveal information about desired epitopes. Based on this information, it is then possible to competitively screen antibodies for binding to the same epitope. One approach to achieve this is to conduct cross-competition studies to find antibodies that bind competitively to each other, ie, the antibodies compete for binding to the antigen. A high throughput process for the "binning" of antibodies based on their cross competition is described in International Patent Application No. WO 03/48731.

As used herein, the term "binning" refers to a method of grouping antibodies based on their antigen binding characteristics. The assignment of bins is somewhat arbitrary depending on how different the observed binding patterns are for all antibodies tested. Therefore, bins do not always correlate with epitopes determined by other means and should not be used to define epitopes.

The term "compete," as used herein with respect to an antibody, means that a first antibody, or an antigen-binding portion thereof, competes for binding to the antigen with a second antibody, or an antigen-binding portion thereof, wherein binding of the first antibody to its related epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, in which the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody may, but need not be. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of the other antibody to its related epitope or ligand, regardless of whether it occurs to the same, greater, or lesser extent, the antibodies are said to "cross-compete" for binding of their respective epitope. (n). Both competing and cross-competing antibodies are included in the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g. steric hindrance, conformational alteration, or binding to a common epitope, or piece thereof, and the like), those skilled in the art will understand, based on the information herein, that such competing and / or cross-competing antibodies are included and may be useful for the methods described herein.

The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of any of these nucleotide types. The term includes single-stranded and double-stranded forms.

The term "isolated polynucleotide" as used herein refers to a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, because of its origin, the "isolated polynucleotide" (1) is not associated with all or part of polynucleotides with which it "Isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not bound in nature, or (3) does not occur in nature as part of a larger sequence.

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, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladate, phosphoramidate, and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14: 9081 (1986); Stee 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)); US Patent No. 5,151,510;

Uhlmann and Peyman, Chemical Reviews 90: 543 (1990), the contents of which are incorporated herein by reference. An oligonucleotide may optionally include a label for detection.

"Operably linked" sequences include expression control sequences located adjacent to the gene of interest, as well as expression control sequences acting in trans or remote to control the gene of interest.

The term "expression control sequence" as used herein refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences with which they have been ligated. Expression control sequences include suitable transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that increase protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include promoter, rihosomal binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequence. The term "control sequences" is intended to include at least all those components, the presence of which is essential for expression and editing, and may also include additional components whose presence provides advantages, for example leader sequences and fusion partner sequences.

The term "vector," as used herein, refers to a nucleic acid molecule with the ability to transport another nucleic acid to which it is attached. In some embodiments, the vector is a plasmid, ie, a circular double-stranded stretch of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into a host cell genome upon introduction into the host cell, thereby replicating together with the host genome. In addition, certain vectors are able to direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. It is noted that "recombinant host cell" and "host cell" mean not only the particular cell in question, but also the progeny of such a cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such offspring may in fact not be identical to the mother cell, but may still be included within the scope of the term "host cell" as used herein.

As used herein, the term "germline" refers to the nucleotide and amino acid sequences of the antibody genes and gene segments as they are passed on by parents to progeny through the germ cells. This germline sequence differs from the nucleotide sequences encoding antibodies in mature B cells that have been modified by recombination and hypermutation events in the course of B cell maturation. An antibody that "uses" a particular germline has a nucleotide or amino acid sequence that aligns best with that germline nucleotide sequence or with the amino acid sequence specified thereby. Such antibodies are often mutated compared to the germline sequence.

The term "percent sequence identity" in the context of nucleic acid sequences means the residues in two sequences that are the same when aligned for maximum match. The length of sequence identity equation may be over a distance of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. A number of different algorithms are known in the art that can be used to measure nucleotide sequence identity. E.g. polynucleotide sequences can be compared using FASTA, Gap or Bestfit programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, that e.g. programs includes FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185 219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71-84 (1998); incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. E.g. percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, incorporated herein by reference.

A reference to a nucleotide sequence includes its complement unless otherwise noted. Thus, a reference to a nucleic acid of a particular sequence is to be understood as its complementary strand, to include its complementary sequence.

The term "substantial similarity" or "substantial sequence history," when referring to a nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%,

97%, 98% or 99% of the nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST or Gap, as discussed above.

The term "percent sequence identity" in the context of amino acid sequences refers to the residues in two sequences that are the same when aligned for maximum match. The length of sequence identity comparison can be over a distance of at least about five amino acids, usually at least about 20 amino acids, more usually at least about 30 amino acids, typically at least about 50 amino acids, more typically at least about 100 amino acids, and more typically about 150, 200 or more amino acids. A number of different algorithms are known in the art that can be used to measure amino acid sequence identity. E.g. amino acid sequences can be compared using FASTA, Gap or Bestfit programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG),

Madison, Wisconsin.

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

The term signal sequence, also called signal peptide, leader peptide, refers to a segment of about 15 to 30 amino acids at the N terminus of a protein that allows the protein to be secreted (passing through a cell membrane). The signal sequence is removed while the protein is secreted.

As used herein, the terms "label" or "labeled" indicate the incorporation of another molecule into the antibody. In one embodiment, the label is a detectable marker, e.g., the inclusion of a radiolabeled amino acid or the attachment to a polypeptide of biotinyl groups detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity, which can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Several methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptides labels include, but are not limited to, the following: radioisotopes or radionuclides (e.g.³H, ¹⁴C, ¹⁵N, ³⁵S, Y, Tc, ⁿⁱIn, ¹²⁵1,¹³¹I), fluorescent labels (e.g. FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter ( e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as whooping cough toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine , vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and its analogs or homologs. In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

The term "primate" refers to a mammal of the order of primates, which includes the anthropoids and prosimians, characterized by refined development of the hands and feet, a shortened snout, and large brain. The mammalian order of the Primates includes humans, apes, monkeys, and prosimians, or lower primates.

Therapeutically effective amount refers to that amount of the administered therapeutic agent, which will relieve to some extent one or more of the symptoms of the disorder being treated. When referring to cancer treatment, a therapeutically effective amount indicates that amount which has at least one of the following effects: reducing the size of the tumor; inhibiting (i.e., slowing to some extent, preferably stopping) tumor metastasis; inhibit to some extent (i.e., delay to some extent, preferably stop) tumor growth, and to some extent relieve (or, preferably, eliminate) one or more symptoms associated with the cancer.

Treatment, treatment and treatment indicate a method of alleviating or eliminating a biological disorder and / or symptoms associated therewith. With regard to cancer, these terms simply mean that the life expectancy of a cancer-affected individual will be increased or that one or more of the symptoms of the disease will be reduced.

Contacting indicates bringing together an antibody or antigen-binding portion thereof of the present invention and a target ALK-1, or epitope thereof, in such a way that the antibody can affect the biological activity of the ALK-1. Such contacting can be accomplished in vitro, ie, in a test tube, a petri dish, or the like. In a test tube, contacting may involve only an antibody or antigen-binding portion thereof and ALK-1 or epitope thereof, or may involve whole cells. Cells can also be maintained or grown in cell culture dishes and contacted in that environment with antibodies or antigen binding pieces thereof. In this context, the ability of a particular antibody or antigen binding piece thereof to influence an ALK-1 related disorder, ie the IC50 of the antibody should be determined before attempting to use the antibody in vivo with more complex living organisms. For cells outside the organism, several methods exist, and are known to those of skill in the art, to contact ALK-1 with its antibodies or antigen-binding stretches.

The acronym “FACS” indicates Fluorescence Activated Cell Sorting. The acronym FACS and flow cytometry are used interchangeably.

Fluorescent labeling allows investigating cell structure and function. Immunofluorescence, the most widely used application, involves staining cells with antibodies conjugated with fluorescent dyes, such as fluorescein and phycoerythrine. This method is often used for labeling molecules on the cell surface, but antibodies can target targets in cytoplasm. In direct immunofluorescence, an antibody to a molecule is directly conjugated with a fluorescent dye, and cells are stained in one step. In indirect immunofluorescence, the primary antibody is unlabeled, and a second fluorescently conjugated antibody specific for the first antibody is added.

Anti-ALK-1 Antibodies

This invention relates to isolated neutralizing antiALK-1 monoclonal antibodies or antigen-binding stretches thereof which bind to primates ALK-1, preferably the ECD of primates ALK-1, more preferably the ECD of human ALK-1. In a preferred embodiment, the invention relates to isolated neutralizing antibodies that are fully human monoclonal antibodies or antigen-binding portions thereof. Preferably, the human antibodies are recombinant human anti-ALK-1 antibodies that have greater affinity for ALK-1 than for ALK-2 to ALK-7. In some embodiments, human anti-ALK-1 antibodies are produced by immunization of a non-human transgenic animal, e.g., a rodent, the genome of which comprises human immunoglobulin genes so that the transgenic animal produces human antibodies. Several aspects of the invention pertain to such antibodies and antigen-binding pieces, and their pharmaceutical compositions, as well as nucleic acids, recombinant expression vectors, and host cells for making such antibodies and antigen-binding pieces. Methods of using the antibodies and antigen binding stretches of the present invention to disable the ALK-1 / TGF-beta-1 / Smadl signaling pathway or detect ALK-1, in vitro or in vivo, are also contemplated by the invention.

An anti-ALK-1 antibody of the invention may comprise a human kappa or a human lambda light chain or an amino acid sequence derived therefrom. In some embodiments that include a kappa light chain, the light chain variable domain (V1) uses a human A27, A2, A1, A3, B3, B2, L1, or L2 V_Kgene. In some embodiments, the light chain uses a human Vk LI gene and a human Jk 4 gene; a human Vk A27 gene and a human Jk 5 gene or a human Jk 4 gene; a human Vk B3 gene and a human Jk 1 gene; a human Vk L2 gene and a human Jk 3 gene; a human Vk A2 gene and a human Jk 1 gene; a human Vk A3 gene and a human Jk 4 gene; a human Vk Al gene and a human Jk 1 gene; a human Vk B2 gene and a human Jk 4 gene; or a human Vk A2 gene and a human Jk 1 gene.

In some embodiments, the V1 of the anti-ALK-1 antibody comprises one or more amino acid substitutions, deletions, or insertions (additions) to the germline V_K amino acid sequence. In some embodiments, the V1 of the anti-ALK-1 antibody comprises 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 amino acid substitutions relative to the germline V_K amino acid sequence. In some embodiments, one or more of the germline substitutions lie in the CDR regions of the light chain. In some embodiments, the V_K amino acid germline substitutions at one or more of the same positions as the germline substitutions found in one or more of the V1 of the antibodies provided herein as shown, for example, in Figure 7. In some embodiments, the amino acid changes lie in one or more of the same positions, but involve a different substitution from the reference antibody.

In some embodiments, germline amino acid substitutions occur at one or more of the same positions as germline substitutions in any of the V1 of antibodies 1.11.1; 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 the substitutions may represent conservative amino acid substitutions at such position (s) relative to the amino acid in the reference antibody. For example, if a particular position in any of these antibodies is altered from the germline and is glutamate, one may use aspartate at that position. Similarly, if an amino acid substitution compared to the germline in an exemplary antibody is serine, one can conservatively substitute threonine for serine at that position. Conservative amino acid substitutions are discussed above.

In some embodiments, the anti-ALK-1 antibody comprises a light chain amino acid sequence of SEQ ID NO: 4. In other embodiments, the light chain comprises the light chain amino acid sequence of antibody 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A); 1.12.KM29I);

1.12.KD19A); 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1.

In some embodiments, the human anti-ALK-1 antibody light chain comprises the V1 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 said amino acid sequence containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions and / or a total of up to 3 non-conservative amino acid has substitutions. In other embodiments, the light chain of the human anti-ALK-1 antibody comprises the V1 amino acid sequence of antibody 1.27.1; 1.29.1 or 1.31.1. In some embodiments, the light chain comprises the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any of the preceding antibodies.

In some embodiments, the light chain may comprise the amino acid sequences of CDR1, CDR2 and CDR3 regions independently selected from the light chain CDR1, CDR2 and CDR3 regions, respectively, of two or more monoclonal antibodies selected from 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A); 1.12.KM29I); 1.12.KD19A); 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 said CDR regions each having less than 3 or less than 2 conservative amino acid substitutions and / or a total of three or less non-conservative amino acid substitutions.

In certain embodiments, the Anti-ALK1 antibody Real chain comprises the amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from 1.11.1; 1.12.1; 1.12.1 (rWT);

1.12.1 (M29I / D19A); 1.12.KM29I); 1.12.KD19A); 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 said CDR regions each having less than 3 or less than 2 conservative amino acid substitutions and / or a total of three or less non-conservative amino acid substitutions.

With respect to the heavy chain, in some embodiments, the variable domain (Vh) uses a human Vh 431, Vh 3-11, Vh 3-15, Vh 3-33, Vh 4-61 or Vh 4-59 gene. In some embodiments, the Vh sequence of the anti-ALK-1 antibody contains one or more amino acid substitutions, deletions, or insertions (additions), collectively, "mutations," to the germline Vh amino acid sequence. In some embodiments, the heavy chain variable domain comprises 1, 2, 3, 4,

5, 6, 7, 8, 9, 10 or 11 mutations with respect to the germline Vh amino acid sequence. In some embodiments, the mutation (s) are non-conservative substitutions compared to the germline amino acid sequence. In some embodiments, the mutations lie in the CDR regions of the heavy chain. In some embodiments, the heavy chain uses a human Vh 3-33 gene, a human D 6-19 gene and a human Jh 3B gene; a human Vh 4-31 gene, a human D 6-19 gene and a human Jh 4B gene; a human Vh 4-61 gene, a human D 6-19 gene and a human Jh 4B gene; a human Vh 4-31 gene, a human D 3-3 gene and a human Jh 3B gene; a human Vh 4-31 gene and a human Jh 3B gene; a human Vh 4-59 gene, a human D 6-19 gene and a human Jh 4B gene; a human Vh 3-11 gene, a human D 3-22 gene and a human Jh 6B gene; a human Vh 3-15 gene, a human D 3-22 gene and a human Jh 4B gene; a human Vh 4-31 gene, a human D 5-12 gene and a human Jh 6B gene; a human Vh 4-31 gene, a human D 4-23 gene and a human Jh 4B gene; a human Vh 4-31 gene, a human D 2-2 gene and a human Jh 5B gene; a human Vh 4-31 gene and a human Jh 6B gene; human Vh 3-15 gene, a human D 1-1 gene and a human Jh 4B gene; a human Vh 3-11 gene, a human D 6-19 gene and a human Jh 6B gene; a human Vh 3-11 gene, a human D 3-10 gene and a human Jh 6B gene; or a human Vh 3-11 gene, a human D 6-6 gene and a human Jh 6B gene.

In some embodiments, amino acid substitutions lie in one or more of the same positions as the germline substitutions in one or more of the V_H of antibodies 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A);

1.12.KM29I); 1.12.KD19A); 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 embodiments, the amino acid changes are in one or more of the same positions but are a different substitution from the reference antibody.

In some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO: 2. In other embodiments, the heavy chain comprises the heavy chain amino acid sequence of antibody 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1 (M29I / D19A); 1.12.KM29I); 1.12.KD19A); 1.13.1; 1.14.1;

1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or

5.59.1. In some embodiments, the heavy chain comprises the Vh amino acid sequence of antibody 1.12.1 (SEQ 1D NOS: 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.KSEQ ID NO: 34); 1.9.KSEQ ID NO: 38); 4.10.1 (SEQ ID NO: 42); 4.24.1 (SEQ ID NO: 46); 4.38.KSEQ ID NO: 50); 4.58.1 (SEQ ID NO: 54); 4.62.1 (SEQ ID NO: 58);

4.68.1 (SEQ ID NO: 62); 4.72.1 (SEQ ID NO: 66); 5.13.1 (SEQ ID NO: 70);

5.34.1 (SEQ ID NO: 74); 5.53.1 (SEQ ID NO: 78); 5.56.1 (SEQ ID NO: 82);

5.57.1 ​​(SEQ ID NO: 86); or 5.59.1 (SEQ ID NO: 90); or said Vh amino acid sequence having up to 1, 2, 3, 4, 6, 8, 9, 10 or 11 conservative amino acid substitutions and / or a total of at most 3 non-conservative amino acid substitutions. In other embodiments, the heavy chain comprises the Vh amino acid sequence of antibody 1.27.1; 1.29.1 or 1.31.1. In some embodiments, the heavy chain comprises the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any of the preceding antibodies.

In some embodiments, the heavy chain comprises the heavy chain CDR1, CDR2 and CDR3 regions of antibody 1.11.1; 1.12.1; 1.12.1 (rWT);

1.12.1 (M29I / D19A); 1.12.KM29I); 1.12.KD19A); 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 said CDR regions each having less than 8, less than 6, less than 4, or less than 3 conservative amino acid substitutions and / or a total of three or less non-conservative amino acid substitutions.

In some embodiments, the heavy chain CDR regions are independently selected from the CDR regions of two or more antibodies selected from antibodies 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1 (M29I / D19A);

1.12.KM29I); 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 embodiment, the antibody comprises a light chain as described above and a heavy chain as described above. In a further embodiment, the light chain CDRs and the heavy chain CDRs are from the same antibody.

In various embodiments, the anti-ALK-1 antibodies have the full-length heavy chain and full-length light chain amino acid sequence (s), the Vh and V1 amino acid sequences, the heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3 amino acid sequences or the heavy chain amino acid sequence from the beginning of the CDR1 to the end of the CDR3 and the light chain amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of an anti-ALK-1 antibody provided herein.

One type of amino acid substitution that can be performed is to modify one or more cysteines in the antibody, which can be chemically reactive, to another residue such as, without limitation, alanine or serine.

In one embodiment, there is a substitution of a non-canonic cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine ​​is canonic.

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

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

In one aspect, the invention provides twenty five inhibitory human anti-ALK-1 monoclonal antibodies and the hybridoma cell lines that produce them. In certain embodiments, antibodies of the present invention IgGs are designated as: 1.11.1; 1.12.1; 1.12.1 (rWT);

1.12.KM29I / D19A); 1.12.KM29I); 1.12.KD19A); 1.13.1; 1.14.1; 1,151.1; 1,162.1;

1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1. In preferred embodiments, the human anti-ALK-1 antibody is antibody 1.12.1, 1.12.1 (M29I / D19A), 1.12.KM29I), 1.12.1 (D19A), 1.27.1,

1.14.1,1.162.1, 1.31.1, 4.62.1 or 4.72.1.

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

Table 1 lists the sequence identifiers (SEQ ID NO) of the nucleic acids encoding the full-length heavy and light chains of

1.12.1 antibody variants and variable domain-containing stretches of antiALK-1 antibodies of the invention, and the corresponding deduced amino acid sequences.

Table 1

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

<td rowspan="3">Antibody</td><td colspan="4">FULL LENGTH</td><td colspan="4">V DOMAIN CONTAINING PIECE</td>

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

<td>DNA</td><td>PROTEIN</td><td>DNA</td><td>PROTEIN</td><td>DNA</td><td>PROTEIN</td><td>DNA</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.KM29IZD 19A)</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> 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>

1.12.1 (M29I / D19A) indicates the anti-ALK-1 antibody containing a specific single amino acid mutation in the heavy chain, where the methionine at position 29 was replaced by isoleucine, and a specific single amino acid mutation in the light chain, replacing the aspartic acid at position 19 with alanine, as described in Example 4.

1.12.1 refers to the mAb 1.12.1 variant isolated from the hybridoma.

1.12.1 (rWT) refers to the mAb 1.12.1 variant expressed as a recombinant mAb described in Example 3.

The invention further provides heavy and / or light chain variants of some of the above-listed human anti-ALK-1 antibodies, comprising one or more amino acid modifications. To indicate the variants, the first letter is the one letter symbol for the amino acid of the naturally occurring antibody chain, the number indicates the position of the amino acid (where position one is the N-terminal amino acid of the FR1), and the second letter the one letter symbol for the variant amino acid.

In still further embodiments, the invention includes antibodies comprising variable domain amino acid sequences of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% sequence identity to a variable domain amino acid sequence of any of the human anti-ALK-1 antibodies listed above.

Class and Subclass of Anti-ALK-1 Antibodies

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 an antibody can be determined using antibodies specific for a particular antibody class and subclass. Such antibodies are commercially available. The 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 antibody constant domains of the heavy and / or light chains, comparing their amino acid sequences with the known amino acid sequences of various immunoglobulin classes and subclasses. , and determine the class and subclass of the antibodies.

The class of an anti-ALK-1 antibody obtained as described above can be switched to another. In one aspect of the invention, a nucleic acid molecule encoding V1 or Vh is isolated using methods known in the art such that it does not include nucleic acid sequences encoding Cl or Ch. "Antibody Engineering" (Kontermann &amp; Dubel, Eds., Springer-Verlag, Berlin (2001)). The nucleic acid molecules encoding V1 or Vh are then operably linked to a nucleic acid sequence encoding a Cl or Ch from a different class of immunoglobulin molecule, respectively. This can be accomplished using a vector or nucleic acid molecule comprising a Cl or Ch chain, as described above. For example, an anti-ALK1 antibody that was originally IgM can be switched from class to an IgG. Furthermore, the switching of class can be used to convert one IgG subclass into another, eg from IgG1 to IgG2. A preferred method of producing an antibody of the invention comprising a desired isotype comprises the steps of isolating a nucleic acid molecule encoding the heavy chain of an antiALK-1 antibody and a nucleic acid molecule encoding the light chain of an anti-ALK- 1 antibody, obtaining the heavy chain variable domain, ligating the heavy chain variable domain with the heavy chain constant domain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and capturing the anti-ALK-1 antibody with the desired isotype.

In some embodiments, the anti-ALK-1 antibody is a monoclonal antibody. The anti-ALK-1 antibody can be an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In a preferred embodiment, the anti-ALK-1 antibody is an IgG and is of an IgG1, IgG2, IgG3, IgG4 subclass. In another preferred embodiment, the antibody is of subclass IgG2.

Identification of ALK-1 Epitopes Recognized by Anti-ALK-1 Antibodies

The invention provides a human anti-ALK-1 monoclonal antibody that binds to ALK-1 and competes or cross-competes with and / or binds 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.KD19A); 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 containing a heavy chain variable domain having the amino acid sequence of the Vh domain in one of SEQ ID NOS: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104, (c) an antibody comprising a light chain variable domain having the amino acid sequence of the V1 domain in one of SEQ ID NOS: and 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 127, (d) an antibody comprising both a heavy chain variable domain as defined in (b) and a light chain variable domain as defined in (c).

One can determine whether an antibody binds to the same epitope or cross-competes for binding with an anti-ALK-1 antibody using methods known in the art. In one embodiment, the antiALK-1 antibody of the invention is allowed to bind to ALK-1 under saturation conditions and the ability of the test antibody to bind to ALK-1 is then measured. If the test antibody can bind to ALK-1 simultaneously with 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 cannot simultaneously bind to ALK1, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the anti-ALK-1 antibody of the invention. This experiment can be performed using ELISA, RIA, BIACORE ™, or flow cytometry. To test whether an anti-ALK-1 antibody cross-competes with an Einder antiALK-1 antibody, the competition method described above can be used in two directions, ie determining whether the known antibody blocks the test antibody and vice versa. In a preferred embodiment, the experiment is performed using BIACORE ™.

Binding affinity of Anti-ALK-1 Antibodies to ALK-1

The binding affinity (Kd) and dissociation rate (calf) of an anti-ALK1 antibody or antigen-binding portion thereof for ALK-1 can be determined by methods known in the art. The binding affinity can be measured by ELISAs, RIAs, flow cytometry, or surface plasmon resonance, such as BIACORE ™. The dissociation rate can be measured by surface plasmon resonance. Preferably, the binding affinity and dissociation rate is measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE ™. It can be determined whether an antibody has substantially the same Kd as an anti-ALK-1 antibody using methods known in the art. Such methods of Kd and calf determination can be used during the initial screening step, as well as during subsequent optimization steps.

Inhibition of ALK-1 Activity by Anti-ALK-1 Antibody

Anti-ALK-1 monoclonal antibodies that inhibit ALK-1 binding can be identified using a number of assays.

For example, neutralizing anti-ALK-1 antibodies can be identified by their inhibition of up-regulation of a specific downstream target gene of ALK-1, Id1, as described in Example 12. Preferred anti-ALK-1 antibodies have an IC50 of no more than

500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 1 nM.

One can also determine the ability of an anti-ALK-1 antibody to

Smadl phosphorylation determined by inhibiting Western Blotting using Odyssey Infrared Imaging System, as described in Example

13. In various embodiments, the anti-ALK-1 antibody has a

IC50 in this assay of no more than 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, nM, 10 nM, or InM.

Inhibition of Angiogenesis by Anti-ALK-1 Antibody

In another embodiment, the anti-ALK-1 antibody or piece of it inhibits angiogenesis of human vessels as demonstrated in a SCID mouse with a human foreskin tissue graft implanted intradermally with human melanoma M24 with tumor cells, as determined by IHC analysis of human CD- 31 signal assay, by a factor of at least 40% compared to a control sample as described in Example 17 and shown in Table 13.

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

Species and Molecular Selectivity

In another aspect of the invention, the anti-ALK-1 antibodies exhibit both species and molecular selectivity. By following the information given in the description, one can determine the species or molecular selectivity for the anti-ALK-1 antibody using methods well known in the art. For example, one can determine the species selectivity by Western blot, surface plasmon resonance, e.g. BIAcore, ELISA, immunoprecipitation or RIA.

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

In other embodiments, the anti-ALK-1 antibody has a selectivity for ALK-1 over ALK-2 to ALK-7. In some embodiments, the anti-ALK-1 antibody shows no appreciable specific binding to any protein other than ALK-1. Preferably, the anti-ALK-1 antibody binds to the ECD of human ALK-1.

Methods of Antibody Production and Antibody Producing

Cell lines

ALK-1 Immunogenic

In some embodiments, the ALK-1 is immunogen or antigen isolated and / or purified ALK-1. In some embodiments, the ALK-1 is immunogenic human ALK-1. In preferred embodiments, the ALK-1 immunogen is the ECD of human ALK-1. Human ALK-1, or antigenic pieces thereof, can be prepared by methods known to those skilled in the art, or may be purchased from commercial vendors. The amino acid and nucleotide sequences of human ALK-1 are known (see e.g. Genbank registration Access Number L17075). ACVRL1 gene encoding a full-length ALK-1 is commercially available from Invitrogen Ine., Clone ID IOH21048, For example, is sold by R&amp;D Systems, Ine., The recombinant human ALK-1 / Fc chimera (Catalog Number 370AL) by expression of a DNA sequence encoding the ECD amino acid residues 1-118 of ALK-1, which DNA sequence was fused to a DNA sequence encoding the F_c region of human IgG via a DNA sequence encoding a polypeptide linker molecule in a murine myeloma cell line. The recombinant mature human ALK-1 / Fc chimera is a disulfide-bound homodimer protein with Asp 22 at the amino terminus. In addition, Example 1 describes the preparation of ALK-1 ECD His-Tag protein used to generate bybridomas producing an anti-ALK- 1 antibody of the present invention.

In other embodiments, the ALK-1 antigen is a cell expressing or overexpressing ALK-1. In other embodiments, the ALK-1 antigen is a recombinant protein expressed by recombinant technology from yeast, insect cells, bacteria such as E. coli, or other sources.

Immunization

In some embodiments, human antibodies are produced by immunizing a non-human, transgenic animal, comprising in its genome part or all of human immunoglobulin heavy chain and light chain loci, with an ALK-1 antigen. In a preferred embodiment, the non-human animal is a XENOMOUSE® animal. (Abgenix, Inc., Fremont, CA).

XENOMOUSE® mice are modified strains of mice comprising large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics 7: 13-21 (1994) and US Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001,6,114,598,6,130,364, 6,162,963 and 6,150,584. See also WO 9U10741, 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.

In another aspect, the invention provides a method of assaying anti-ALK-1 antibodies from non-human, non-mouse animals by immunizing non-human transgenic animals comprising human immunoglobulin loci with an ALK-1 antigen. Such animals can be produced using the methods described in the documents cited above. The methods described in these documents can be modified as described in US Patent 5,994,619, which is hereby incorporated by reference. US Patent 5,994,619 describes methods for producing new cultured internal cell mass (CICM) cells and cell lines from pigs and cows, and transgenic CICM cells in which heterologous DNA has been inserted. CICM transgenic cells can be used to produce cloned transgenic embryos, fetuses and offspring. The '619 Patent also describes methods of producing transgenic animals capable of transmitting the heterologous DNA to their offspring. In preferred embodiments of the present invention, the non-human animals are mammals, especially rats, sheep, pigs, goats, livestock, horses or chickens.

XENOMOUSE® mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies. In some embodiments, the XENOMOUSE® mice contain approximately 80% of the human antibody V gene repertoire by introducing germline configuration fragments of megabase size from the human heavy chain locus and kappa light chain loci in yeast artificial chromosome (YAC). In other embodiments, XENOMOUSE® mice further contain about everything from the human lambda light chain locus. See Mendez et al., Nature Genetics 15: 146-156 (1997), Green and Jakobovits, J. Exp. Med. 188: 483-495 (1998), and WO 98/24893, the contents of which are incorporated by reference herein.

In some embodiments, the non-human animals comprising human immunoglobulin genes are animals which possess a human immunoglobulin "minilocus". In the minilocus approach, an exogenous Ig locus is simulated by including individual genes from the Ig locus. Thus become one

... woo · »· 17.

Onion UL1W1 ""

Vh genes, one or more Dh genes, one or more

Jh genes, a mu constant domain, and a second constant domain (preferably a gamma constant domain) formed into a construct for insertion into an animal. This approach is described, inter alia, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and 5,643,763, incorporated herein by reference.

In another aspect, the invention provides a method of making humanized anti-ALK-1 antibodies. In some embodiments, non-human animals are immunized with an ALK-1 antigen as described below under conditions that allow antibody production. Antibody-producing cells are isolated from the animals, and nucleic acids encoding the heavy and light chains of an anti-ALK-1 antibody of interest are isolated from the isolated antibody-producing cells or from an immortalized cell line produced from such cells. These nucleic acids are then modified using techniques known to those skilled in the art and as further described below to reduce the amount of non-human sequence, ie to humanize the antibody to reduce the immune response in humans.

Immunization of animals can be by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, livestock and horses are well known in the art.

See, e.g., Harlow and Lane, supra, and U.S. Patent 5,994,619. In a preferred embodiment, the ALK-1 antigen is administered with an adjuvant to stimulate the immune response. Examples of adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immune stimulating complexes). Such adjuvants can protect the polypeptide from rapid spread by sequestering it in a local depot, or they may contain substances that stimulate the host to secrete factors chemotactic to macrophages and other components of the immune system. Preferably, when a polypeptide is administered, the immunization schedule will involve two or more administrations of the polypeptide over several weeks. Example 2 exemplifies a method for producing anti-ALK-1 monoclonal antibodies in XENOMOUSE® mice.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with an ALK-1 antigen, antibodies and / or antibody-producing cells can be obtained from the animal. In some embodiments, anti-ALK-1 antibody-containing serum is obtained from the animal by drawing blood or killing the animal. The serum can be used as obtained from the animal, an immunoglobulin fraction can be obtained from the serum, or the antiALK-1 antibodies can be purified from the serum.

In some embodiments, antibody-producing cell lines are prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized in any manner known in the art. Methods of cell immortalization include, but are not limited to, transfection with oncogenes, infection with an oncogenic virus and culturing under conditions that select for immortalized cells, subjection to carcinogenic or mutant compounds, fusion with an immortalized cell, e.g. a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. Preferably, when fusion with myeloma cells is used, the myeloma cells do not secrete immunoglobulin polypeptides (a non-secretory cell line).

Immortalized cells are screened using ALK-1, or a portion thereof. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay. An example of ELISA screening is given in WO 00/37504, incorporated herein by reference.

Anti-ALK-1 antibody-producing cells, e.g., hybridomas, are selected, cloned and further screened for desired characteristics, including robust growth, high antibody production and desired antibody characteristics, as discussed further below. Hybridomas can be expanded in vivo in syngenic animals, in animals lacking an immune system, e.g. naked mice, or in cell culture in vitro.

Methods for selecting, cloning and expanding hybridomas are well known to those skilled in the art.

In a preferred embodiment, the immunized animal is a non-human animal expressing human immunoglobulin genes and the spleen B cells are fused to a myeloma cell line from the same species as the non-human animal. In a more preferred embodiment, the immunized animal is a XENOMOUSE® mouse and the myeloma cell line is a non-secretory murine myeloma. In an even more preferred embodiment, the myeloma cell line is P3-X63-Ag8.653 (American Type Culture Collection). See eg Example 2.

Thus, in one embodiment, the invention provides methods of producing a cell line that produces a human monoclonal antibody or a fragment thereof directed against ALK-1, comprising (a) immunizing a non-human transgenic animal described herein with ALK-1, a piece of ALK-1 or a cell or tissue expressing ALK-1; (b) causing the transgenic animal to initiate an immune response to ALK-1; (c) isolating antibody-producing cells from the transgenic animal; (d) immortalizing the antibody-producing cells; (e) creating individual monoclonal populations of the immortalized antibody-producing cells; and (f) screening the immortalized antibody-producing cells to identify an anti-ALK-1 antibody.

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

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

In another embodiment, phage display techniques can be used to provide pools containing a repertoire of antibodies with varying affinities for ALK-1. It is not necessary to immortalize the B cells from the immunized animal for the production of such repertoires. Rather, the primary B cells can be used directly as a source of DNA. The mixture of cDNAs obtained from B cells, e.g. from spleens, is used to prepare an expression library, for example a phage display library transfected into E. coli. The resulting cells are tested for immunoreactivity against ALK-1. Techniques for identifying high affinity human antibodies from such pools have been 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, which are incorporated by reference. Finally, clones from the library are identified that produce binding affinities of a desired size for the antigen and the DNA encoding the product responsible for such binding is recovered and manipulated for standard recombinant expression. Phage display pools can also be constructed using similarly engineered nucleotide sequences. In general, the cDNAs encoding heavy and light chains are supplied or linked independently to form Fv analogs for production in the phage library.

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

Nucleic acids. Vectors, Host Cells and Recombinant Methods for It

Making Antibodies

Nucleic acids

The present invention also includes nucleic acid molecules encoding anti-ALK-1 antibodies or an antigen-binding fragments thereof. In some embodiments, different nucleic acid molecules encode an anti-ALK-1 immunoglobulin heavy chain and light chain. In other embodiments, the same nucleic acid molecule encodes an anti-ALK-1 immunoglobulin heavy chain and light chain.

In some embodiments, the nucleic acid molecule encoding the light chain variable domain (V1) uses a human A27, A2, A1, A3, B3, B2, L1 or L2 V_K gene, and a human Jk5, JkI, Jk3 or Jk4 gene. In some embodiments, the nucleic acid molecule uses a human A27 Vk gene and a human Jk5 gene. In other embodiments, the nucleic acid molecule uses a human A2 gene and a human JkI gene. In some embodiments, the nucleic acid molecule encoding the light chain encodes an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions to the germline amino acid sequence (s). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a V1 amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions and / or 1 , 2, or 3 non-conservative substitutions compared to germline Vk and Jk sequences. Substitutions can be in the CDR regions, the framework regions, or in the constant domain.

In some embodiments, the nucleic acid molecule encodes a V1 amino acid sequence comprising one or more mutations compared to the germline sequence that are identical to the germline mutations found in the V1 of any of the antibodies 1.11.1; 1.12.1; 1.12.1 (rWT);

1.12. KM29I / D19A); 1.12.KM29I); 1.12.KD19A); 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1.

In some embodiments, the nucleic acid molecule encodes at least three amino acid substitutions compared to the germline sequence that are identical to the germline mutations found in the VL of any of the antibodies 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A);

1.12. KM29I); 1.12.KD19A); 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1;

1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7.11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91 or 126, encoding the V1 amino acid sequence of monoclonal antibody

1.12.KM29I / D19A), 1.11.1, 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; 5.59.1 or 1.12.1.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 127. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3 or a portion thereof. In some embodiments, the nucleic acid encodes the light chain amino acid sequence of one, two, or all three CDRs of said antibody. In some embodiments, said stretch encodes an adjacent region from CDR1-CDR3 of the light chain of an anti-ALK-1 antibody.

In some embodiments, the nucleic acid molecule encodes a V1 amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the V1 amino acid sequence of any of the antibodies 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1 (M29I / D19A); 1.12.KM29I);

1.12.KD19A); 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 to the amino acid sequence of the V1 region of SEQ ID NO: 4. Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, or which are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a nucleic acid encoding the amino acid sequence are the V1 region of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 126 or to a nucleic acid comprising the V1 region nucleotide sequence of SEQ ID NO: 4.

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

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

In some embodiments, the nucleic acid molecule encodes a Vh sequence comprising one or more amino acid mutations compared to the germline Vh sequence that are identical to amino acid mutations found in the Vh of one of the monoclonal antibody 1.11.1; 1.12.1; 1.12.1 (rWT);

1.12. KM29I / D19A); 1.12.KM29I); 1.12.1 (D19A); 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1. In some embodiments, the nucleic acid encodes at least three amino acid mutations compared to the germline sequences, which are identical to at least three amino acid mutations found in any of the monoclonal antibodies listed above.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5.13.17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, or 103, encoding the Vh amino acid sequence of monoclonal antibody

1.12. KM29I / D19A), 1.11.1, 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; 5.59.1 or 1.12.1.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of one of SEQ ID NOs: SEQ ID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104. In various preferred embodiments, the nucleic acid molecule comprises at least part of the nucleotide sequence of SEQ ID NOS: 1 or 95. In some embodiments, said stretch encodes the Vh region, a CDR3 region, all three CDR regions, or an adjacent region that includes CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a Vh amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the Vh amino acid sequence in any of SEQ ID NOS: SEQ ID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104. Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, or that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical are a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42;

46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90, 100 or 104, or on a V._H region thereof, or to a nucleic acid comprising the nucleotide sequence of SEQ ID NOs: 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 the nucleotide sequence encoding a Vh region thereof.

In another embodiment, the nucleic acid encodes a full length heavy chain of an antibody selected from the group consisting of 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A); 1.12.1 (M29I); 1.12.KD19A); 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. Furthermore, the nucleic acid may comprise the nucleotide sequence of SEQ ID NOs: 1 or 95.

A nucleic acid molecule encoding the heavy or light chain of an anti-ALK-1 antibody or pieces thereof can be isolated from any source producing such an antibody. In various embodiments, the nucleic acid molecules are isolated from a B cell that produces an anti-ALK-1 antibody, isolated from an animal immunized with ALK-1 or from an immortalized cell derived from such a B cell. Methods of isolating nucleic acids encoding an antibody are known in the art. See, e.g., Sambrook J. &amp; Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000). mRNA can be isolated and used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In a preferred embodiment, the nucleic acid molecule is isolated from a hybridoma which, as one of its fusion partners, has a cell from a non-human transgenic animal, which cell produces a human immunoglobulin. In an even more preferred embodiment, the cell producing human immunoglobulin is isolated from a XENOMOUSE® animal. In another embodiment, the cell producing the human immunoglobulin is isolated from a non-human, non-mouse transgenic animal, as described above. In another embodiment, the nucleic acid is isolated from a non-human, non-transgenic animal. For example, the nucleic acid molecules isolated from a non-human, non-transgenic animal. are used for humanized antibodies comprising one or more amino acid sequences from a human anti-ALK-1 antibody of the present invention.

In some embodiments, a heavy chain nucleic acid encoding an anti-ALK-1 antibody of the invention may comprise a nucleotide sequence encoding a Vh domain of the invention linked by a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding a light chain of an anti-ALK-1 antibody of the invention may comprise a nucleotide sequence encoding a V1 domain of the invention linked by a nucleotide sequence encoding a light chain constant domain from any source.

In a further aspect of the invention, nucleic acid molecules encoding the variable domain of the heavy (Vh) and / or light (V1) chains are "converted" into full-length antibody genes. In one embodiment, nucleic acid molecules encoding the Vh or V1 domains are converted into full-length antibody genes by insertion into a

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chain encodes constant (Cl) domains such that the Vh segment is operably linked to the Ch segment (s) in the vector, and / or the V1 segment is operably linked to the C1 segment in the vector. In another embodiment, nucleic acid molecules encoding the Vh and / or V1 domains are converted into full-length antibody genes by connection, e.g. ligation, of a nucleic acid molecule encoding a Vh and / or V1 domains with a nucleic acid molecule encoding a Ch and / or Cl domain using standard molecular biology techniques.

Nucleic acid sequences from human heavy and light chain immunoglobulin constant domain genes are known in the art. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.,

NIH Publ. No. 91-3242,1991. Nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed from a cell into which they have been introduced and the anti-ALK-1 antibody isolated.

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

In another embodiment, a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence. E.g. the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer for amplifying DNA regions that, inter alia, could be used to isolate additional nucleic acid molecules encoding variable domains of anti-ALK-1 antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encode all or part of one or more of the CDRs of antibodies 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1 (M29I / D19A); 1.12.1 (M29I); 1.12.KD19A); 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

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

In some embodiments, the anti-ALK-1 antibodies of the invention or antigen-binding stretches are expressed by inserting DNAs encoding partial or full-length light and heavy chains obtained as described above into expression vectors such that the genes are operably linked to necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences in the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In a preferred embodiment, both genes are inserted in the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g. ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present).

A useful vector is a coding for a functionally complete human Ch or Cl immunoglobulin sequence, with suitable restriction sites constructed such that any Vh or V1 sequence can be easily inserted and expressed as described above. In such vectors, splicing normally occurs between the spbce donor site in the inserted J region and the splice acceptor site that precedes the human C domain, and also at the splice regions that occur in the 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 a host cell. The antibacterial chain gene can be cloned into the vector such that the signal peptide is linked to the amino terminus of the immunoglobin chain in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobin protein).

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

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

Non-Hybridoma Host Cells and Methods of Recombinant Production of

Protein

Nucleic acid molecules encoding anti-ALK-1 antibodies and vectors comprising these nucleic acid molecules can be used to transfect an appropriate mammalian, plant, bacterial or yeast host cell. Transformation can be accomplished by 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 dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference). Methods of transforming plant cells are known in the art, including, e.g., Agrobacterium-mediated transformation, Holistic transformation, direct injection, electroporation and viral transformation.

Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, and a number of other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high levels of expression. 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 long enough for expression of the antibody in the host cells or, rather, secretion of the antibody to the culture medium in which the host cells are grown, possible. Antibodies can be recovered from the culture medium by standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc.

Bacterial host cells include E. coli and Streptomyces species.

Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

Furthermore, the expression of antibodies of the invention by production cell lines can be enhanced by a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a widely used approach for increasing expression under certain conditions. The GS system is being discussed in whole or in part in connection with European Patent Nos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841.

Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation. However, any antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, are part of the present invention regardless of the glycosylation of the antibodies.

Transgenic Animals and Plants

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

In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding an anti-ALK-1 antibody of the invention into the animal or plant by standard transgenic techniques. See Hogan and U.S. Patent 6,417,429, supra. The transgenic cells used to make the transgenic animal can be embryonic stem cells or somatic cells or a fertilized egg. The transgenic non-human organisms can be chimeric, non-chimeric heterozygotes, and non-chimeric homozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2^nd 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), all of which are incorporated herein by reference. In some embodiments, the transgenic non-human animals have a targeted disruption and replacement by a targeted construct encoding a heavy and / or light chain of interest. In a preferred embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that bind specifically to ALK-1, preferably human ALK-1. In some embodiments, the transgenic animals include nucleic acid molecules encoding a modified antibody such as a single chain antibody, a chimeric antibody or a humanized antibody. The anti-ALK-1 antibodies can be made in any transgenic animal. In a preferred embodiment, the non-human animals are mice, rats, sheep, pigs, goats, livestock or horses. The non-human transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other body fluids.

Phage Display Collections

The invention provides a method of producing an antiALK-1 antibody or antigen-binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with ALK-1 or an antibody-binding member thereof, isolating phage that bind ALK-1, and obtaining the antibody from the phage. For example, a method of preparing the pool of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with ALK-1 or an antigenic stretch thereof to create an immune response. extraction of antibody-producing cells from the immunized animal; isolation of RNA encoding heavy and light chains of antibodies of the invention from the extracted cells, reverse transcription of the RNA to produce cDNA, amplification of the cDNA using primers, and inserting the cDNA into a phage display vector containing antibodies expressed on the phage. In this way, recombinant anti-ALK-1 antibodies of the invention can be obtained.

Recombinant human anti-ALK-1 antibodies of the invention can be isolated by screening a recombinant combinatorial antibody pool. Preferably, the library is a scFv phage display library generated using human V1 and Vh cDNAs prepared from mRNA isolated from B cells. Methods for the preparation and screening of such collections are known in the art. Kits for generating phage display pools are commercially available (eg, the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SuriZAP ™ Phage Display Kit, Catalog No. 240612). There are also other methods and reagents that can be used in the generation and screening of antibody display pools (see, e.g., U.S. Patent No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio / Technology 9: 1370-1372 (1991); Hay 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: 13731377 (1991); Hoogenboom et al., Nuc. Acid Res. 19: 4133-4137 (1991); and Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991), all incorporated herein by reference.

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

Once initial human V1 and Vh domains are selected, mix and match experiments are performed in which different pairs of the initially selected Vl and Vh segments are screened for ALK-1 binding to select preferred Vl / Vh pair combinations. In addition, to further improve the quality of the antibody, the Vl and Vh segments of the preferred Vl / Vh pair (s) can be randomly mutated, preferably in the CDR3 region of Vh and / or Vl, in a process analog to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be achieved by amplification of Vh and Vl domains using PCR primers complementary to the Vh CDR3 or Vl CDR3, respectively, which primers are "spiked" with any mixture of the four nucleotide bases at certain positions such that the resulting PCR products encode Vh and V1 segments into which random mutations have been introduced in the Vh and / or V1 CDR3 regions. These randomly mutated Vh and V1 segments can be rescreened for binding to ALK-1.

Following the screening and isolation of an anti-ALK-1 antibody of the invention from a recombinant immunoglobulin display pool, nucleic acids encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and in other expression vectors subcloned by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention, as described below. To express a recombinant human antibody isolated by screening a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described above.

Demunized Antibodies

In another aspect of the invention, the antibody can be de-immunized to reduce its immunogenicity, using the techniques described in, e.g., PCT Publication Nos. WO98 / 52976 and WOOO / 34317 (incorporated by reference).

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and host cells can be used to make mutated anti-ALK-1 antibodies. The antibodies can be mutated in the variable domains of the heavy and / or light chains, e.g., to alter a binding property of the antibody. For example, a mutation can be made in one or more of the CDR regions to increase or decrease the Kd of the antibody for ALK-1, increase or decrease calf, or change the binding specificity of the antibody.

Site-directed mutagenesis techniques are well known in the art.

See, e.g., Sambrook et al. And Ausubel et al., Supra. In another embodiment, one or more mutations are made at an amino acid residue of which change is known compared to the germline in monoclonal antibody 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A); 1.12.1 (M29I);

1.12.KD19A); 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. The mutations can be made in a CDR region or framework region of a variable domain, or in a constant domain. In a preferred embodiment, the mutations are made in a variable domain. In some embodiments, one or more mutations are made at an amino acid residue of which change is known, compared to the germline in a CDR region or framework region of a variable domain of an amino acid sequence SEQ ID NO: 2, 4, 6, 8, 10, 12 , 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 , 64, 66,

68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 or 127, or the nucleic acid sequence of which is shown 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.

In another embodiment, the framework region is mutated such that the resulting framework region (s) possess the amino acid sequence of the corresponding germline gene. A mutation can be made in a framework region or constant domain to increase the half-life of the antiALK-1 antibody. See, e.g., PCT Publication No. WO 00/09560, incorporated herein by reference. A mutation in a framework region or constant domain can also be made to alter the immunogenicity of the antibody, provide a site for covalent or non-covalent binding to another molecule, or such properties as complement fixation, FcR binding and antibody dependent alter cell-mediated cytotoxicity (ADCC). According to the invention, a single antibody may have mutations in one or more of the CDRs or framework regions of the variable domain or in the constant domain.

In some embodiments, there are from 1 to 13, including any number in between, amino acid mutations in the Vh or V1 domains of the mutated anti-ALK-1 antibody compared to the anti-ALK-1 antibody before mutation. In any of the above embodiments, the mutations can occur in one or more CDR regions. Furthermore, any of the mutations can be conservative amino acid substitutions. In some embodiments, there are no more than 5, 4, 3, 2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin can be made comprising all or part of an antiALK-1 antibody of the invention coupled to another polypeptide. In a preferred embodiment, only the variable domains of the anti-ALK-1 antibody are linked to the polypeptide. In another preferred embodiment, the Vh domain of an anti-ALK-1 antibody is linked to a first polypeptide, while the V1 domain of an anti-ALK-1 antibody is linked to a second polypeptide that associates with the first polypeptide in such a way that the Vh and Vl domains interact with each other to form an antigen binding site. In another preferred embodiment, the Vh domain is separated from the V1 domain by a linker molecule such that the Vh and V1 domains can interact (see below under Single Chain Antibodies). The VH linker molecule VL antibody is then coupled to the polypeptide of interest. In addition, fusion antibodies can be created in which two (or more) single chain antibodies are linked together. This can be useful if you want to create a divalent or polyvalent antibody on a single polypeptide chain, or if you want to create a his-specific antibody.

To create a single chain antibody (scFv), the DNA fragments encoding Vh and V1 are operably linked to another fragment encoding a flexible linker molecule, e.g. encoding the amino acid sequence (Gly * -Ser) 3, such that the Vh and V1 sequences can be expressed as a contiguous single chain protein, with the V1 and Vh domains linked by the flexible linker molecule. 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). The single chain antibody can be monovalent if only a single Vh and V1 are used, bivalent if two Vh and V1 are used, or polyvalent if more than two Vh and V1 are used. Bispecific or polyvalent antibodies can be generated that bind specifically to ALK-1 and another molecule.

In other embodiments, other modified antibodies can be prepared using anti-ALK-1 antibody-encoding nucleic acid molecules. E.g. may "Kappa bodies" (Π1 et al., Protein Eng. 10: 949-57 (1997)), "Minibodies" (Martin et al., EMBO J. 13: 53039 (1994)), "Diabodies" (Holliger et. al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J. 10: 3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7: 51-52 (1992)) are prepared using standard molecular biology techniques by following the directions in the description.

Bispecific antibodies or antigen binding fragments can be produced by a variety of methods including fusion of hybridomas or coupling of Fab 'fragments. See, e.g., Songsivilai &amp; Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In addition, bispecific antibodies such as "diabodies" or "Janusins" can be generated. In some embodiments, the bispecific antibody binds to two different epitopes of ALK-1. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibody 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.KM29I / D19A); 1.12.KM29I);

1.12.KD19A); 1.13.1; 1.14.1; 1,151.1; 1,162.1; 1,183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1 and an additional antibody heavy chain and light chain. In some embodiments, the additional light chain and heavy chain are also one of the above-identified monoclonal antibodies, but are different from the first heavy and light chains.

In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions from a human antiALK-1 monoclonal antibody provided herein.

Derivatized and Labeled Antibodies

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

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those which are heterobifunctional, with two different reactive groups separated by a suitable spacer (e.g. mmaleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g. disuccinimidyl suberate). Such left molecules are available from Pierce Chemical Company, Rockford, II.

Another type of derivatized antibody is a labeled antibody. Useful detection means capable of derivatizing an antibody or antigen-binding piece of the invention include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine naphthalenesulfonyl chloride, phycoerythrine, lanthanide phosphors and the like. An antibody can also be labeled with enzymes 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 used by the enzyme to produce a reaction product that can be distinguished. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product, which is detectable. An antibody may also be labeled with biotin and detected by indirect measurement of avidin or streptavidin binding. An antibody can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

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

Pharmaceutical Compositions and Administration

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

The antibodies and antigen-binding pieces of the present invention can be incorporated into pharmaceutical compositions suitable for administration to an individual. The pharmaceutical composition typically includes an antibody or antigen binding piece of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retardants and the like which 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 preferred that isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride be included in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which increase the storage time or effectiveness of the antibody.

The compositions of this invention can exist in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., by injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of solutions for injection or infusion, such as compositions similar to those used for passive immunization in humans. The preferred mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative. The compositions can take forms such as suspensions, solutions, or emulsions in oily or aqueous carriers, and can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable carrier, e.g. sterile pyrogen-free water, before use.

Therapeutic compositions should typically be sterile and stable under the conditions of preparation 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 incorporating the anti-ALK-1 antibody in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying to give a powder of the active ingredient plus any additional desired ingredient from a pre-sterile filtered solution thereof. The correct fluidity of a solution can e.g. are maintained by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Longer lasting absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

The antibodies or antibody pieces of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications the preferred route / route of administration is subcutaneous, intramuscular, or intravenous infusion. As the skilled person will understand, the route and / or route of administration will vary depending on the desired results.

In certain embodiments, the antibody compositions of the present invention can be prepared with a carrier that will protect the antibody from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are generally 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, which is incorporated herein by reference.

Additional active compounds can also be included in the compositions. In certain embodiments, an inhibitor antiALK-1 antibody of the invention is formulated and / or co-administered with one or more additional therapeutic agents. These agents include, without limitation, antibodies that bind other targets, anti-tumor agents, anti-angiogenesis agents, signal transduction inhibitors, anti-proliferative agents, chemotherapeutic agents, or peptide analogs that inhibit anti-ALK-1. Such combination therapies may require lower doses of the inhibitory anti-ALK-1 antibody as well as the agents co-administered therewith, thus avoiding possible toxicities or complications associated with the various monotherapies.

As noted above, the compositions of the present invention may optionally further comprise a pharmaceutically acceptable antioxidant in addition to a chelating agent. Suitable antioxidants include, but are not limited to, methionine, sodium thiosulfate, catalase, and platinum. For example, the composition may contain methionine at a concentration ranging from 1 mM to about 100 mM and in particular is about 27 mM. For example, an aqueous formulation may be: 10 mg / mL anti-ALK-1 antibody, 20 mM Histidine, pH 5.5, 84 mg / mL Trehalose dihydrate, 0.2 mg / mL Polysorbate 80, 0.05 mg / mL disodium EDTA, 0.1 mg / mL L-Methionine.

The compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen binding stretch of the invention. A "therapeutically effective amount" means an amount which, at dosages and for periods of time required, has the effect of achieving the desired therapeutic result. A therapeutically effective amount of the antibody or antigen-binding piece may vary depending on factors such as the disease condition, age, sex, and weight of the individual, and the ability of the antibody or antibody piece to elicit a desired response in the individual. . A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antigen binding stretch are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount which, at dosages and for periods of time required, has the effect of achieving the desired prophylactic result. Since a prophylactic dose is used in individuals before they are ill, or at an earlier stage of the disease, the prophylactically effective amount may typically be less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). E.g. a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally as indicated by the requirements of the therapeutic situation. It is especially preferred to formulate parenteral compositions in the form of a dosage unit for ease of administration and dosage uniformity. Dosage unit form as used herein denotes physically discrete units suitable as unitary dosages for the mammalian individuals to be treated; each unit containing a predetermined amount of active compound which, according to calculation, produces the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly depend on (a) the unique characteristics of the anti-ALK-1 antibody or piece thereof and the respective therapeutic or prophylactic effect to be achieved, and (b) the inherent limitations in the formulation of such an antibody for the treatment of susceptibility in individuals.

An example of a non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody piece of the invention is 0.025 to 50 mg / kg, more preferably 0.1 to 50 mg / kg, more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg / kg. In some embodiments, a formulation contains 5 mg / mL of antibody in a buffer

100 of 20mM sodium citrate, pH 5.5, 140mM NaCl, and 0.2mg / mL polysorbate 80. It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated. It should further be noted that for each particular individual, specific dosage regimens should be established over time according to the individual need and professional judgment of the person administering or supervising administration of the compositions, and that the dosage ranges shown herein are only as serve as an example and are not intended to limit the scope or practice of the claimed composition.

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

Diagnostic Methods of Use

The anti-ALK-1 antibodies or antigen-binding pieces thereof can be used in diagnostic methods for detecting ALK-1 in a biological sample in vitro or in vivo. E.g. the anti-ALK-1 antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot, or immunoprecipitation. The anti-ALK-1 antibodies of the invention can be used to detect ALK-1 in humans. The anti-ALK-1 antibodies can also be used

101 used to detect ALK-1 in other primates, e.g. cynomolgus monkeys.

The invention provides a method of detecting ALK-1 in a biological sample comprising contacting the biological sample with an anti-ALK-1 antibody of the invention and detecting the bound antibody. In one embodiment, the anti-ALK-1 antibody is directly labeled with a detectable label. In another embodiment, the anti-ALK-1 antibody (the first antibody) is unlabeled and a second antibody or other molecule capable of binding the anti-ALK-1 antibody is labeled. As is well known to those skilled in the art, a second antibody is selected which can specifically bind the particular species and class of the first antibody. For example, if the anti-ALK-1 antibody is a human IgG, the secondary antibody could be an anti-human IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are commercially available, e.g., from Pierce Chemical Co.

Suitable labels for the antibody or secondary antibody have been previously 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 umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrine; an example of a luminescent material includes luminol; and examples of suitable radioactive material ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In other embodiments, ALK-1 in a biological sample can be determined by a competition immunoassay using ALK-1 standards labeled with a detectable substance and an unlabeled anti-ALK-1 antibody. In this assay, the biological sample is labeled

102

ALK-1 standards and the anti-ALK-1 antibody are combined and the amount of labeled ALK-1 standard bound to the unlabeled antibody is determined. The amount of ALK-1 in the biological sample is inversely proportional to the amount of labeled ALK-1 standard bound to the anti-ALK-1 antibody.

The immunoassays described above can be used for a number of purposes. For example, the anti-ALK-1 antibodies can be used to detect ALK-1 in cultured cells. In a preferred embodiment, the anti-ALK-1 antibodies are used to determine the amount of ALK-1 produced by cells treated with various compounds. This method can be used to identify compounds that modulate ALK-1 protein levels. According to this method, one sample of cells is treated with a test compound for a time while another sample is left untreated. If the total level of ALK-1 is to be measured, the cells are lysed and the total ALK-1 level is measured using one of the immunoassays described above. The total level of ALK-1 in the treated versus untreated cells is compared to determine the effect of the test compound.

A preferred immunoassay for measuring total ALK-1 levels is flow cytometry or immunohistochemistry. Methods such as ELISA, RIA, flow cytometry, Western blot, immunohistochemistry, cell surface labeling of integral membrane proteins and immunoprecipitation are well known in the art. See, e.g., Harlow and Lane, supra. In addition, the immunoassays can be scaled up for high throughput screening to test a wide variety of compounds for activation or inhibition of ALK-1 expression.

The anti-ALK-1 antibodies of the invention can also be used to determine the levels of ALK-1 in a tissue or in tissue-derived cells. In some embodiments, the tissue is a diseased tissue. In some embodiments of the method, a

103 tissue or biopsy from a patient. The tissue or biopsy is then used in an immunoassay to determine e.g. total ALK-1 levels or localization of ALK-1 by the methods discussed above.

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

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

104

Therapeutic Methods of Use

In another embodiment, the invention provides a method of inhibiting ALK-1 activity by administering an anti-ALK-1 antibody to a patient in need. Any of the antibodies or antigen-binding pieces thereof described herein can be used therapeutically. In a preferred embodiment, the anti-ALK-1 antibody is a human, chimeric or humanized antibody. In another preferred embodiment, the anti-ALK-1 antibody is human antibody, and the patient is a human patient. Alternatively, the patient may be a mammal expressing 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., a cynomolgus monkey) for veterinary purposes or as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficiency of antibodies of this invention.

In another embodiment, an anti-ALK-1 antibody or antibody piece thereof can be administered to a patient expressing inappropriately high levels of ALK-1. The antibody can be administered once, but it is preferable to be administered several times. The antibody can be administered from three times a day to once every six months or longer. Administration may be on a schedule such as 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 every month, once every two months, once every three months and once every six months. The antibody can also be administered continuously through a mini pump. The antibody can be administered by a mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, or intratumor route. The antibody can be administered once, at least twice or for at least the time until the condition is treated, soothed or cured. The antibody will be in it

105 generally administered as long as the condition is present. The antibody will generally be administered as part of a pharmaceutical composition as described above. The antibody dosage will generally be in the range of 0.1 to 100 mg / kg, more preferably 0.5 to 50 mg / kg, more preferably 1 to 20 mg / kg, and even more preferably 1 to 10 mg / kg . The serum concentration of the antibody can be measured by any method known in the art.

In one embodiment, the antibody is administered in a formulation as a sterile aqueous solution having a pH ranging from about 5.0 to about 6.5 and containing about 1 mg / ml to about 200 mg / ml of antibody, about 1 millimolar to about 100 millimolar of histidine buffer, about 0.01 mg / ml to about 10 mg / ml polysorbate 80, about 100 millimolar to about 400 millimolar trehalose, and about 0.01 millimolar to about 1.0 millimolar of disodium EDTA dihydrate.

Furthermore, it is contemplated by the present invention that any of the compositions herein can be administered to a patient susceptible to or suffering from a condition associated with increased angiogenesis ("an angiogenic condition").

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

For example, the present invention relates to methods of treating or preventing conditions associated with ocular neovascularization using any of the compositions / methods herein. Conditions associated with ocular neovascularization include, but are not limited to, diabetic retinopathy, age-related macular

106 degeneration ("ARMD"), rubeotic glaucoma, interstitial keratitis, retinopathy of prematurity, ischemic retinopathy (eg sickle cell), pathological myopia, ocular histoplasmosis, pterygia, punitiate internal choroidopathy, and the like.

Treatment of Abnormal Cell Growth

This invention 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 stretch thereof, such as described herein, which is effective in the treatment of abnormal cell growth.

In one embodiment of this method, the abnormal cell growth is cancer, including, but not limited to, mesothelioma, liver bile (liver and bile duct), a primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC ), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal), breast cancer, uterine cancer, fallopian tube carcinoma, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the kidney pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, non hodgkins's lymphoma, spinal cord tumors, brain stem glioma pituitary adenoma, adrenocortical cancer, gallbladder cancer, multiple myeloma,

107 cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the previous cancers.

In a preferred embodiment of the present invention, the cancer is selected from lung cancer (NSCLC and SCLC), cancer of the head or neck, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, breast cancer, kidney or ureter cancer renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, non hodgkins's lymphoma, spinal cord tumors, or a combination of one or more of the previous cancers.

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

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

This invention also relates to a method of treating abnormal cell growth in a mammal comprising administering to said mammal an amount of an anti-ALK-1 antibody or antigen-binding portion thereof as described herein which is effective in the treatment of abnormal cell growth in combination with an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, antibiotic intercalation, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics, anti-hormones, and anti-androgens.

The invention also relates to a pharmaceutical composition for the treatment of abnormal cell growth in a mammal, including a human, comprising an amount of an anti-ALK-1 antibody or antigen-binding portion thereof as described herein, which is effective in the

108 treatment of abnormal cell growth in combination with a pharmaceutically acceptable carrier and an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalation antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, and anti-androgens.

The invention also relates to a method of treating a hyperproliferative disorder in a mammal comprising administering to said mammal a therapeutically effective amount of an anti-ALK-1 antibody or antigen binding stretch thereof as described herein, in combination with an anti-tumor agent selected from the group consisting of anti-proliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-I inhibitors, cox-II inhibitors, mitotic inhibitors, alkylating agents, anti-metabolites, antibiotic intercalation, growth factor inhibitors, radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics, anti-hormones, statins, and anti-androgens.

In one embodiment of the present invention, the anti-tumor agent has been used in conjunction with an anti-ALK-1 antibody or antigen binding portion thereof, and pharmaceutical compositions described herein, an antiangiogenesis agent, kinase inhibitor, pan kinase inhibitor or growth factor inhibitor. Preferred pan kinase inhibitors include Sutent (Pfizer Inc., SU11248), described in U.S. Patent No. 6,573,293 (Pfizer, Inc, NY, USA).

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

109

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 under PTK-787, ZK- 222584: Novartis &amp; Schering AG), Macugen (pegaptanib octosodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862 (Cytran Inc. of Kirkland, Washington, USA); and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California) and combinations thereof. VEGF inhibitors useful in the practice of the present invention are described in U.S. Patent Nos. 6,534,524 and 6,235,764, both of which are incorporated herein in their entirety for all purposes. Particularly preferred VEGF inhibitors include CP-547,632, AG13736, Vatalanib, Macugen and combinations thereof.

Additional VEGF inhibitors are described, for example, in WO 99/24440 (published May 20, 1999), PCT International Application PCT / IB99 / 00797 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995), WO 99/61422 ( published December 2, 1999), US Patent 6, 534,524 (describes AG13736), US Patent 5,834,504 (issued November 10

1998), WO 98/50356 (published November 12, 1998), US Patent 5,883,113 (issued March 16, 1999), US Patent 5,886,020 (issued March 23, 1999), US Patent 5,792,783 (issued August 11, 1998), US Patent No. US 6,653,308 (issued November 25, 2003), WO 99/10349 (published March 4, 1999), WO 97/32856 (published September 12, 1997), WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 3, 1997) 1998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8, 1999), and WO 98/02437 (published January 22, 1998), all of which are incorporated herein by reference in their entirety.

Other antiproliferative agents that can be used with the antibodies, or antigen-binding portions thereof, of the present invention include inhibitors of the enzyme famesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFr, including the compounds described and claimed in the following US Patent Applications:

110

09/221946 (filed December 28, 1998); 09/454058 (filed December 2

1999); 09/501163 (filed February 9, 2000); 09/539930 (filed March 31

2000); 09/202796 (filed May 22, 1997); 09/384339 (filed August 26, 1999); and 09/383755 (filed August 26, 1999); and the compounds described and claimed in the following US Provisional Patent Applications: 60/168207 (filed November 30, 1999); 60/170119 (filed December 10, 1999); 60/177718 (filed January 21, 2000); 60/168217 (filed November 30, 1999), and 60/200834 (filed May 1, 2000). Each of the foregoing Patent Applications and Provisional Patent Applications is hereby incorporated by reference in its entirety.

For additional PDGRr inhibitors, see W001 / 40217, published July 7, 2001 and W02004 / 020431, published March 11, 2004, the contents of which are incorporated herein in their entirety for all purposes. Preferred PDGFr inhibitors include Pfizer's CP-868,596 and its pharmaceutically acceptable salts.

Preferred GARF inhibitors include Pfizer's AG-2037 (pelitrexol and its pharmaceutically acceptable salts). GARF inhibitors useful in the practice of the present invention are disclosed in U.S. Patent No. 5,608,082, which is incorporated herein in its entirety for all purposes.

Examples of useful COX-II inhibitors that can be used in conjunction with an Anti-ALK-1 antibody or antigen binding portion thereof, as described herein, and pharmaceutical compositions described herein include CELEBREX ™ (celecoxib), parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189 (Lumiracoxib), BMS 347070, RS 57067, NS-398, Bextra (valdecoxib), paracoxib, Vioxx (rofecoxib), SD-8381, 4Methyl-2- (3,4-dimethylphenyl ) -1- (4-sulfamoyl-phenyl) -1H-pyrrole, 2- (4Ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole, T-614, JTE-522, S2474, SVT-2016, CT-3, SC-58125 and Arcoxia (etoricoxib). For additional COXII inhibitors, see U.S. Patent Application Nos. 10 / 801,446 and 10 / 801,429, the contents of which are incorporated herein in their entirety for all purposes.

111

In one preferred embodiment, the anti-tumor agent is celecoxib, see

US Patent No. 5,466,823, the content of which is incorporated herein by reference in its entirety for all purposes. The structure for Celecoxib is shown below

<img file="NL1032452C2_D0002.tif" />

^CF3 celecoxib

CAS Ho. 169590-42-5

5,466,823

C-2779

SC-50635

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

<img file="NL1032452C2_D0003.tif" />

valdecoxib

CAS NO. 181695-72-7

5,633,272

C-2865

SC-65872

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

<img file="NL1032452C2_D0004.tif" />

In one preferred embodiment, the anti-tumor agent is deracoxib, see 20 US Patent No. 5,521,207, the contents of which are incorporated by reference herein in their entirety for all purposes. The structure for deracoxib is shown below:

112

<img file="NL1032452C2_D0005.tif" />

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

<img file="NL1032452C2_D0006.tif" />

In one preferred embodiment, the anti-tumor agent is ABT-963, see 10 International Publication No. WO 2002/24719, the contents of which are incorporated by reference herein in its entirety for all purposes. The structure for ABT-963 is shown below:

<img file="NL1032452C2_D0007.tif" />

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

<img file="NL1032452C2_D0008.tif" />

In one preferred embodiment, the anti-tumor agent is MK-663 (etoricoxib), see International Publication No. WO 1998/03484, of which the

113 content is incorporated herein by reference in its entirety for all purposes. The structure for etoricoxib is shown below:

<img file="NL1032452C2_D0009.tif" />

In one preferred embodiment, the anti-tumor agent is COX-189 (Lumiracoxib), see International Publication No. WO 1999/11605, the contents of which are incorporated by reference herein in its entirety for all purposes. The structure for Lumiracoxib is shown below:

<img file="NL1032452C2_D0010.tif" />

Novartis WO 99/11605

In one preferred embodiment, the anti-tumor agent is BMS-347070, see U.S. Patent No. 6,180,651, the contents of which are incorporated by reference herein in their entirety for all purposes. The structure for BMS347070 is shown below:

<img file="NL1032452C2_D0011.tif" />

BMS 347070

CAS No. 197438-48-5 6,180,651

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

114

<img file="NL1032452C2_D0012.tif" />

NS-39S

CAS No. 123653-11-2

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

O

<img file="NL1032452C2_D0013.tif" />

RS 57067

CAS NO. 17932-91-3

In one preferred embodiment, the anti-tumor agent is 4-Methyl-2 (3,4-dimethylphenyl) -1- (4-sulfamoyl-phenyl) -1H-pyrrole. The structure for 410 Methyl-2- (3,4-dimethylphenyl) -1- (4-sulfamoyl-phenyl) -1H-pyrrole is shown below:

<img file="NL1032452C2_D0014.tif" />

In one preferred embodiment, the anti-tumor agent is 2- (415 Ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole. The structure for 2- (4

Ethoxyphenyl) -4-methyl-1- (4-sulfamoylphenyl) -1H-pyrrole is shown below:

<img file="NL1032452C2_D0015.tif" />

so₂nh₂

115

In one preferred embodiment, the anti-tumor agent is meloxicam. The structure for meloxicam is shown below:

<img file="NL1032452C2_D0016.tif" />

Other useful inhibitors as anti-tumor agents used in conjunction with antibodies of the present invention and pharmaceutical compositions described herein include aspirin, and non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit the enzyme that makes prostaglandins (cyclooxygenase I and II), resulting in lower levels of prostaglandins, including but not limited to the following,

Salsalate (Amigesic), Diflunisal (Dolobid), Ibuprofen (Motrin), Ketoprofen (Orudis), Nabumetone (Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn), Diclofenac (Voltaren), Indomethacin (Indocin) ), Tolmetin (Tolectin), Etodolac (Lodine), Ketorolac (Toradol), Oxaprozin (Daypro) and combinations thereof. Preferred COX-I inhibitors include ibuprofen (Motrin), nuprin, naproxen (Aleve), indomethacin (Indocin), nabumetone (Relafen) and combinations thereof.

Targeted agents used in conjunction with an anti-ALK-1 antibody or antigen binding stretch thereof, as described herein, and pharmaceutical compositions thereof as described herein include EGFr inhibitors such as Iressa (gefitinib, AstraZeneca), Tarceva (erlotinib or OSI-774 ,

OSI Pharmaceuticals Inc.), Erbitux (cetuximab, Imclone Pharmaceuticals, Inc.), EMD-7200 (Merck AG), ABX-EGF (Amgen Inc. and Abgenix Inc.), HR3 (Cuban Government), IgA antibodies (University of Erlangen25 Nuremberg), TP-38 (IVAX), EGFR fusion protein, EGF vaccine, anti-EGFr immunoliposomes (Hermes Biosciences Inc.) and combinations thereof.

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

116

The present invention also relates to anti-tumor agents selected from pan erb receptor inhibitors or ErbB2 receptor inhibitors, such as CP-724,714 (Pfizer, Ine.), Cl-1033 (canertinib, Pfizer, Ine.), Herceptin (trastuzumab, Genentech Inc.), Omitarg (2C4, pertuzumab, Genentech Ine.), TAK-165 (Takeda), GW-572016 (lonafamib, GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth), PKI-166 (Novartis ), dHER2 (HER2 Vaccine, Corixa and GlaxoSmithKline), APC8024 (HER2 Vaccine, Dendreon), anti-HER2 / neu bispecific antibody (Decof Cancer Center), B7.her2.IgG3 (Agensys), AS HER2 (Research Institute for Rad Biology &amp; Medicine), tri-functional bispecific antibodies (University of Munich) and mAB AR-209 (Aronex Pharmaceuticals Inc) and mAB 2B-1 (Chiron) and combinations thereof. Preferred erb selective anti-tumor agents include Herceptin, TAK-165, CP-724,714, ABX-EGF, HER3 and combinations thereof. Preferred pan erbb receptor inhibitors include GW572016, CI-1033, EKB-569, and Omitarg and combinations thereof.

Additional erbB2 inhibitors include those in WO 98/02434 (published January 22, 1998), WO 99/35146 (published July 15, 1999), WO 99/35132 (published July 15, 1999), WO 98/02437 (published January 22, 1998), WO 97/13760 (published April 17, 1997), WO 95/19970 (published July 27, 1995), US Patent 5,587,458 (issued December 24, 1996), and US Patent 5,877,305 (issued March 2, 1999), each of which is herein incorporated by reference in their are completely included. For additional ErbB2 receptor inhibitors useful in the present invention, see U.S. Patent Nos. 6,465,449, and 6,284,764, and International Application No. WO 2001/98277, each of which is herein incorporated by reference in its entirety.

In addition, other anti-tumor agents may be selected from the following agents, Sorafenib (Onyx Pharmaceuticals Inc .; BAY-43-9006), Genasense (Augmerosen, Genta), Panitumumab (Abgenix / Amgen), Zevalin (Schering), Bexxar (Corixa / GlaxoSmithKline), Abarelix, Alimta, EPO 906 (Novartis), Discodermolide (XAA-296), ABT-510 (Abbott), Neovastat (Aetema), etcastaurin (Eli Lilly), Combrestatin A4P (Oxigene), ZD-6126 (AstraZeneca ),

117 flavopiridol (Aventis), CYC-202 (Cyclacel), AVE-8062 (Aventis), DMXAA (Roche / Antisoma), Thymitaq (Eximias), Temodar (Temozolomide, Schering Plow) and Revilimd (Occasion) and combinations thereof.

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

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

Further anti-tumor agents can be selected from the following agents, CeaVac (CEA), NeuTrexin (trimetresate glucuronate) and combinations thereof. Additional anti-tumor agents can be selected from the following agents, OvaRex (oregovomab), Osidem (IDM-1), and combinations thereof. Additional anti-tumor agents can be selected from the following agents, Advexin (ING 201), Tirazone (tirapazamine), and combinations thereof. Additional anti-tumor agents can be selected from the following agents, RSR13 (efaproxiral), Cotara (1311 chTNT 1 / b), NB1-3001 (IL-4) and combinations thereof. Additional anti-tumor agents can be selected from the following agents, Canvaxin, GMK vaccine, PEG Interon one, Taxoprexin (DHA / paclitaxel) and combinations thereof. Other preferred anti-tumor agents include Pfizer's MEK1 / 2 inhibitor PD325901, Array Biopharm's MEK inhibitor ARRY-142886, Bristol Myers' CDK2 inhibitor BMS387,032, Pfizer's CDK inhibitor PD0332991 and AstraZeneca's AXD-5438 and combinations thereof. In addition, mTOR inhibitors can also be used such as CCI-779 (Wyeth) and rapamycin derivatives RAD001 (Novartis) and AP118

23573 (Ariad), HD AC inhibitors SAHA (Merck IncJAton Pharmaceuticals) and combinations thereof. Additional anti-tumor agents include Aurora 2 inhibitor VX-680 (Vertex), Chkl / 2 inhibitor XL844 (Exilixis).

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

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

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

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

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

The following topoisomerase I inhibitors can be used as anti-tumor agents: camptothecin, irinotecan HCl (Camptosar), edotecarin,

119 orathecin (Supergen), exatecan (Daiichi), BN-80915 (Roche) and combinations thereof. Particularly preferred toposimerase II inhibitors include epirubicin (Ellence).

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

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

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, cytarabine ocphosphate, enocitabine, Slrexed, Timrex , LY231514, MTA), Gemzar (gemcitabine, Eli Lilly), fludarabin, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflomithine, ethynylcytidine, cytosine arabinoside, hydroxyurea, TS-1, melphalan, nelarabine, nolatrexed, ocphosphate, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, vinorelbine; or, for example, one of the preferred anti-metabolites described in European Patent Application No. 239362 such as N- (5- [N- (3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl) -Nmethylamino] -2- thenoyl) -L-glutamic acid and combinations thereof.

120

Antibiotics include antibiotic intercalation but are not limited to: aclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, bleomycin, daunorubicin, doxorubicin, elsamitrucine, epirubicin, galarubicin, idarubicin, mitomubicin cinepomubycin C, pepomubycin C, pepinomycin streptozocin, valrubicin, zinostatin and combinations thereof.

For example, plant-derived anti-tumor substances include those selected from mitotic inhibitors, for example, vinblastine, docetaxel (Taxotere), paclitaxel, and combinations thereof.

Cytotoxic topoisomerase inhibitors include one or more agents selected from the group consisting of aclarubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan HCl (Camptosar), edotecarine, epirubicin (Ellirocicin) gimatecan, lurtotecan, mitoxantrone, pirarubicin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan, and combinations thereof.

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), etoposide, SN-38, topotecan, and combinations .

Immunological agents include interferons and numerous other immune enhancers. Interferons include interferon alfa, interferon alfa-2a, interferon, alfa-2b, interferon beta, interferon gamma-la, interferon gamma-lb (Actimmune), or interferon gamma-nl and combinations thereof. Other means include filgrastim, lentinan, sizofilan, TheraCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograstim, lentocin OncoVAXCL, sargramostim, tasonermin, tecleukin, thymalasin, tositumomab, Virulizin,

121

Z-100, epratuzumab, mitumomab, oregovomab, pemtumomab (Y-muHMFGl), Provenge (Dendreon) and combinations thereof.

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

Other anti-cancer agents include alitretinoin, ampligen, atrasentan bexarotene, bortezomib. Bosentan, calcitriol, exisulind, finasteride, photemustine, ibandronic acid, miltefosine, mitoxantrone, 1asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazarotne, Telcyta (TLK-286, Telik Ine.), Velcade and combinations thereof.

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

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

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

Other anti-tumor agents include mitoxantrone, 1-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pentostatin, tretinoin and combinations thereof.

Anti-tumor agents that can enhance anti-tumor immune responses, such as CTLA-4 (cytotoxic lymphocyte antigen 4) antibodies, and other agents that can block CTLA-4 can also be used, such as MDX-010 (Medarex) and CTLA-4 compounds described in US Patent No. 6,682,736; and anti-proliferative agents such as other famesyl protein transferase inhibitors, for example the famesyl protein transferase

122 inhibitors. For additional specific CTLA-4 antibodies that can be used in the present invention, see U.S. Patent Application 60 / 113,647 (filed December 23, 1998), U.S. Patent No. 6,

682,736, both of which are hereby incorporated by reference in their entirety. For example, another anti-CTLA-4 antibody that can be used in accordance with the present invention is ticilimumab, which truncates the sequence of monoclonal antibody 11.2.1 in US Patent 6,682,736.

For specific IGF1R antibodies that can be used in the present invention, see International Patent Application No. WO 2002/053596, which is hereby incorporated by reference in its entirety.

For specific CD40 antibodies that can be used in the present invention, see International Patent Application No. WO 2003/040170, which is hereby incorporated by reference in its entirety.

Gene therapy agents can also be used as anti-tumor agents such as TNFerade (GeneVec), which express TNFalfa in response to radiotherapy.

In one embodiment of the present invention, statins can be used in conjunction with an Anti-ALK-1 antibody or antigen binding portion thereof, as described herein, and pharmaceutical compositions thereof. Statins (HMG-CoA reducatase 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 Ine .), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin and Niacin (Advicor, Kos Pharmaceuticals), derivatives and combinations thereof.

In a preferred embodiment, the statin is selected from the group consisting of Atovorstatin and Lovastatin, derivatives and combinations thereof.

Other agents useful as anti-tumor agents include Caduet.

For any of the methods of treatment of a hyperproliferative disorder or abnormal cell growth as described herein using

123 a combination of an anti-ALK-1 antibody or antigen binding stretch with at least one additional therapeutic agent, the anti-ALK-1 antibody can be conjugated, or derivatized, with the additional therapeutic agent. The at least one additional therapeutic agent can also be administered separately, or in a non-derivatized or non-conjugated manner. When the at least one additional therapeutic agent is not derivatized or conjugated with the antibody, it can be administered in the same pharmaceutical formulation as the antibody, or it can be administered in a separate formulation.

Treatment of Vision Loss

The compounds of the invention and pharmaceutical compositions containing them are useful for treating severe 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, retinitis pigmentosa, and the like.

For example, the compositions of the invention can be used to form a drug depot behind the eye and may comprise one or more pharmaceutically active agents in addition to one or more inactive excipients as described herein. Examples of pharmaceutically active agents useful in the compositions of the invention include anti-infectives, including, without limitation, antibiotics, antivirals, and antifungals; antiallergenic agents and mast cell stabilizers; steroidal and nonsteroidal anti-inflammatory drugs (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, without limitation, adrenergics, beta-adrenergic blocking agents, alpha-adrenergic agonists, parasypathomimetic agents, cholinesterase inhibitors, carbonic acid

124 anhydrase inhibitors, and prostaglandins; combinations of anti-glaucoma agents; antioxidants; dietary supplements; drugs for the treatment of cystoid macular edema including, without limitation, non-steroidal anti-inflammatory agents; drugs for the treatment of age-related macular degeneration (AMD) including nonexudative (dry) and exudative (wet) AMD, including, without limitation, angiogenesis inhibitors, including angiogenesis inhibitors that inhibit protein kinase receptors, including protein kinase receptors that are VEGF receptors; and nutritional supplements; medicines to treat herpetic infections and CMV ocular infections; drugs for the treatment of proliferative vitreoretinopathy including, without limitation, antimetabolites and fibrinolytics; wound modulation agents, including, without limitation, growth factors; anti-metabolites; neuroprotective drugs, including, without limitation, eliprodil; and angiostatic steroids for the treatment of posterior segment 26 diseases or conditions, including, without limitation, age-related macular degeneration (AMD) including nonexudative (dry) and exudative (wet) AMD, choroidal neovascularization, retinopathies, retinitis, uveitis, macular edema, and glaucoma. For additional information on such angiostatic steroids, see U.S. Patent Nos. 5,679,666 and 5,770,592. A non-steroidal anti-inflammatory agent for the treatment of cystoid macular edema is nepafenac.

For administration to the eye, a compound of the present invention is delivered in a pharmaceutically acceptable ophthalmic carrier such that the compound is kept in contact with the ocular surface for a sufficient time to allow the compound to penetrate the cornea and / or the eye skirt and internal areas of the eye, including, for example, the anterior chamber, the posterior chamber, the vitreous, aqueous humor, vitreous humor, cornea, iris / ciliary, lens, choroid / retina and eye skirt. The pharmaceutically acceptable

125 ophthalmic carrier can be an ointment, vegetable oil, or an encapsulation material. A compound of the invention can also be injected directly into the vitreous humor or aqueous humor.

Furthermore, a compound can also be administered by well known, acceptable methods, such as sub-Tenon and / or subconjunctival injections. As known in the ophthalmic field, the macula primarily includes retinal cones and is the region of maximum visual acuity in the retina. A Tenon's capsule or Tenon's membrane is located on the eye skirt. A conjunctiva covers a short area from the bulb from the posterior eye to the limbus (the bulbar conjunctiva) and folds up (the upper cul-de-sac) or out (the lower cul-de-sac) to the inner regions of resp. . cover the upper eyelid and lower eyelid. The conjunctiva is on top of Tenon's capsule. The eye skirt and Tenon's capsule define the exterior surface of the eyeball. For the treatment of ocular diseases such as age-related macular degeneration (AMD) including nonexudative (dry) and exudative (wet) AMD, choroidal neovascularization, retinopathies (such as diabetic retinopathy, prematurity retinopathy), diabetic macular edema, retinitis, uveitis, cystoid macular edema (CME), glaucoma, and other diseases or conditions of the posterior segment of the eye, it is preferred to leave a deposit of a specific amount of an ophthalmically acceptable pharmaceutically active agent directly on the outer surface of the eye skirt and below Tenon's capsule. In addition, in cases of age-related macular degeneration (AMD) including nonexudative (dry) and exudative (wet) AMD and CME, it is highly preferable to deposit directly on the outer surface of the eye skirt, below Tenon's capsule, and generally above leave the macula behind.

The compounds can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) intramuscular injection or by the above-mentioned Sub-Tenon or intravitreal injection.

126

Alternatively, the active ingredient can be in powder form for constitution with a suitable carrier, e.g. sterile pyrogen-free water, before use.

In particularly preferred embodiments of the invention, the compounds can be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in the form of eye drops. The solution or suspension can be prepared in its pure form and administered several times a day. Alternatively, the present compositions prepared as described above can also be administered directly to the cornea.

In preferred embodiments, the composition is prepared with a mucoadhesive polymer that binds to the cornea. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

A pharmaceutical carrier for hydrophobic compounds is a cosolvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system can be a VPD cosolvent system. VPD is a solution of 3% w / v benzyl alcohol, 8% w / v of the non-polar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300, by volume in absolute ethanol. The VPD cosolvent system (VPD: 5W) contains VPD diluted 1: 1 with a 5% dextrose in water solution. This cosolvent system dissolves hydrophobic compounds well, and itself produces low toxicity when administered systemically. Of course, the proportions of a cosolvent system can be varied considerably without destroying its solubility and toxicity properties. Furthermore, the identity of the cosolvent components can be varied: e.g. other low-toxicity non-polar surfactants may be used in place of polysorbate 80; the fraction size of polyethylene glycol can be varied; may be other biocompatible

127 polymers replace the polyethylene glycol, e.g. polyvinylpyrrolidone; and other sugars or polysaccharides can be substituted for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be used. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide can also be used, although usually at the cost of greater toxicity. In addition, the compounds can be delivered using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Several sustained release materials have been established and are known to those skilled in the art. Sustained-release capsules, depending on their chemical nature, can release the compounds for several weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional protein stabilization strategies can be used.

The pharmaceutical compositions may also include 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.

Any of the compositions can be formulated for administration to an individual. Preferably, an individual of the present invention is a mammal, or more preferably a human.

The pharmaceutical formulations herein may further comprise a therapeutic agent selected from the group consisting of: an antineoplastic agent, an anti-inflammatory agent, an antibacterial agent, an antiviral agent, an angiogenic agent, and an anti-angiogenic agent. Examples of such agents are described herein.

128

For example, an antineoplastic agent can 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; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Eddatrexate; Eflomithine Hydrochloride;

Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized oil 1131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Phloxuridine; Fludarabine phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxy urea; Idarubicin Hydrochloride; Ifosfamide; Imofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta-la; Interferon Gamma-Ib; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin

129

Sulfate; Perphosphamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porphimer sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;

Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl, Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;

Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribin Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate;

Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.

Anti-angiogenic agents are any agents that inhibit angiogenesis, whether described herein or known in the art. In preferred embodiments, an anti-angiogenic agent is an anti-VEGF agent, such as

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

Pharmaceutical compositions can be formulated by standard techniques using one or more suitable carriers, excipients, and diluents. See kijv. Remington's Pharmaceutical Sciences, (19^thEd. Williams &amp; Wilkins, 1995) (incorporated herein by all-purpose reference).

Formulations suitable for parenteral administration include aqueous and non-aqueous formulations that are isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may comprise suspending systems designed to direct the compound to blood components or one or more organs. The formulations can be presented in unit dose or multi-dose closed

130 containers, for example, ampoules or vials. Unit dosages are preferred for intraocular formulations because the formulation does not contain preservatives. Preservative may be used for other parenteral formulations which would allow multi-dose containers.

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

Particular parenteral administrations contemplated by the present invention include intraocular and intravitreal ocular administrations. Pharmaceutical formulations for intraocular and intravitreal administrations include phosphate buffered saline (PBS) and balanced isotonic saline (BSS) with or without excipients such as mannitol or sorbitol as protein stabilizers.

Generally, water, suitable oil, saline, aqueous dextrose (glucose), or related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizers and, if necessary, buffering agents. Antioxidants, such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Also used are citric acid salts, or sodium EDTA. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, or chlorobutanol. Suitable pharmaceutical carriers are described in Remington, cited supra.

In any of the embodiments herein, a composition or pharmaceutical formulation herein may be lyophilized.

In any of the embodiments herein, the pharmaceutical formulations preferably have less than about 10, more preferably less

131 than about 5, more preferably less than about 3, or more preferably less than about 1 endotoxin unit (s) per milligram of therapeutic agents.

in some embodiments, the methods of treatment described herein further comprise administering to an individual suffering from an angiogenic condition one or more therapeutic agents selected from the group consisting of antineoplastic agents, antiviral agents, anti-inflammatory agents, antibacterial agents, angiogenic agents, or antiangiogenic agents.

Such combination treatments can be accomplished either by administering to an individual a co-formulation of the compositions herein with the additional therapeutic agent (s) or by administering the compositions herein and the therapeutic agent (s) as two separate pharmaceutical formulations. In embodiments in which more than one composition / therapeutic agent is administered to an individual, lower dosages of the compositions and / or therapeutic agent (s) may be used due to the synergistic effect of both active ingredients.

Antineoplastic agents that can be administered to an individual include, but are not limited to Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Antramycin; 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; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene

132

Citrate; Dromostanolone Propionate; Duazomycin; Eddatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized oil 1131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;

Fenretinide; Phloxuridine; Fludarabine phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxy urea; Idarubicin Hydrochloride; Ifosfamide;

Imofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl;

Interferon Alfa-n3; Interferon Beta-la; Interferon Gamma-Ib; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Meiengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perphosphamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porphimer sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl, Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribin Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;

133

Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.

Antibacterial agents that can be administered to an individual include, but are not limited to, penicillins, aminoglycosides, macrolides, monobactams, rifamycins, tetracyclines, chloramphenicol, clindamycin, lincomycin, imipenem, fusidic acid, novobiocin, phosphomycin, fusinycininate polymyxinin, sodium, neinocycin , colistimethate, colistin, gramicidin, minocycline, doxycycline, vanomycin, bacitracin, kanamycin, gentamycin, erythromicin and cephalosporins.

Anti-inflammatory agents that can be administered to an individual include, but are not limited to NSAIDS (eg aspirin (salicylamide), sodium salicylamide, indoprofen, indomethacin, sodium indomethacin trihydrate, Bayer ™, Bufferin ™, Celebrex ™, diclofenac, Ecotrin ™, diflunisal, fenoprofen, naproxen, sulindac, Vioxx ™), corticosteroids or corticotropin (ACTH), colchicine, and anecortave acetate.

Antivirals that can be administered to an individual include, but are not limited to, α-methyl-β-adamantane methylamine, 1, D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9- [2-hydroxyethoxylmethylguanine, adamantanamine , 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, CD4, 3'-azido-3'deoxythymidine (AZT), 9- (2-hydroxyethoxymethyl) -guanine (acyclovir), phosphonomic acid, 1-adamantanamine, peptide T, and 2 ', 3'-dideoxycytidine.

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

For example, compositions of the present invention can be administered systemically or locally by any means known in the art (e.g., oral, intraocular, intravascular (iv), intradermal, intramuscular, transdermal, transmucosal, enterical, parental, by inhalation spray, rectal, or topical)

134 dosage unit formulations and containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.

As used herein, the term intraocular includes intravitreal, subretinal, and the like.

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

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

For systemic administration, the anti-ALK-1 antibody or antigen-binding portion thereof of the present invention and / or one or more additional therapeutic agents are preferably administered at a dose 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 embodiments, the polypeptides (preferably dimers or homodimers) and / or small molecules herein are administered systemically at a dose of 0.1-100 mg / kg, more preferably 0.5-50 mg / kg, more preferably 130 mg / kg body weight , or more preferably 5-20 mg / kg.

For localized administration, the anti-ALK-1 antibody or antigen-binding portion thereof of the present invention and / or one or more additional therapeutic agents are preferably administered at a dose of at least 50 pg, 100 pg, 150 pg, 200 pg , 250 pg, 300 pg, 350 pg, 400 pg, 450

135 pg, 500 pg, 550 pg, 600 pg, 650 pg, or 700 pg. In other embodiments, the polypeptides (preferably dimers or homodimers) and / or small molecules herein are administered locally at a dose of 50-1000 pg, more preferably 100-800 pg, more preferably 200-500 pg, or even more preferably 300 -400 pg per place.

For example, for dermal administration, the anti-ALK-1 antibody or antigen-binding portion thereof of the present invention and / or peptidomimetics and / or one or more additional therapeutic agents is administered at a dose of 50-1000 pg / cm², more preferably 100-800 pg / cm², or more preferably 200-500 pg / cm². In another example, for ocular administration, the polypeptides and / or peptidomimetics and / or small molecules of the present invention are administered at a dose of 50-1000 pg / eye, more preferably 100-800 pg / eye, or more preferably 200-500 pg / eye.

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

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

136

In terms of weight percent, the formulations of the present invention will preferably comprise the active agent, e.g. an anti-ALK-1 antibody, in an amount from about 0.0001 to about 10 wt%, more preferably from about 0.001 to about 1 wt% %, more preferably from about 0.05 to about 1 wt%, or more preferably from about 0.1 wt% to about 0.5 wt%.

Gene Therapy

The nucleic acid molecules encoding the antibodies and antibody pieces of the present invention can be administered to a patient in need thereof through gene therapy. Therapy can be in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding a heavy chain and a light chain are administered to a patient. In a more preferred embodiment, the nucleic acid molecules are administered such that they stably integrate into chromosomes of B cells, because these cells are specialized for the production of antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and transplanted back into a patient in need. In another embodiment, precursor B cells or other cells are infected in vivo using a virus known to infect the cell type of interest. Typical vectors used for gene therapy include liposomes, plasmids, and viral vectors. Examples of viral vectors are retroviruses, adenoviruses and adeno-associated viruses. Following in vivo or ex vivo infection, levels of antibody expression can be monitored by taking a sample from the treated patient and using an immunoassay known in the art or discussed herein.

In a preferred embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain, or an antigen-binding portion thereof, of an anti-ALK-1

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

Method for Screening ALK-1 Antagonists or Agonists

In one embodiment, the present invention provides a method of determining whether a substance inhibits upregulation of a specific downstream target gene of ALK-1, Id1, such as, for example, the Taqman Assay for Idl described in Example 12. The method comprises contacting a sample of cells expressing Id1 with the substance and determining whether Id1 expression is inhibited, in which a reduced level of Id1 expression in the sample of cells contacted with the substance, compared to a control sample of cells is indicative of inhibition of Id1 expression by said substance. In one specific embodiment, the substance is an antibody that binds to the extracellular domain of ALK-1. In another embodiment, the substance is a small molecule. According to the invention, the cells can inherently express both ALK-1 and Id1, such as HUVECs described in Example 12, or which have been transformed or transfected with DNA encoding one or both thereof.

138

Expression of Idl can be determined, e.g., using the Taqman Assay for Idl described in Example 12.

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

In order to better understand this invention, the following examples are given. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

Examples

In the following examples and preparations, "MW" means molecular weight; "His-Tag" means C-terminal polyhistidine (6xHis) tag for rapid purification with nickel chelating resin and detection with an anti-His (C-term) antibody; “BSA means bovine serum albumin;

"EDTA" means ethylenediamine tetraacetic acid; "DMSO" means dimethyl sulfoxide; "MOPS" means 3- (N-morpholino) propanesulfonic acid; "MES" means 2- (N-Morfolino) ethanesulfonic acid; "PBS" means phosphate buffered saline; "DPBS" means Dulbecco's phosphate buffered saline; "HEMA" means 2-hydroxyethyl methacrylate; "DMEM" means Dulbecco's modified eagle's medium; "FBS" means fetal bovine serum; "NEAA" means non-essential amino acids; "HEPES" means N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; and "DMF" means dimethylformamide.

Example 1. ALK-1 Immunogenic Preparation

The ECD of ALK-1 was cloned from full-length human ALK-1 ORF clone (Invitrogen, clone ID IOH21048) by PCR using the forward 5'-ACGGCCCAGCCGGCGGACCCTGTGAAGGCGTCT (SEQ ID NO: 96) and reverse 5'ACTAAGGGGTGTCTGGGGTGATGGGTGTGATGGGTGTGATGGGGGGTGATGGGGG

139

ID NO: 97) primers. The PCR product was purified, treated with the Sfil and Hind III restriction enzymes, and cloned into the Sfil / Hind III site of a mammalian expression vector pSecTag2 / Hygro (Invitrogen Inc., Catalog No. V910-20). The clone was used for transient transfection of 293T cells with Fugene 6 transfection reagent (Roche Applied Science, Catalog No. 1814443) according to the manufacturer's instructions. Cell culture supernatant containing the secreted target protein was harvested 72 hours post-transfection and allowed to bind to Ni-NTA resin (QIAGEN, Catalog No. 30430) overnight at 4 ° C. 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 buffer containing 20 mM Tris pH 8.0, 300 mM imidazole and 300 mM sodium chloride. A CM Sepharose cation exchange resin was used to further purify the protein in 20 mM sodium phosphate (pH 7.0), and the unbound fraction containing the target protein was collected. The protein was buffered for PBS or 10 mM HEPES, pH 7.4, plus 150 mM sodium chloride by dialysis and concentrated to 0.2-1 mg / mL with&gt; 90% final purity, assessed by SDS PAGE gel stained with Coomassie blue. The ALK-1 ECD His-Tag protein was heavily glycosylated with an apparent MW of 26 KDa, compared to a theoretical MW for the 11 KDa protein. The ALK-1 ECD His-Tag protein (SEQ ID NO: 98) has been used to generate hybridomas producing anti-ALK-1 antibody as described in Example 2.

Human ALK-1 ECD His-Tag protein:

gene sequence (the lowercase part is the secretion signal): atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgcggccc agccggccGACCCTGTGAAGCCGTCTCGGGGCCCCGCTGGTGACCTGCACGTG

TGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACA

GTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCT

GCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGT

140

CAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGC TGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCA GCATCATCATCATCATCAT (SEQ ID NO: 99) protein sequence:

DPVKPSRGPLVTCTCESPHCKGPTCRGAWCTWLVREEGRHPQEHR GCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDG QHHHHHH (SEQ ID NO: 98)

Example 2. Formation of Hybridomas Producing Anti-ALK-1 Antibody

Eight to ten week old XENOMOUSE® mice were immunized in the foot pads of their hind legs with 10 µg / mouse of either recombinant human ALK-1 / Fc chimera (R&amp;D Systems, Ine.,

Catalog No. 370-AL) or with the ALK-1 ECD His-Tag protein described in Example 1. This dose was repeated five to seven times over a three to five week period. Mice were given a final injection of the immunogen in PBS three or four days before fusion. The lymph node lymphocytes from immunized mice were fused to the non-secretory myeloma P3-X63-Ag8.653 cell line via electrocell fusion (ATCC Cat. CRL 1580), and these fused cells were subjected to HA-DMEM selection as described previously (DMEM / 15% FBS / 1% 200 mM L-glutamine / 1% 100X Non-essential amino acids / 1% 100X Pen / Strep / 10 U / ml IL-6/1 vial / liter OPI media supplement plus 0.5x HA (Azaserine-Hypoxanthine, Sigma, Cat. # A9666)). A panel of hybridomas was collected which secretes all ALK-1 specific human IgG2 antibodies.

An ELISA assay was used to detect antibody binding. Immunogen was coated overnight at 4 ° C with 4pg / mL in 50 mM sodium bicarbonate buffer on the 96-well Immulon microtiter plate (NUNCImmuno ™ plate MaxiSorp ™ surface, Nalge Nunc International, Cat. No. 4339454). Plates were washed and then blocked with PBS

141 with the addition of 0.1% Tween-20 and 0.5% bovine serum albumin. Antibodies were added to the blocked ELISA plates, incubated for 1 hour and washed with PBS with Tween-20. The binding was detected by anti-human IgG horseradish peroxidase (Pierce, Catalog No. 31420) followed by the addition of ABTS (Pierce, Catalog No. 37615). Colorimetric measurements were performed at 405 nm in a micro-plate reader (SpectraMax Plus 384, Molecular Devices).

Twenty-five hybridomas were selected for further study. These were cloned single cell by limiting dilution and were digested with 1.11.1; 1.12.1; 1.12.1 (rWT); 1.12.1 (M29I / D19A); 1.12.KM29I);

1.12.KD19A); 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.

Mouse Hybridoma Cell Line LN 15916 (the hybridoma 1.12.1) was deposited on June 21, 2005 under the Budapest Convention terms with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209. The hybridoma 1.12.1 is assigned the following Access Number: PTA-6808.

Example 3. Sequences of Anti-ALK-1 Antibodies

To analyze the structure of antibodies produced according to the invention, nucleic acids encoding heavy and light chain fragments from anti-ALK-1 monoclonal antibody-producing hybridomas were cloned. Cloning and sequencing were performed by standard means.

Poly (A)⁺ mRNA was isolated using a Fast-Track ™ kit (Invitrogen) from approximately 2 X 10⁵ hybridoma cells for each of the ALK-1 antibodies. cDNA was synthesized from the mRNA using random primers. The random primed cDNA was amplified by PCR using human Vh or human Vk family specific variable domain primers together with primers specific for human

142

Cy2 constant region, or a Ck constant region, to amplify the variable region of the antibody including all framework regions (FRs) and complementarity determining regions (CDRs). Nucleic acid sequences encoding human heavy and kappa light chain transcripts from the anti-ALK-1 producing hybridomas were obtained by direct sequencing of both strands of PCR products. Sequences were analyzed using software owned by Abgenix and publicly available sequence information for human VH and Vk genes, the "V BASE sequence directory", Tomlinson et al., MRC Center for Protein Engineering, Cambridge, UK). Identical results could be obtained by one skilled in the art using publicly available sequence alignment software using MacVector and Geneworks software programs.

Specifically, full-length ALK-1 antibody 1.12.1 was cloned into expression vectors as follows: Poly (A)⁺ mRNA was isolated using an RNeasy Mini Kit (Qiagen) and cDNA synthesized from the mRNA with the Advantage RT-for-PCR kit (BD Biosciences) using oligo (dT) priming. The oligo (dT) primed clone cDNA

1.12.1 was amplified using primers listed in Table 2. Amplification was performed using the Pfu Ultra polymerase (Stratagene) and a PTC-200 DNA Engine (MJ Research) with the following cycles: 3 '@ 95 ° C; 25x (20 "@ 95 ° C, 30" @ 52 ° C, l'20 "@ 72 ° C); 10 ° @ 72 ° C. Clones were sequence verified using Grills 16^thBDTv3.1 / dGTP chemistry (Applied Biosystems Ine) and a 3730x1 DNA Analyzer (Applied Biosystems Ine). In the method of cloning 1.12.1 Vh, the 8^e codon introduced a silent mutation, converting “GGC” into a “GGT.” All sequences were analyzed by alignments with the * V BASE sequence director} / (Tomlinson, et al, J. Mol. Biol., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-B60 ( 1994); EMBO J., 14, 4628-4638 (1995).)

143

Table 2

Heavy and Light Chain Amplification Primers Used for Full-Length Cloning 1.12.1

<td>Primer Name</td><td>Primer Sequence</td><td>SEQ ID NO</td>

<td> 4-61</td><td>5 'tcttcaaRcttRatatctctagaaficcgccaccATGAAACACCTGTGGTTCTTCCTCC 3'</td><td> 105</td>

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

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

<td>K_FL_R</td><td>5 ' ttctttgatcagaattctcaCTAACACTCTCCCCTGTTGAAGCTCTTT G3 '</td><td> 108</td>

Lowercase miet hybridizing bases

Example 4. Gene Use Analysis and CDR Analysis

From the nucleic acid sequence and predicted amino acid sequence of the antibodies, the gene use for each antibody chain was identified. Table 3 shows the gene use of selected hybridoma clones of antibodies of the invention.

Table 3

Heavy and Light Chain Gene Use

<td>Clone</td><td colspan="4">Heavy Chain Germline</td><td colspan="3">Kappa Light Chain Germline</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>LI</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>

144

<td>Clone</td><td colspan="4">Heavy Chain Germline</td><td colspan="3">Kappa Light Chain Germline</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> 1.9.1</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> 5.34.1</td><td> 73</td><td> 4-31</td><td></td><td>JH6B</td><td> 75</td><td>Already</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>JII6B</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>

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

Table 4

Mutagenic Oligonucleotides (Sequences 5 * to 3 '):

<td>Primer</td><td>Sense</td><td>Antisense</td>

<td>1.12.1 (D19A)</td><td>CTCCAGGGGAAAGAGCCACCCTCTCCTGTA GG (SEQ ID NO: 109)</td><td>CCTACAGGAGAGGGTGGCTCTTTGCCCTGG AG (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>

Mutations are in bold and underlined.

Nucleic acid molecules encoding the heavy chain variable domain (SEQ ID NO: 5) and the light chain variable domain (SEQ ID: 7)

145 chain of the 1.12.1 (M29I / D19A) antibody were deposited on July 14, 2005 under Budapest Treaty terms with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 201102209. Deposits include following Accession Numbers Assigned: 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 V_K (D19A): UC 25503.

A number of anti-ALK-1 specific human antibodies showed a common pattern in CDR1 of the heavy chain variable domain. These leading molecules use the 4-31 or 4-61 heavy chain V gene segments. The FR1 and CDR1 sequences corresponding to these antibody heavy chains are shown in Table 4A aligned against the germline sequences. A dash (-) in the alignment indicates a residue, which is identical to the germline. In all cases, the GYYWS (SEQ ID NO: 136) pattern at the end of CDR1 has undergone somatic mutations, resulting in a new sequence pattern, turning the G residue into an acid residue (D or E) and the last S residue in an N has been changed to 9 out of 12 examples. Sequence diversity in other regions of VH indicates that these are likely independent somatic mutation events leading to the same sequence pattern at the end of VH CDR1.

Table 4A

ALK-1 Antibody Heavy Chain Sequence Patterns

<td>Clone</td><td>V gene f</td><td>D gene lemline</td><td>J gene</td><td>FR1 QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 122)</td><td>CDR1 GGSISSGGYYHS (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--- yy</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 --- N</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>

146

1.12.1 VH4-31 D6-19 JH4B

--M --- E --- N

Germline

1.13.1 VH4-61 D6-19

1.14.1 VH4-61 D6-19

QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 124)

GGSVSSGGYYWS (SEQ ID NO: 125)

JH4B

JH4B

--D --- N

--D --- N

Example 5. Preparation of 1.12.1 Fab Molecules

Fab fragment of 1.12.KM29I / D19A) was prepared by digestion of

1.12.1 (M29I / D19A) IgGl using papain. Protein A purified full-length 1.12.1 (M29I / D19A) IgGl was incubated with papain (VWR) in 1:50 ratio (papaine: protein) in buffer containing 30 mM sodium phosphate (pH 7.0), 2 mM EDTA and 2 mM cysteine ​​at 37 ° C for 2-3 hours. The digestion mixture was then placed on a protein A mini column to remove undisclosed full length protein and Fc fragment. Unbound Fab was collected in the flow-through liquid. A size exclusion column (Superdex 200, Amersham Pharmacia Biotech) was then used to further purify the Fab protein and exchange the buffer in PBS. Endotoxin was removed by applying the protein solution through Detoxi gel (PIERCE) and then Vivapure Mini Q ion exchange column (VivaScience). The protein was filtered with a 0.2 µm syringe filter and the endotoxin level was tested with a LAL pyrogen kit (Cambrex). The final purified protein was at a concentration of 2-3 mg / mL, with endotoxin level of &lt;0.1 EU / mg and purity of&gt; 95%.

1.12.1 (M29I / D19A) Fab fragment has a molecular weight of 47.347 under non-reduced condition as shown by electron spray mass spectrometry. Edman N-terminal sequence analysis confirmed the light chain N-termini sequence of EIVLTQSPG (SEQ ID NO: 113) and heavy chain sequence of QVQLQESG (SEQ ID NO: 114), respectively.

Example 6. Determination of Avidity Values ​​of Fully Human AntiALK-1 Monoclonal Antibodies by Surface Plasmon Resonance (SPR) Using BIACORE ™

147

Avidity measurements of purified anti-ALK-1 antibodies by surface plasmon resonance using the BIACORE ™ 3000 instrument were performed using the manufacturer's protocols as follows.

To perform kinetic analyzes, recombinant human ALK-1 / Fc fusion protein (hALK-1 / Fc) and cynomologus ALK-1 / Fc fusion protein (cALK-1 / Fc) were immobilized on separate flow cells of a CM5 BIAcore sensor chip with using routine amine coupling. Surfaces were prepared using 10 mM acetate buffer, pH 5.0 as the immobilization buffer, and protein densities of 300 and 150 RU were achieved for the hALK-1 / Fc and cALK-1 / Fc fusion proteins, respectively. Deactivation of unreacted N-hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride, pH 8.5. Antibody samples in running buffer were prepared at concentrations ranging from 0.125 to 2 nM (a 0 nM solution containing running buffer alone was included as a zero reference). Samples were randomized and injected in duplicate over 10 cells each over 10 minutes using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as running buffer. Values ​​were observed to be independent of flow rates from 1 to 100 µL / min, indicating that there was no mass transport limitation. A flow rate of 25 µL / min was used to determine avidity values. The dissociation of the antibody was monitored for 10 minutes, the surface regenerated by injection of 100 mM H3PO4 (25 µL / min) for 12 seconds. The raw data was processed using the Scrubber (© Biologic Software) software package and analyzed using the CLAMP (© Biologic Software) software package. Multiple sets of data from a single surface, six sets of data at a time, were globally fitted into a simple 1: 1 Langmuir binding model simultaneously using a common variable Rmax value. Table 5 lists avidity values ​​for representative anti-ALK-1 antibodies of the

148 present invention. The data shown indicates that the antibodies prepared according to the invention have high affinities and strong binding constants for human ALK-1.

Table 5

Determination of Avidity Value by Surface Plasmon Resonance (BIAcore)

<td>Clone</td><td>HALK-1 / Fc Avidity (pM)</td><td>HALK-1 / Fc calf (1 / s)</td><td>cALK-l / Fc avidity (pM)</td>

<td>1.12.KM29I / D19A)</td><td> &lt;6.8</td><td>&lt;5.0x10-®</td><td> 27</td>

<td> 1.14.2</td><td> 76</td><td>5.6 x 10⁵</td><td> 280</td>

<td> 1.27.3</td><td> 2.9</td><td>1.9 x 10⁵</td><td> 60</td>

<td> 1.31.1</td><td> &lt;13</td><td>&lt;5.0 x 10- «</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-®</td><td> 1800</td>

<td> 4.24.2</td><td> 70</td><td>4.4 x 10⁵</td><td> 430</td>

<td> 4.38.1</td><td> 100</td><td>4.0 x ÏO-®</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 LFR®</td><td> 19</td>

<td> 4.68.2</td><td> 86</td><td>3.8 x 10⁵</td><td> 320</td>

<td> 4.72.2</td><td> 73</td><td>3.4x10-5</td><td> 280</td>

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

1.12.KM29I / D19A) indicates the mAb 1.12.1 variant which was expressed recombinant mAb with two specific amino acid mutations in it (replace methionine at position 29 in the heavy chain with isoleucine and replace aspartic acid at position 19 in the light chain by alanine).

Example 7. Determination of Affinity Constants (Kp) of Variants of

Full Human Anti-ALK-1 Monoclonal Antibody 1.12.1 by Surface Plasmon Resonance (SPR) using BIACORE ™

Affinity measurements of purified anti-ALK-1 antibodies by surface plasmon resonance using the BIACORE ™ 3000

149 instrument were performed using the manufacturer's protocols as follows.

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

150

Table 6

Determination of mAb 1.12.1 variant affinity constant. Kd. by surface plasmon resonance (BIAcore)

<td>Antibody</td><td>on value (M¹ s¹)</td><td>Off value (s)¹)</td><td>Kn (nM)</td>

<td> 1.12.1</td><td>1.9 x 103</td><td>7.4 x 10⁵</td><td> 39</td>

<td>1.12.1 (rWT)</td><td>2.2 x 103</td><td>5.8 x 10-3</td><td> 26</td>

<td>1.12.KD19A)</td><td>2.6 x 10³</td><td>4.4 x 10⁵</td><td> 17</td>

<td>1.12.KM29I)</td><td>2.4 x 10³</td><td>S.lxlO-⁵</td><td> 38</td>

<td>1.12.KM29I / D19A) (1) *</td><td>2.2 x 10³</td><td>9.5 x 10-5</td><td> 43</td>

<td>1.12.KM29I / D19A) (2) *</td><td>2.3 x 10³</td><td>3.4 x 10-6</td><td> 37</td>

* The two affinity constants for 1.12.KM29I / D19A) (1) and (2) were obtained using two separate surfaces.

1.12.1 refers to the mAb 1.12.1 variant isolated from the hybridoma.

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

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

1.12.KD19A) refers to the mAb 1.12.1 variant which was expressed recombinant mAb containing a specific single amino acid mutation in which the aspartic acid at position 19 in the light chain was replaced by alanine.

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

151

Example 8. Determination of Affinity Constants (Kd) from Representative

Fully Human Anti-ALK-1 Monoclonal Antibodies by Surface

Plasmon Resonance (SPR) using BIACORE ™

Affinity measurements (Kd and calf) of purified anti-ALK-1 antibodies by surface plasmon resonance using the BIACORE ™ 3000 instrument were performed using the manufacturer's protocols as follows.

To perform kinetic analyzes, affinity purified mAbs were immobilized on the dextran layer of a CM5 biosensor chip using amine coupling. Surfaces were prepared using 10 mM acetate buffer pH 5.0 as the immobilization buffer and protein densities of 200-400 RU were achieved. Deactivation of unreacted N-hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride pH 8.5. Samples of monomeric ALK-ECD in running buffer were prepared at concentrations ranging from 3,125-400 nM (a 0 nM solution including running buffer alone was included as a zero reference). Samples were randomized and injected in duplicate for 2 minutes over all 4 flow cells using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as running buffer. On values ​​were observed to be independent of flow rates from 1 to 100 µL / min, indicating that there was no mass transport limitation. A flow rate of 25 µL / min was used to determine affinity constants. Dissociation of monomer ALK-ECD was monitored for 10 minutes, the surface regenerated by a 12 second injection of 100 mM H3PO4 (25 µL / min). The raw data was processed using the Scrubber (© BioLogic Software) software package and analyzed using the CLAMP (© BioLogic Software) software package. The data were globally fitted into a simple 1: 1 Langmuir binding model. Table 7 lists affinity measurements for representative anti-ALK-1 antibodies of the present invention:

152

Table 7

Determination of Affinity Constant. Kd. for Representative Monoclonal

Antibodies by Surface Plasmon Resonance (BIAcore)

<td>mAb</td><td>on value (M '¹ s-¹)</td><td>off value (s¹)</td><td>Ko (nM)</td>

<td>1.12.KM29I / D19A)</td><td>3.3 x 10⁴</td><td>8.2 x 1 (H</td><td> 25</td>

<td> 1.31.1</td><td>3.2 x 10⁴</td><td>1.9 x 1 (H</td><td> 6.0</td>

<td> 4.72.1</td><td>3.2 x 10⁴</td><td>2.5 x 10-6</td><td> 0.8</td>

<td>Fab 1.12.KM29I / D19A)</td><td>3.8 x 10⁴</td><td>8.2 x 10-⁴</td><td> 22</td>

The monomeric ALK ECD used to generate the data in Example 8 was a different preparation from that used to generate the data in Example 7.

1.12.KM29I / D19A) indicates the mAb 1.12.1 variant which was expressed recombinant mAb containing two specific amino acid mutations mentioned above (aspartic acid at position 19 in the light chain replaced by alanine and methionine at position 29 in the heavy chain replaced by isoleucine).

Fab 1.12.KM29I / D19A) indicates the Fab fragment of mAb 1.12.1 (M29I / D19A) prepared by digestion of 1.12.KM29I / D19A) IgG1 using papain.

Example 9. Identification of Epitope Selectivity of Anti-ALK-1

Antibodies

Cross competition experiments were performed using the BIACORE ™ 3000 instrument (Biacore International AB, Uppsala, Sweden and Piscataway, NJ), according to the manufacturer's protocols.

Recombinant human ALK-1 / FC chimera was immobilized on the dextran layer of a CM5 biosensor chip using amine coupling. Chips were prepared using 10 mM acetate buffer pH 5.0 as the immobilization buffer and a protein density of 940 RU

153 realised. Deactivation of unreacted N-hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride, pH 8.5.

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

After regeneration, the primary antibody was reinjected over the flow cell at a rate of 10 µL / min for 600 seconds. After the injection was completed, another secondary antibody was chosen and injected at the rate of 10 µL / min over the same fluid for 600 seconds. Once the entire panel of 14 antibodies was used as the secondary antibody, a new primary antibody was selected and the procedure with the new primary antibody was repeated. These procedures were performed until all possible combinations of primary and secondary antibodies were injected over the flow cell. Binding of the secondary antibody was considered to have occurred if the overall response observed after injection of both antibodies was greater than that observed for both possible threshold values. Threshold values ​​were determined using the same antibody as both the primary and secondary antibodies. Shown in Table 8, a response matrix was created based on whether binding was observed: - indicates no binding of the secondary antibody, x indicates binding was observed (response was greater than the thresholds for the individual antibodies). Grouping of the clones that have the same reactivity pattern

- 154 results in two different epitope bins with 1.11.1 in one bin and all other antibodies in the other bin.

155

Table 8. BIAcore epitope binning response matrix

<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.KM29I / D19A)</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> 4.68.2</td><td> 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>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>X</td><td>X</td>

<td>1.12.KM29I / D19A)</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> 1.27.3</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> 4?' ^J, f, Y</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>M</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></td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 4.58.2</td><td></td><td>X</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td></td><td>J »</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>f</td>

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Example 10. Isolation of Cvnomolgus Monkey ALK-1 Gen

Cynomolgus monkey C'Cyno ”) ALK-1 gene was extracted from Cyno lung tissue. Based on the published gene sequence for human ALK1 (Genebank registration L17075), primers were designed for PCR amplification of the full-length Cynomolgus ALK-1. mRNA was prepared from frozen excised cynomolgus lung tissue (ca. 1 g) using the mRNA purification kit (Ambion, Catalog No. 1915) according to the manufacturer's instructions. 200 ng of the mRNA was reverse transcribed and PCR amplified using the OneStep RT-PCR kit (Qiagen, Catalog No. 210210) using gene specific oligos: 5'-AGCGGGCCCAGAGGGACCATG (Seq ID NO: 115) (forward ) and 5'-CAGAAAGGAATCAGGTGCTCCTGGGCTA (Seq ID NO: 116) (reverse) at an annealing temperature of 61 ° C. A properly sized RT-PCR product (~ 1.5 Kb) was excised and purified from a 0.9% agarose gel after electrophoresis, then cloned with TOPO-TA into the pCR4-T0P0 vector (Invitrogen, Catalog No. K4575-01). The insertion sequence was determined to obtain the ORF nucleotide sequence of Cynomolgus ALK-1. The nucleotide and predicted translated amino acid sequences are shown in SEQ ID NOs: 93 and 94, respectively. While the cytoplasmic stretch of the gene encodes identical protein sequences between Cyno and human, there are 5 amino acid differences in the extracellular domain (ECD, which includes positions 22-118) and 1 amino acid difference in the transmembrane domain of the protein. ECD sequence identity between human and Cyno is 94.8%. An alignment of the human and primate ECD is shown in Figure 2.

A few primers were used (forward primer: 5'-GATTATGGCCTTGGGCTCCCCCAGGAAA (Seq ID NO: 117) and reverse primer: ö'-GGGCTCTTTATATCACTTTAGGCTTCTCTGGACTGTTG) (Seq ID NO: 118) for full-length PCR amplification ALK Cynom 1 gene .

157

Example 11. Determination of Cell Surface Binding Characteristics and

Primates Cross hybridization by Flow CTometry (FACS)

To generate ALK-1 overexpressed cell lines, which can be used to test anti-ALK-1 binding affinity using flow cytometry (FACS), full-length human, Cyno, and rat ALK-1 genes were included in Invitrogen's (Catalog No. K6510-20) pcDNA5 / FRT / To TOPO vector cloned and transfected into 293 Flp-In Τ-Rex host cell (Invitrogen, Catalog No. R780-07), respectively. Selections were made using hygromycin to obtain the final stable cell lines. Overexpression of the respective full-length ALK-1 proteins was accomplished by tetracycline (2 µg / mL) induction at 37 ° C / 5% CO2 for 24 hours.

Anti-ALK-1 mAbs were tested for their binding affinities for cell surface ALK-1 using FACS assay, using 293 stable cells with overexpression of ALK-1 proteins. The cells were detached using trypsin-EDTA and washed with cold PBS-SA. After aliquoting into 96-well plates, the cells were serum blocked and incubated with different concentrations of specific mAb for 1 hour at 4 ° C. Cells were then washed and incubated with an anti-human κ secondary antibody conjugated to the R-PE fluorophore before analysis using a FACSCalibur flow cytometer (BD Biosciences). 10000 events were collected for each sample without applying any entry requirement. Shown in Table 9, the geometric mean of each sample histogram was plotted as a function of the mAb concentration and Kd was calculated for each mAb after fitting into a two-state equilibrium model. Examples of equivalent human and primate FACS experiments are shown in Figure 3.

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Table 9

Mean Binding Affinity (Kp) Results of Anti-ALK-1 Monoclonal Antibodies for Cell Surface Humans or Cyno ALK-1 Measured by FACS

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

<td>Human</td><td>Cyno</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>

In addition, the FACS assay demonstrated that 1.12.1 has very limited crossover to the rat (Kd&gt; 100 nM) and is predicted to have very low crossover to the mouse, given the 74% and 68%, respectively.

ECD sequence identity between rat / human and mouse / human ALK-1).

FACS assay was also used to determine Kd of the recombinant 1.12.1 mAb variants. Shown in Table 10, the results indicate similar binding affinity of the recombinant antibody.

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Table 10

Mean Binding Affinity (Kd) Results of 1.12.KM29I / D19A)

Variants for Cell Surface Humans or Cvno ALK-1 Measured by FACS

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

<td>Human</td><td>Cyno</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>ND</td>

1.12.1 refers to the mAb 1.12.1 variant isolated from the hybridoma.

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

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

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

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

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Fab 1.12.1 (M29I / D19A) indicates the Fab fragment of mAb 1.12.1 (M29I / D19A) prepared by digesting 1.12.1 (M29I / D19A) IgG1 using papain.

Example 12. Taoman Assay for Idl

HUVECs (Biowhittaker, Cat. # CC-2519) were seeded on 24-well plates, 12000 cells / well in 600 µL of complete HUVEC medium (EGM-2 Bullet kit, Biowhittaker, Cat. # CC-3162), and overnight opportunity given to grow. The next day, the cells were typically 50% confluent. Complete Medium was removed and 200 µL of Starvation Medium (EBM-2 with only 0.2% FBS) was added. Cells were incubated for 2 hours. The cells were then treated with 40 µL of antibody solution in PBS. Lyophilized Ab was reconstituted with sterile PBS.

Finally, the cells were treated with 1% FBS / Basal medium (final concentrations) for 30 minutes, the medium was removed and the cells were lysed in 400 µL of RTL Buffer (Rneasy 96 kit, Qiagen, Cat. # 74182), according to the protocols from the manufacturer. RNA was then prepared using RNeasy kit (according to manufacturer's instructions). The RNA was eluted and quantitated with RiboGreen® RNA Quantitation Kit (Molecular probes, Cat. # R-11490). An equal amount of total RNA was used for real time PCR analysis to detect Id1 RNA expression (ABI 7900 instrument). PCR was performed using the Taqman One Step PCR Master Mix Kit (ABI, Cat. # 4309169) and the ID1 primer / probe sequences listed below. PCR was performed using 40 cycles of the following annealing and amplification conditions: 95 ° C, 15 seconds; 60 ° C, 1 min.

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

Name: IDl-Probe

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

119)

Taqman PCR Primers:

161

Name: ID1-F

Sequence: 5 'AAGGTGAGCAAGGTGGAGATTC 3' (Seq ID NO: 120) Name: ID1-R

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

A summary of mean IC50 values ​​for this assay is shown in Table 11. All IC50 determinations were performed in triplicate.

Example 13. Smadl Phosphorylation Detected by Odyssey Infrared

Imaging System from LI-COR Biosciences (24-well plate)

HUVECs (Biowhittaker, Cat. # CC-2519) were seeded on 24-well plates, 18000 cells / well in 600 µL of complete HUVEC medium (EGM-2 Bullet kit, Biowhittaker, Cat # CC-3162), and overnight opportunity given to grow. The next day, the cells were typically 50% confluent. Complete Medium was removed and 200 µL of Starvation Medium was added (Starvation Medium: EBM-2 with only 0.2% FBS). Cells were incubated for 2 hours. The cells were then treated with 40 µL of antibody solution in PBS for 3 hours. Finally, the cells were treated with 0.3X Complete Medium (final concentration) for 35 minutes. The medium was removed and the cells lysed in 80 µL of 1.1X Monster Buffer (Invitrogen, Cat, No. NP0007). Phosphorylated Smadl was determined by Western Blotting using X Cell Surelock Mini-Cell &amp; Blot Module (Invitrogen, Cat. # EI0002). Phosphorylated Smadl was detected using rabbit anti-phosphorus Smadl antibody (Cell Signaling,

Cat. No. 9511), which is then detected by IRDye ™ 800 conjugated Anti-RABBIT IgG (Rockland Immunochemicals, Cat. No. 611-732-127). The amount of phosphorylated Smadl was quantitated using Odyssey Infrared Imager (Li-Cor). Actin (Santa Cruz, # sc-8432) was used for normalization (anti-mouse Alex 680, Molecular Probe, Cat. No. A-162 21058). A summary of mean IC50 values ​​for this assay is shown in Table 11. All IC50 determinations were performed in triplicate.

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Table 11

<td>Clone</td><td>ID1 Taqman IC50 nM</td><td>nSmadl Western [C50 nM</td>

<td> 1.11.1</td><td>nd</td><td>nd</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> 4.72.1</td><td> 21</td><td> 35</td>

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

Example 14. Internalization Characteristics of Anti · ALK-1 Monoclonal

Antibodies

FACS was used to monitor the time course of the residual surface receptor ALK-1 as well as the neutralizing antibody. Remaining cell surface ALK-1 is monitored by a marker antibody capable of binding cell surface ALK-1, but recognizes a different epitope than the neutralizing antibody. A mouse anti-human ALK-1 ECD mAh (R&amp;D systems, Cat. No # AF310) was identified and used in the study as the marker antibody.

The time course of internalization was studied using endothelial cell lines HUVEC and HUAEC. The cells were grown at 37 ° C

164 with 5% CO2 in 24-well plates containing 200 µL of complete culture medium per well. At each of 11 time points over the 48 hour course, 2 µL of 1 mg / mL antibody solution was added to one well and mixed (final neutralizing antibody concentration is 10 µg / mL). The plate was then returned to the 37 ° C incubator until the time of 0 hours when the plate was placed on ice to stop the internalization process. At this time, marker antibody was added to the wells (10 µg / mL final concentration) and incubated on ice for 1 hour. The cells were then washed with PBS detached by trypsination and transferred to a 96-well plate. Cells were then washed, blocked, and treated with secondary antibodies carrying various flurophores to monitor both neutralizing antibody and receptor ALK-1 remaining on the cell surface. The samples were tested on a FACSCalibur flow cytometry instrument, counting 3000-5000 events / sample. The geometric mean of each sample in the specific fluorescence channel was calculated and plotted as a function of time. The data was fitted into a modified radio-decay equation to obtain the half-life (t1 / 2) of internalization as well as the percentage of neutralizing antibody or receptor ALK-1 remaining on the cell surface when the internalization has reached the stable state. As shown in Figure 6, mAb internalizes 1.12.1 (M29I / D19A) at the same rate and to the same extent as the cell surface receptor ALK-1.

Half-life of the 1.12.1 (M29I / D19A) internalization is ~ 2 hours. An equilibrium was reached when 50% of the antibody was internalized. A polyclonal antibody purchased from R&amp;D systems (Cat. No. # AF370) internalizes at 11/2 of 1 hour and reaches the steady state when ~ 70% of the receptor is internalized (Figure 6). Similar internalization characteristics were observed with other human anti-ALK-1 mAbs of the invention (not shown).

165

Example 15. Establishment of Human Foreskin SCID Chimera

Mice

Significant modification of the surgical procedure was applied to a procedure previously published by HC Yan, et al. "Human / Severe Combined Immunodeficient Mouse Chimeras, An Experimental In Vivo Model System to Study the Regulation of Human Endothelial Cell-Leukocyte Adhesion Molecules", J. Clin. Invest. 91: 986, 1993; J. Varner “Regulation of Angiogenesis in Vivo by Ligation of Integrin a5bl with the Central Cellbinding 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. After arriving from National Disease Research Institute and Cooperative Human Tissue Network, pieces of human foreskin were cut from unhealthy areas and transferred to RPMI media (Cellgro / Mediatech, Cat # MT-15-040-CV supplemented with Penicillin and Streptamycin (Gibco / Life Tech , Cat # 15070-063) (add 5 mLs of the pen / strep stock solution in 500mLs of RPMI).

Using a scalpel and cutting in a sterile petri dish, the skins were cut into an oval shape of approximately 8 x 13 mm with cleaning of any frayed ends and connective tissues, and stored on wet ice before surgery. The appropriate volume (4 pL / gram of animal) of 100 mg / mL Ketamine (Ketaset ™, Fort Dodge Animal Health) / 1 mg / mL medetomidine (Pfizer Animal Health - Dormitor) solution was injected intraperitoneally into the abdomen of scid mice ( i.e at an angle of 45 °, under the skin but not too deep inside). After anesthesia, the mice were given eye lubricant, a subcutaneous injection of Ketoprofen (10 mg / kg, Fort Dodge Animal Health) and she was shaved at the site of the procedure. The surgical region was surgically scrubbed three times using the Clorahexiderm (Butler, Chclo-Scrub 40, cat # WAB20109) and then the alcohol in a circular motion that started from the center of the surgical site and out of a dirty area going back to

166 avoided a clean area. The mice were transferred to the prepared surgical hood and placed on the heated water pad (Gaymar Industries, cat # TP500 T / Pump) kept at 37 ° C. The mice were then placed under isofluorin anesthesia for the duration of the procedure. The back of a mouse was covered with a surgical gown cut to reveal the site of the surgery. The mouse skin was picked up with forceps and an oval shaped skin tissue was cut out with curved scissors in one movement. An appropriately sized human foreskin was placed on the mouse. Human and mouse skin were sutured together using the Ethilon suture (Ethicon cat # 697H.), Starting at the top of the oval, then the bottom, then the right-most and then the left-most side. More sutures were placed in between to further bond the fabrics together. About 8 sutures were made at equal distances around the skin. During the procedure, a syringe with sterile saline was used to irrigate the skin / mouse surgical wound when it became dry. A Bandaid was placed over the wound. A transparent dressing (3M Tegaderm ™) was then used to wrap loosely around the bandage. The dressing was trimmed to cover a slightly larger area than the Bandaid. The mouse then received Atipamezole (50-100 µL, Pfizer Animal Health - Antisedan) and the mouse recovered in a heated cage in 5-10 min. The dressing and bandages were removed within 7-10 days and by 15^e most skins looked like scabs. Complete healing took place between 21-28 days after which the skins were ready to be inoculated with tumor cells. Shown in Figure 8 is an example of the histological (H&amp;E Staining) analysis of a section of the skin graft post surgery. The histology of the skin graft closely mimics the characteristics of human skin implanted in mice described by Tahtis, et al “Expression and Targeting of Human Fibroblast Activation Protein in a Human Skin / Severe Combined Immunodeficient Mouse Breast

167

Cancer Xenograft Model ”Mol. Cancer. Ther. 2 (8): 729, 2003. he: human epidermal layer; hd: human dermal layer.

Example 16. Collagen Model in Human Foreskin SCID Chimera

Mice

Collagen I stock solution (cat # 354236, Becten-Dickinson) was diluted to 4 mg / mL with 0.02 N acetic acid and kept on ice before implantation. The acidic collagen solution (8 parts) was mixed with 10X M199 (Sigma, Cat # M9163) (1 part) and human plasma fibronectin (Fn) (cat # 354008, Becten-Dickinson) to give a final Fn concentration of 90 µL / mL to achieve; NaOH (1.0 N) was added to adjust the pH to ~

7.2. The Collagen / Fn mixture was kept on ice until use. The implant mix was prepared using the above Collagen / Fn mixture plus the angiogenic inhibitor of interest with or without human macrovescular endothelial cells (HMVEC), (Cascade Biologies, Cat # C-010-5C). The HMVECs were prepared as 6 x 10⁶cells / mL in PBS. 50-100 µL of the implant mixture was injected intradermally into the foreskin into the scid chimera mouse. 7-14 days later, the collagen plugs were harvested, embedded in the OCT compound (cat # 4583, Sajura Finetek, CA) and frozen quickly for immunohistochemistry analysis. The foreskin collagen plug was identified with the Trichrome Kit (cat # KC1641, Mater Tech, CA) as blue staining as shown in Figure 9 (A). Human vessels were identified for human P-CAM staining using the anti-human CD-31 antibody (Clone 13.3, Vector Laboratories) (Figure 9 (B)). Table 12 summarizes the human vessel staining and quantification in the collagen model in the foreskin SCID chimera mice.

168

Table 12. Summary of the results of the Collagen model

<td>Matrix</td><td>HMVEC in Matrix</td><td>Treatment (Rx)</td><td>Days of Rx</td><td>Study end point</td><td>Human vessels scoring (1 x 10³)</td><td>% of control (human vessels)</td>

<td>1.6 mg / ml Collagen</td><td>No</td><td>no treatment</td><td> 4</td><td></td><td> 0.036 ±0.001</td><td> 40</td>

<td>1.6 mg / ml Collagen</td><td>7x 10³</td><td>no treatment</td><td> 4</td><td></td><td> 0.071 ± 0.022</td><td> 78</td>

<td>1.6 mg / ml Collagen</td><td>1.4 x 10 «</td><td>no treatment</td><td> 4</td><td rowspan="2">human CD-31 staining</td><td> 0.063 ± 0.016</td><td> 69</td>

<td>2.4 mg / ml Collagen</td><td>No</td><td>no treatment</td><td> 4</td><td> 0.091 ± 0.056</td><td> 100</td>

<td>2.4 mg / ml Collagen</td><td>7x 10³</td><td>no treatment</td><td> 4</td><td></td><td> 0.067 ± 0.049</td><td> 74</td>

<td>2.4 mg / ml Collagen</td><td>1.4 x 10 "</td><td>no treatment</td><td> 4</td><td></td><td> 0.062 ± 0.047</td><td> 68</td>

<td>3.0 mg / ml Collagen</td><td>8.8 x 10³</td><td>no treatment</td><td> 4</td><td></td><td> 54 ±9</td><td> 100</td>

<td>3.0 mg / ml Collagen</td><td>8.8 x 10³</td><td>Isotype control antibody 100 µg / ml mixed in gel</td><td> 4</td><td rowspan="2">human CD-31 staining</td><td> 52 ± 13</td><td> 96</td>

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

<td>3.0 mg / ml Collagen</td><td>no</td><td>no treatment</td><td> 4</td><td rowspan="2">human CD-31 staining</td><td> 0.112 + 0.026</td><td> 100</td>

<td>5.0 mg / ml Collagen</td><td>no</td><td>no treatment</td><td> 4</td><td> 0.031 + 0.012</td><td> 28</td>

<td>3.0 mg / ml Collagen</td><td>no</td><td>Isotype control antibody 100 pg / ml, id. Injection</td><td> 4</td><td></td><td> 75 ± 15</td><td> 100</td>

<td>3.0 mg / ml Collagen</td><td>no</td><td>1.12.1 (M29I / D19A) antibody 100 pg / ml, id. injection</td><td> 4</td><td>human CD-31 staining</td><td> 39 ± 11</td><td> 52</td>

<td>3.0 mg / ml Collagen</td><td>no</td><td>1.14.1 antibody 100 pg / ml, id injection</td><td> 4</td><td></td><td> 44 ±28</td><td> 59</td>

169

Example 17. M24 with Tumor Model in Human Foreskin SCID Chimera

Mice

Typically, a graft aged between 5-10 weeks post surgery was used in these studies. The M24met cell line was described by Mueller and collaborators in “Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells,” Cancer Research, 55 (8): 1629-32, 1995. An M24 with cell suspension was prepared as follows: 80% confluent M24 with cells were washed, trysonized using Trypsin / EDTA (Gibco, Cat # 25200-056) and collected in the PRMI (Cellgro / Mediatech, cat # MT-15-040- CV) media supplemented with 10% FBS (Cellgro / Mediatech, Cat # AKD-11775) and 2 mM L-glutamine (Cellgro / Mediatech, Cat # 25-005-C1). The cells were centrifuged at 600 rpm for 5 min, resuspended in sterile PBS. Cell counts were estimated using a Coulter Counter (Beekman Coulter, Model Z2). The cells were centrifuged at 600 rpm for 5 min and resuspended in Collagen / and Fn (3 mg / ml) mixture to make a 4x10⁷ cells / ml of cell suspension for implantation.

For inoculation, 2x10⁶ of the cells above injected intradermally (50 μΐ of 4x10⁷ cells / ml) in the human skin transplanted into the mouse. On day 5-7 post implantation, the tumors would be palpable and the mice were randomized into the control and treatment groups before dosing. The control group is dosed as one in which the animals would receive either no dose, a dose of the vehicle in which the anti-ALK-1 antibody was established, or a dose of the isotype-matched IgG2 human monoclonal antibody anti-KLH (Pfizer Ine) . The treatment group is defined as one in which the animals would receive a dose of the anti-ALK-1 antibody 1.12.1 (M29I / D19A).

Example 18. Human and Mouse CD-31 Immunofluorescence (IF) Double Staining

170

The frozen tissue sections were air-dried and fixed in acetone (Fisher, Cat # A16S-4) at -20 ° C, or 10 min. The samples were air-dried again and washed three times in PBS each for 5 min.

The samples were blocked in 5% rabbit serum (Vector Laboratories, Cat # S-5000) in PBS for 30 min at room temperature. Primary antibody mixture was prepared in 5% rabbit serum with the anti-human CD-31 antibody (Santa Cruz, Cat # SC1505) and the anti-mouse CD-31 (Pharmingen, Clone Meel 3.3, Cat # 0195IA) at 1: 100 and 1: 150 dilutions. The antibody mixture above was added to the tissue samples for 1 hour at RT. The blocks were washed three times for 5 min each in PBS before incubating with the secondary antibody mixture for 1 hour at RT. The secondary antibody mixture was prepared in PBS / 0.05% Tween-20 (Sigma, Cat #

P1379), Texas Red rabbit anti-goat antibody (Jackson Labs, Cat # 305-075003) and FITC rabbit anti-rat antibody (Jackson Labs, Cat # 312-095-003).

The antibodies were diluted to 1:50 when using frozen antibodies or to 1: 100 when using fresh antibodies. The plates were rewashed in PBS three times for 5 min each before mounting in Vectashield (Hard Set, Mounting medium with DAPI, Vector Lab, CA, Cat # H-1500). The plates were kept in the dark and at 4 ° C until image analysis followed. The image analysis was performed using an Olympus BX60 fluorescence microscope and pictures were taken using an Olympus microfire digital color camera. Photos were taken of 3-5 hot spots / plate, one plate / animal, 4-7 animals / group and the areas of vessels as indicated by positive staining of anti-human CD-31 were quantitated by three individuals using Image Pro Plus v4.5 (MediaCybernetics). The pharmacodynamic endpoint (group mean) was expressed either as the percentage of human CD-31 inhibition compared to the control group or as a total region of human vessels. Statistical significance was determined by ANOVA. Shown in Figure 10 is an immunofluorescent image

171 of human (red) and mouse (green) vessels of the M24 with tumor in the human foreskin SCID chimera mouse.

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

The frozen tissue sections were air dried and fixed for 10 min at -20 ° C in acetone. The samples were air dried again and washed twice in PBS for 5 min each. The samples were incubated in 0.075% H 2 O 2 / methanol (Fisher Cat # A433-4) for 15 min and washed 3 times in PBS 3 times each. The samples were blocked for 5 min in 5% rabbit serum / PBS and applied for 1 hour at RT with anti-human CD-31 antibody 1: 100 (Santa Cruz, Cat # SC1505) in 5% rabbit serum. The samples were washed twice in 5 min each time in PBS and applied in rabbit anti-goat at 1: 200 (Vector Labs, Cat # BA5000) in 5% rabbit serum for 35 min at RT. The plates were then washed twice in 5 min each in PBS and freshly made streptavidin (Vector Labs, ABC Elite kit, Cat # PK-6100) was added. The slides were again washed twice in 5 min each in PBS and then developed in diaminobenzidine (DAB) (Vector Labs, Cat # SK-4100). The plates were washed twice in PBS for 5 min each followed by Mayer's hematoxylin (Sigma, Cat # HHS-32) for 5 seconds. The samples were rinsed well in dilLO and dipped briefly twice in the diluted (5ml stock in 1L of diH2O) ammonium hydroxide solution (Sigma, Cat # a-6899) ​​and rinsed again in dilLO. The samples were then dehydrated for 1 minute in 70%, 90% and then 100% alcohol (Harleco, Cat # 65347/85) and finally in xylene (JT Baker, Cat # 516.09). The slides were mounted with Cytoseal 60 (Stephens Scientific, Cat # 8310-4,) and covered with coverslips for image analysis. Shown in Figure 11 is the IHC image of human vessels (brown) from the M24 with tumor in the human foreskin SCID chimera mouse.

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Example 20, Therapeutic Treatment with the Anti-ALK-1 Antibody

1.12.1 (M29I / D19A)

For treatment, the dose was performed either subcutaneously (sc) or intravenously (iv). Typically, one dose of it

1.12.1 (M29I / D19A) antibody given for each study. The second dose of the ALK-1 antibody, if necessary, was administered on days 9 or 10. Sometimes multiple dose levels, ie 1, 5, 10, 50 mg / kg, were administered to investigate dose-dependent inhibition of human vessel growth . Animals were monitored daily and tumors were measured three times a week with calipers. By day 14-17, tumors were between 250-350 mm³ and were removed from the mice, embedded in OCT and frozen for IF or IHC analysis. Shown in Figure 12 are representative immunofluorescence images of human (red) and mouse (green) vessels from the control and 1.12.1 (M29I / D19A) treated (10 mg / kg) M24 with tumors in the human foreskin scid chimera mouse. Dose-dependent inhibition of human tumor vessels by 1.12.1 (M29I / D19A) in the human foreskin SCID chimera mouse model is shown in Figure 13 and a summary of related studies is presented in Table 13.

Table 13.

Summary of in vivo model characterization and inhibition of growth of human vessels of the M24 tumors in the SCID chimera model

<td colspan="5">Protocol Parameters</td><td colspan="2">Endpoints</td><td rowspan="2">General remarks</td>

<td>Tumor</td><td>Drug</td><td>Do- sis</td><td>Route</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day from Study</td>

<td>MCF-7</td><td>no</td><td>after</td><td>after</td><td>after</td><td>not quantified</td><td> 19</td><td>Tumors implanted intradermally. Tested with and without estradiol and collagen implant. The tumors grew slowly and expressed little human CD31</td>

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<td colspan="5">Protocol Parameters</td><td colspan="2">Endpoints</td><td rowspan="2">General remarks</td>

<td>Tumor</td><td>Drug</td><td>Do- sis</td><td>Route</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day from Study</td>

<td>M24 with</td><td>no</td><td>after</td><td>after</td><td>after</td><td>not quantified</td><td> 19</td><td>Tumors implanted intradermally. Tested with and without collagen / FN matrix. With matrix superior tumor growth found, all future studies will include matrix supplements. Tumors showed good human CD31 staining</td>

<td>M24 with (small)</td><td>no</td><td>after</td><td>after</td><td>after</td><td>not quantified</td><td> 9</td><td>Tumor size &lt;100 mm³. Little human CD31</td>

<td>M24 with (medium)</td><td>no</td><td>after</td><td>after</td><td>after</td><td>not quantified</td><td> 12</td><td>Tumor size &lt;100-200 mm³. Single human CD31</td>

<td>M24 with (big)</td><td>no</td><td>after</td><td>after</td><td>after</td><td>not quantified</td><td> 12</td><td>Tumor size &lt;200 mm³. Large M24 with tumors have superior larger numbers of human vessel staining, future studies will be conducted with larger tumors</td>

<td rowspan="2">M24 with</td><td>Not- Specifically human IgG</td><td>10 mg / kg</td><td>IV</td><td rowspan="2">2 doses (day 5 &amp; 9)</td><td> 0</td><td rowspan="2"> 15</td><td rowspan="2">First screening study. 1.12.1 (M29I / D19A) showed significant reduction in human CD31 staining. 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">M24 with</td><td>Not- Specifically human IgG</td><td>10 mg / kg</td><td>IV</td><td rowspan="2">2 doses (day 5 &amp; 10)</td><td> 0</td><td rowspan="2"> 14</td><td rowspan="2">Second screening study. Confirmed results of GW-366. No tumor growth inhibition was observed</td>

<td>1.12.1 (M29I / D1 9A)</td><td>10 mg / kg</td><td>IV</td><td> 40</td>

<td rowspan="4">M24 with</td><td>Not- Specifically human IgG</td><td>10 mg / kg</td><td>SC</td><td rowspan="4">2 doses (day 5 &amp; 10)</td><td> 0</td><td rowspan="4"> 14</td><td rowspan="4">First test of dose-dependent activity of 1.12.1 (M29I / D19A) against human CD31. Some dose dependent effect observed. PK results that single dose will be sufficient for significant reduction in CD31. No tumor growth inhibition was observed</td>

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

<td>1.12.1 (M2 9I / D19A)</td><td>1 mg / kg</td><td>SC</td><td> 50</td>

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

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<td colspan="5">Protocol Parameters</td><td colspan="2">Endpoints</td><td rowspan="2">General remarks</td>

<td>Tumor</td><td>Drug</td><td>Do- sis</td><td>Route</td><td>Scheme</td><td>CD31 (% inhibition compared to control)</td><td>Day from Study</td>

<td rowspan="6">M24 with</td><td>no dose</td><td>0 mg / hg</td><td>after</td><td>after</td><td></td><td rowspan="6"> 16</td><td rowspan="6">Second test of dose-dependent activity of 1.12.1 (M29I / D19A) against human CD31. Clear dose dependent antiCD31 effect was observed. No tumor growth inhibition was observed</td>

<td>Isotype matched IgG</td><td>10 mg / kg</td><td>SC</td><td rowspan="5">one dose (day 5)</td><td> 0</td>

<td>Not- Specifically human IgG</td><td>10 mg / kg</td><td>SC</td><td>UB</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="7">M24 with</td><td>Isotype matched IgG</td><td>10 mg / kg</td><td>SC</td><td rowspan="7">one dose (day 5)</td><td> 0</td><td rowspan="7"> 14</td><td rowspan="7">Final test with wide dose range for 1.12.1 (M29I / D19A). Study showed good dose-dependent effects. Inclusion data for a sigmoidal dose response curve gives an IC 50 of 93 nM. No tumor growth inhibition was observed.</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 9I7D19A)</td><td>10 mg / kg</td><td>SC</td><td> 73</td>

<td>1.12.1 (M2 9I / D19A)</td><td>50 mg / kg</td><td>SC</td><td> 70</td>

Example 21. In vivo EC50 Assay

Human foreskin SCID chimera mice were intradermally treated with

M24 implanted with cells and treated (sc) with anti-ALK-1 antibody 1.12.1 (M29I / D19A) at 1, 3, 5, 7.5, 10 and 50 mg / kg or isotype-matched anti-human KLH antibody (10 mg / kg). At the end of the experiment, the area of ​​human vessels in each tumor was quantitated as described above. Mouse plasma concentrations of anti175

ALK-1 antibody 1.12.1 (M29I / D19A) were measured using the method described as follows: Serum samples from mice were analyzed for anti-ALK-1 antibody 1.12.1 (M29I / D19A) concentration by an ELISA (enzyme- Connected Immunosorbent Assay). ELISA plates were coated with 10 µg / ml goat anti-human IgG Fc specific antibody (Pierce, cat # 31123) in PBS, incubated overnight at 4 ° C, then incubated with StartBlock blocking buffer (Pierce, cat # 37542) for 1 hour at room temperature. ) blocked. Serum samples were diluted 100 and 1000 fold in StartBlock blocking buffer before analysis. Two sets of standards were prepared in the blank serum diluted 100 and 100-fold. Standards and diluted serum samples were incubated on the plate for 1 hour. Bound anti-ALK-1 antibody 1.12.1 (M29I / D19A) was detected using horseradish peroxidase (HRP) -labelled goat anti-human IgG (Fab specific) antibody (Sigma, cat # A0293). The substrate used was 3.3 ', 5.5'-tetramethyl benzidine (Sigma, cat # T8665). Absorbance was read at 450nm on a Vmax plate reader (Molecular Devices, Menlo Park, CA). A standard curve was fitted by non-linear regression. The limit of detection of this assay was 10 ng / ml of anti-ALK-1 antibody 1.12.1 (M29I / D19A).

SCID mouse plasma concentration of anti-ALK-1 antibody 1.12.1 (M29I / D19A) is shown in Figure 15.

Figure 15 shows the estimated EC50 for 1.12.1 (M29I / D19A) in the M24 With Foreskin SCID chimera Model. The human vessel area was plotted against the mean plasma PK over the study period (14 days) for each treatment group. A fitted curve was produced by the Sigmoidal Dose Dependent program in the Graphpad (Prizm). EC50 of 93 ng / ml (EC50 is defined as the plasma concentration required for a 50% reduction in the area of ​​human vessels in the control group) was derived from the curve fit.

- 176 All publications, patents, and patent applications cited in this description are incorporated herein by reference, as if any individual publication or patent application would be specifically and individually designated as being incorporated by reference. While the foregoing invention has been described in some detail by way of illustration and as an example for purposes of clear understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications may be made therein without to leave the spirit or scope of the appended claims.

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Text in Figures

<td>5 human in Figures 2, 3A expressed in Figure 7A germline in Figures 7A, 7B, 7C, 7D</td><td>human expressed germline</td>

Text in Sequence liist

<td>human in Sequences 1-92, 95, 98-104,</td><td></td>

<td> 113-114, 122-136</td><td>man</td>

<td>monkey in Sequences 93-94</td><td>monkey</td>

<td>artificial in Sequences 96, 105-112,</td><td></td>

<td> 15 115-121</td><td>artificial</td>

<td>the forward ... in Sequence 96</td><td>use the forward primer for cloning of ECD from ALK-1</td>

<td>the reverse ... in Sequence 96</td><td>the reverse primer</td>

<td> 20</td><td>used for cloning of ECD from ALK-1</td>

<td>amplification ... in Sequences 105-108</td><td>amplification primer used for full-length cloning 1.12.1</td>

<td>25 mutagenic in Sequences 109-112</td><td>mutagen</td>

<td>forward primer in Sequences 115, 117 reverse primer in Sequences 116, 118</td><td>forward primer reverse primer</td>

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