WO2006017759A2 - Tumor endothelial marker-1 (tem1) binding antibodies and uses thereof - Google Patents

Abstract

The present invention relates to antibodies that bind tumor endothelial marker 1 (TEM1) and effectively inhibit angiogenesis, tumor growth, or both. Pharmaceutical compositions and uses thereof for the treatment of angiogenesis-associated diseases including cancer, polycystic kidney disease, diabetic retinopathy, age-related macular degenerations, rheumatoid arthritis, and psoriasis are also disclosed. Also disclosed are methods of identifying such antibodies by assessing (1) the inhibitory activity of candidate antibodies in proliferation and tube formation assays using endothelial cells, endothelial precursor cells and pericytes, and (2) the antibody-dependent cellular cytotoxic, phagocytosis, and complement-dependent cytotoxic activities of candidate antibodies.

Description

TUMOR ENDOTHELIAL MARKER-I (TEMl) BINDING ANTIBODIES

AND USES THEREOF

Cross Reference to Related Application

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 60/599,359, filed August 5, 2004. The contents of this application are incorporated by reference in its entirety.

Technical Field

[0002] The present invention relates to the field of immunology. More particularly, the present invention relates to antibodies that bind tumor endothelial marker 1 (TEMl) antigen and are useful in the therapy of diseases such as cancer and pathologic angiogenesis.

Background Art

[0003] Angiogenesis encompasses the generation of blood vessels in a tissue or organ. Under normal physiological conditions, angiogenesis occurs in very specific situations such as wound healing, fetal development, and the formation of the corpus luteum, endometrium and placenta. The process of angiogenesis is highly regulated through a system of naturally occurring stimulators, e.g., angiopoietin-1, IL-8, platelet-derived endothelial cell growth factor (PD-ECGF), and vascular endothelial growth factor (VEGF), and inhibitors, e.g., thrombospondin, interferon, and metalloproteinase inhibitors.

[0004] Angiogenesis also can occur as a significant factor in a number of disease states. In fact, uncontrolled angiogenesis directly contributes to the pathological damage associated with many diseases. This uncontrolled or excessive angiogenesis occurs when an imbalance in the angiogenic factors and angiogenic inhibitors occurs, e.g., when an excessive amount of angiogenic factor is produced. Insufficient angiogenesis also contributes to certain disease states. For example, inadequate blood vessel growth contributes to the pathology associated with coronary artery disease, stroke, and delayed wound healing.

[0005] Excessive angiogenesis occurs in diseases such as diabetic retinopathy, age-related macular degeneration, atherosclerosis, and inflammatory conditions such as rheumatoid arthritis and psoriasis. For example, in rheumatoid arthritis, the blood vessels in the synovial lining of the joints undergo inappropriate angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction, and thus may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. See, e.g., Bodolay, E., et ah, J. Cell MoI. Med. 6: 357-76 (2002). Similarly, in osteoarthritis, the activation of the chondrocytes by angiogenic-related factors may contribute to the destruction of the joint. See, e.g., Walsh, D. A., et ah, Arthritis Res. 3: 147-53 (2001).

[0006] Angiogenesis plays a decisive role in the growth and metastasis of cancer. See, e.g., B.R. Zetter, Ann. Rev. Med. 49: 407-24 (1998), J. Folkman, Sent. Oncol. 29: 15-18 (2002). First, angiogenesis results in the vascularization of a primary tumor, supplying necessary nutrients to the growing tumor cells. Second, the increased vascularization of the tumor provides access to the blood stream, thus enhancing the metastatic potential of the tumor. Finally, after the metastatic tumor cells have left the site of primary tumor growth, angiogenesis must occur to support the growth and expansion of the metastatic cells at the secondary site.

[0007] Normal and disease-related angiogenesis seem to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Disease or injury induces the production of pro-angio genie factors. Initially, these factors activate endothelial cells, vascular smooth muscle cells, and leukocytes to release enzymes that erode or dissolve the basement membrane of the existing blood vessels. The activated endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Pro-angiogenic stimulants then induce the endothelial cells to migrate through the compromised basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells begin to proliferate. The endothelial sprouts merge with each other to form capillary loops using adhesion factors, creating new blood vessels. Additional enzymes, e.g., matrix metalloproteinases, then digest the tissue at the tip of the sprouting vessel, permitting active tissue remodeling around the new vessel. The newly formed vessels are stabilized by the pericytes (i.e., a type of specialized smooth muscle cells). Once stabilized, the new vessels support blood flow.

[0008] Vascular/perivascular cells ("pericytes") are defined by their location in vivo. The pericyte is a small ovoid shaped cell with many finger-like projections that parallel the capillary axis and partially surrounds an endothelial cell in a vessel. Pericytes share a common basement membrane with the endothelial cell. They are elongated cells with contractile properties having a variety of functional characteristics. For example, pericytes regulate endothelial cell proliferation and differentiation, contact in a manner that either exacerbates or reduces endothelial cell junctional inflammatory leakage, synthesize and secrete various vasoactive autoregulating agonists, and synthesize and release structural constituents of the basement membrane and extracellular matrix. See, e.g., Shepro et al, FASEB J. 7: 1031-8 (1993). Thus, pericytes have thus been implicated as playing a role in vasoconstriction as well as a role in capillary blood flow, the formation of blood vessels, the immune response (particularly in the central nervous system), the extrinsic coagulation pathway, and the regulation of the function of the glomerulus. See, e.g., U.S. Application Publication No. 2002/0173464. Through these mechanisms, pericytes play a role in a variety of pathologies including hypertension, atherosclerosis, complications of diabetes (both insulin-dependent and non-insulin-dependent), ovarian failure, multiple sclerosis, and tumor vascularization, as well as in normal aging.

[0009] Numerous compounds have been identified as angiogenesis inhibitors. Exemplary compounds include protamine (Taylor et al, Nature 297:307 (1982)), heparin, steroids (Folkman et al, Science 221:719 (1983) and U.S. Pat. Nos. 5,001,116 and 4,994,443), Avastin™ (anti-VEGF) (Ferrara et al, Nature DrugDiscov. 3:391-400 (2004)), thalidomide (RJ. D'Amato, et al. Proc. Natl. Acad. ScL U.S.A. 91: 4082-85 (1994)), TNP-470 (Ingber, et al, Nature 348: 555-57 (1990)), and carboxyamidotriazole (CAI) (Kohn, et al, Cancer Res. 56: 569-73 (1996). Endogenously produced inhibitors include interferon alpha (IFN-α) (White et al, NewEnglandJ. Med. 320:1197-1200 (1989), Sidky et al, Cancer Res. 47:5155-61 (1987), angiostatin (O'Reilly, et al, Cell 79: 315-28 (1994)), endostatin (O'Reilly, et al, Cell 88: 1-20 (1997)), and thrombospondin (Volpert et al, Proc Nat 'I Acad. Sd. U.S.A. 26:6343-48 (1998)).

[0010] The limitation of many of these compounds is in their generic nature of anti- angiogenic activity. In other words, most compounds lack the capacity to distinguish between normal angiogenesis and disease-associated angiogenesis. As a result, significant toxicity limits the usefulness of many of the identified anti-angiogenic agents clinically because both normal and disease-related angiogenesis are inhibited. Therefore, an angiogenic inhibitor that selectively targets disease-associated angiogenesis offers significant advantages in decreased toxicity and potentially increased long-term efficacy. Disclosure of the Invention

[0011] While the importance of pericytes and endothelial cells in angiogenesis is well known, effective antibodies that specifically target areas of undesirable angiogenesis have been difficult to develop to date. Two cell types critical in angiogenesis are the endothelial cell and the pericyte. The endothelial cell and/or the pericyte express tumor endothelial marker 1 (TEMl). Using a selection paradigm that assesses functional neutralization activity as well as cytotoxic activity in vitro, antibodies that specifically bind TEMl with therapeutic efficacy are identified herewith. The TEMl antibodies also are active in in vivo animal models. Exemplified antibodies were generated from a mouse that can produce human antibodies. See, e.g., WO 02/043478. Thus, these antibodies have a discrete set of physiologically relevant characteristics as well as reduced immunogenicity, making them ideal therapeutic agents.

[0012] The present invention relates to monoclonal antibodies that can bind TEMl . In one embodiment, the present invention provides monoclonal antibodies that inhibit the proliferation of endothelial cells and/or pericytes. In another embodiment, the present invention provides monoclonal antibodies that are useful in the treatment of cancer, angiogenesis-related diseases, or pericyte disorder-related diseases. The monoclonal antibodies can be human antibodies. More particularly, provided herein is a monoclonal antibody, or biologically active fragment thereof, wherein the variable region sequences comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 (or SEQ ID NO:9a) and SEQ ID NO: 10 (or SEQ ID NO: 10a), SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 15 and SEQ ID NO: 17. In some embodiment, alternative heavy chain FRl regions as set forth in SEQ ID NO: 18-28 may be employed with the above heavy chain sequences. Also provided herein is a monoclonal antibody, or a biologically active fragment thereof, wherein the antibody is originally isolated from a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEM 1-#187(ATCC PTA-6066). The antibody of the present invention includes the antibody produced by the hybridoma as well as an antibody of the same amino acid sequence produced by recombinant means, hi one embodiment, the antibody, or a biologically active fragment thereof, comprises the same sequence of amino acids of the variable region as the antibody originally produced by the hybridomas TEM1-#87(ATCC PTA-6064), TEM1-#132(ATCC PTA-6065), or TEMl- #187(ATCC PTA-6066). In one embodiment, the antibody is an IgG antibody. The subclass of IgG can be selected from IgG₁, IgG₂, IgG₃ and IgG₄. In the case that antibody-dependent cell cytotoxicity (ADCC), phagocytosis, and/or complement-dependent cytotoxicity (CDC) activity is necessary or expected for therapeutic efficacy, IgG₁, IgG₂ and IgG₃ are preferred isotypes. When both ADCC and CDC activity are necessary or expected for therapeutic efficacy, IgG₁ is the more preferred isotype. In some embodiments, at least one amino acid of the heavy chain is deleted, added, or substituted with an amino acid different from the original amino acid sequence of the antibody produced by the hybridomas TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), or TEM1-#187(ATCC-6O66).

[0013] It is also contemplated that the antibody of the present invention may be conjugated to an antitumor agent or an antiangiogenic agent, or co-administrated with an antitumor agent, an antiangiogenic agent, or radiation therapy. In some embodiments, the antitumor agent or antiangiogenic agent is a radionuclide.

[0014] hi one embodiment, the antibody inhibits angiogenesis, particularly where the angiogenesis promotes or causes diseases such as cancer, polycystic kidney disease, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and psoriasis.

[0015] In one embodiment, the antibody inhibits tumor growth. The rumor can be an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma. Additionally, the tumor may be a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, soft tissue, spleen, testis, thymus, thyroid, or uterus.

[0016] Also provided herein is a pharmaceutical composition comprising an antibody, or a biologically active fragment thereof, of the TEMl antibody and a suitable excipient, wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 (or SEQ ID NO:9a) and SEQ ID NO:10 (or SEQ ID NO: 10a), SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:15 and SEQ ID NO:17.

[0017] hi another embodiment, the pharmaceutical composition of the present invention comprises a TEMl antibody originally isolated from a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl- #187(ATCC PTA-6066), and a suitable excipient. In yet another embodiment, the pharmaceutical composition of the present invention comprises an antibody comprising a variable region with the same amino acid sequence as the TEMl antibody originally isolated from a hybridonia selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEM1-#132(ATCC PTA-6065), and TEMl -#187(ATCC PTA-6066), and a suitable excipient.

[0018] Further provided herein is a monoclonal antibody that binds to TEMl, wherein the antibody mediates a high degree of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and/or phagocytosis activity against TEMl expressing cells.

[0019] Also provided herewith are the hybridoma strains designated TEMl -#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEM 1-#187(ATCC PTA-6066).

[0020] The antibodies of the present invention are useful in methods of inhibiting tumor growth. Thus, provided herein is a method of inhibiting tumor growth, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the variable region sequences comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 (or SEQ ID NO:9a) and SEQ ID NO: 10 (or SEQ ID NO:10a), SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO: 16, or SEQ ID NO:15 and SEQ ID NO:17, whereby the antibody inhibits tumor growth. Also provided herein a method of inhibiting tumor growth, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl -#187(ATCC PTA-6066), whereby the antibody inhibits tumor growth. In some embodiments, the antibody of the method is one with the same variable region sequence as that of the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEM1-#132(ATCC PTA-6065), and TEM 1-#187(ATCC PTA- 6066). It is contemplated that the subject expresses the antigen bound by the antibody. The antibody can be an intact antibody molecule, scFv, a Fab fragment, or a F(ab')₂ fragment. In some embodiments, the antibody is conjugated to an antitumor agent or antiangiogenic agent, or co-administered with an antitumor agent, an antiangiogenic agent, or radiation therapy. In some embodiments, the agent is a radionuclide. The tumor treated by this method can be an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma. In some embodiments, the rumor is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, soft tissue, spleen, testis, thymus, thyroid, or uterus.

[0021] The antibodies of the present invention are also useful in methods of inhibiting angiogenesis. Also provided herein is a method of inhibiting angiogenesis, comprising: administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the variable region sequences comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 (or SEQ ID NO:9a) and SEQ ID NO: 10 (or SEQ ID NO: 10a), SEQ ID NO:11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 15 and SEQ ID NO: 17, whereby the antibody inhibits angiogenesis. Also provided herein a method of inhibiting angiogenesis, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the antibody was originally produced by a hybridoma selected from the group consisting of TEMl -#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl -#187(ATCC PTA-6066), whereby the antibody inhibits angiogenesis. In some embodiments, the antibody of the method is one with the same variable region sequence as that of the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl -#187(ATCC PTA- 6066). The subject treated by this method expresses the antigen bound by the antibody. In some embodiments, the angiogenesis treated by this method is neoangiogenesis. The antibody can be an intact antibody molecule, a scFv, a Fab fragment, or a F(ab')₂ fragment. In some embodiments, the antibody is conjugated to an antiangiogenic agent or an antitumor agent or co-administered with an antitumor agent, antiangiogenic agent or radiation therapy. In some embodiments, the agent is a radionuclide. In some specific embodiments, the subject treated by this method has a disease such as cancer, polycystic kidney disease, diabetic retinopathy, rheumatoid arthritis, or psoriasis.

[0022] Also provided herein is the use of any of the antibodies, or a biologically active fragment thereof, that binds TEMl and is disclosed herewith, in the preparation of a medicament for a treatment to inhibit tumor growth or to inhibit angiogenesis.

[0023] The present invention also relates to methods of identifying TEMl antibodies with therapeutic efficacy. Provided herein is a method of identifying a TEMl antibody as inhibitory for tumor growth or angiogenesis comprising: contacting a TEMl -expressing cell with a candidate antibody; assessing the inhibitory activity of the antibody in a tube formation assay in vitro, wherein the tubes are formed by endothelial cells, endothelial precursor cells, or pericytes; assessing the inhibitory activity of the antibody in a proliferation assay in vitro, wherein the proliferating cell is an endothelial cell, endothelial precursor cells, or pericytes; and assessing the activity of the ADCC assay, phagocytosis assay, and/or a CDC assay, whereby the candidate antibody which inhibits the tube formation by endothelial cells, endothelial precursor cells, and pericytes, proliferation of endothelial cells, endothelial precursor cells and pericytes, and mediates ADCC, phagocytosis, and/or CDC is identified as an antibody which inhibits tumor growth or angiogenesis. Preferably, the antibody is a human antibody. In one embodiment, the method comprises contacting a TEMl -expressing cell with a candidate antibody; assessing the inhibitory activity of the antibody in a tube formation assay in vitro at concentrations <20 μg/ml, wherein the tubes are formed by endothelial cells, endothelial precursor cells or pericytes; assessing the inhibitory activity of the antibody in a proliferation assay in vitro at concentrations <20 μg/ml, wherein the proliferating cell is an endothelial cells, endothelial cells or a pericyte; and assessing the activity of the antibody in an antibody-dependent cell cytotoxicity assay, phagocytosis, and/or a complement-mediated cytotoxicity assay at concentrations <5 μg/ml, whereby the candidate antibody which inhibits the tube formation by endothelial cells, endothelial precursor cells, and pericytes, inhibits the proliferation of endothelial cells, endothelial precursor cells, and pericytes, and mediates ADCC, phagocytosis, and/or CDC activity is identified as an antibody which a candidate antibody which inhibits tumor growth or angiogenesis.

Modes of Carrying Out the Invention

[0024] The antibodies disclosed herein relate to the growth and function of endothelial cells and/or pericytes in vitro and in vivo. More particularly, these antibodies are specific for tumor endothelial marker TEMl . The disclosed antibodies likely modulate endothelial cell and/or pericyte activity in vivo {e.g., as a therapeutic agent) through one or more of several different mechanisms. First, the antibody can act directly on its target cell by modulating or inhibiting key signal transduction pathways. For example, an antibody can bind a critical ligand or its receptor and inhibit a necessary positive stimulus or, alternatively, induce a negative signal {e.g., one eliciting apoptosis). In this way, the antibody may inhibit cellular proliferation, migration, and/or tube formation by commandeering the cell's signal transduction machinery, or by simply trapping the TEMl -expressing cell. Second, the antibody can engage other components of the immune system to act on the target cell. For example, the antibody can be used to trigger antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDC). Third, the antibody can have intrinsic cytotoxicity. Additionally, the antibody can act indirectly on the target cell by simply binding its target and delivering a bioactive agent as its conjugate, e.g., a radionuclide or a toxin, with therapeutic benefit.

[0025] Targeting blood vessels has several unique advantages for tumor therapy. First, there is a minimal barrier between the luminal surface of blood vessels and intravenously administered agents, enhancing the efficiency of delivery to the targeted cells. Second, localized damage of a few blood vessels cells can lead to vascular occlusion, allowing the localized damage to achieve a more global effect. Third, blood vessels cells may not develop resistance to the antibody, permitting repeated administration of the antibody without any decrease in antibody efficacy.

TEMl antibodies

[0026] The target molecule for each of the disclosed antibodies is TEMl. TEMl, also known as endosialin, is a 165 kDa glycoprotein. Rettig, et al, Proc. Nat 'I Acad. Sd. U.S.A. 89: 10832-36 (1992), Christian, et al., J. Biol. Chem. 276: 7408-14 (2001). The sequence of TEMl was identified as that of the endosialin protein by St. Croix and colleagues in Science 289: 1197-1202 (2001). TEMl is a C-type lectin-like, type I membrane protein with a signal leader peptide, five globular extracellular domains, followed by a mucin-like region, a transmembrane segment and a short cytoplasmic tail. The N-terminal shows homology to thrombomdulin, a receptor involved in regulating blood coagulation and to complement receptor CIqRp. Webster, et al, J. Leuk. Biol. 67: 109-16 (2000). Murine and human TEMl share 77.5% amino acid identity with 100% identity in the transmembrane region. Opavsky, et al, J. Biol. Chem. 276: 38795-807 (2001). TEMl has a signal sequence at amino acids 1-17 and its transmembrane domain at amino acids 686-708. Its extracellular domain is at residues 1-685. TEMl expression varies with cell density (or cell cycle). Opavsky et al, supra (2001). TEMl is maximally expressed in confluent (G₀) cells, the most relevant phase of the cell cycle in vivo. The DNA sequence of TEMl is disclosed as SEQ ID NO. 196 in U.S. Serial No. 09/918715 (Publication No. 20030017157).

[0027] Thus, the antibodies of the present invention selectively bind TEMl . As used herein, the term "selective binding" refers to the preferential binding of the antibody to TEMl. In a preferred embodiment, the antibody is specific for TEMl . Antibodies can be tested for selective and/or specific binding to TEMl by comparing binding to TEMl to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to TEMl at least 2, 5, 7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific. Selective binding of an antibody to human TEMl is also shown by Kd value. In a specific embodiment, a human TEMl antibody has a Kd value of 10^"6M to 10^"9M, preferably 10^"10M or less. In one embodiment, the antibody is one that binds human TEMl , but does not bind non-human TEMl , e.g., murine TEMl . hi another embodiment, the TEMl antibody binds to a TEMl antigen of other species, e.g., murine TEMl antigen.

[0028] Provided herein is a monoclonal antibody, or biologically active fragment thereof, wherein the variable region sequences comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NOrIO, SEQ ID NOiI l and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:15 and SEQ ID NO:17. Also, provided herein is a monoclonal antibody, or a biologically active fragment thereof, wherein the antibody is originally isolated from or produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6055), and TEMl -#187(ATCC PTA-6066). The term "antibody" includes any polypeptide or protein comprising an antibody antigen binding domain present in the antibody of the present invention. Antibodies can include a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site for the target antigen. Such polypeptides or proteins include, but are not limited to Fab, scFv, Fv, diabodies, Fd, minibodies, and nanobodies. An antibody that is originally isolated from a hybridoma is one that has the amino acid sequence of the antibody produced by the hybridoma. This antibody can be produced by any suitable means, including recombinant production.

[0029] Variable region sequences of antibodies produced by the hybridoma TEM 1 -3 , TEMl -70, TEMl -#87, TEMl -97, TEMl -#132 and TEMl -#187 have been determined. The results were shown in Tables 1 and 3. In initial sequencing analysis, the variable region sequences of TEM1-170 and TEM1-184 were similar to that of TEM1-#187. The sequences for TEM1-#187H and TEMl -#187Hl represent sequences with variations in the framework regions of the antibody. Tables 2 and 4 provide the subclass identity of the antibodies disclosed herein. Table 1 Amino Acid Sequence of TEMl Antibody Light Chains

SEQ ID Clone

NO : No : Frame Region 1 -CDRl- Frame region 2

1 3K i EIVLTQSPAT LSLSPGERAT LSCRASQSVS S--YLAWYQQ KPGQAPRLLI

3 7 OK i EIVLTQSPGT LSLSPGERAT LSCRASQSVS SS-YLAWYQQ KPGQAPRLLI

5 87K 1 EIVLTQSPAT LSLSPGERAT LSCRASQSVS -S-YLAWYQQ KPGQAPRLLI

7 97K 1 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SS-YLAWYQQ KPGQAPRLLI

9 132K 1 EIVLTQSPAT LSLSPGERAT LSCRASQSVS -S-YLAWYQQ KPGQAPRLLI

11 170K 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS -SSYLAWYQQ KPGQAPRLLI 13 184K 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS -SSYLAWYQQ KPGQAPRLLI 15 187K 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS -SSYLAWYQQ KPGQAPRLLI

SEQ ID Clone NO : _No . CDR2— __ Frame Region 3

1 3K 49 YDASNRATGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC QQRSNWPPTF

3 7 OK 50 YGASSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSPLTF

5 87K 49 YDASNRATGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC QQRSNWPYTF

7 97K 50 YGASSRATGI PDRFGGSGSG TDFTLTISRL EPGDFAVYYC QQYGSSPITF

9 132K 49 YDASNRATGI PARFSGSGSG TDFTLTISSL EPEDFAVYYC QQRSNWPLTF

11 170K 50 YGASSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSPWTF 13 184K 50 YGASSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSPWTF 15 187K 50 YGASSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSPWTF

SEQ ID _{c lone}

NO : No : JK

1 3K 99 GQGTKVEI

3 7OK 100 GGGTKVEIK

5 87K 99 GQGTKLEIK

7 97K 100 GQGTRLEIK

9 132K 99 GGGTKVEIK

9a 132K 99 GQGTKVEIK

11 170K 100 GQGTKVEIK

13 184K 100 GQGTKVEIK

15 187K 100 GQGTKVEIK

Table 2 Subclass Identity of TEMl Antibody Light Chains

SEQ ID NO: Clone VK JK Locus

1 3 3 1 L6

3 70 3 4 A27

5 87 3 2 L6

7 97 3 5 A27

9 132 3 4 L6

11 170 3 1 A27

13 184 3 1 A27

15 187 3 1 A27

Table 3 Amino Acid Sequence of TEMl Antibody Heavy Chains

SEQ

ID Clone

NO: No: Frame Region 1 rcpiP1 TPv""πne Region 2_

2 3H 1 QVQVQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE

4 7OH 1 QVQLQESGPG LVKPSETLSL TCTVSGGSIS YYYWSWIRQP PGKGLEWIGY

6 87H 1 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE

8 97H 1 QVQLIQSGPE LKKPGASVKV SCKASGYTFT SYGLNWVRQA PGQGLEWMGW

10 132H 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SSSMNWVRQA PGKGLEWVSY

10a 132H 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SSSMNWVRQA PGKGLEWVSY

12 170H 1 QVQLQESAPG LVKPSETLSL TCTVSGGSIR SYYWSWIRQP PGKGLEYIGY

14 184H 1 QVQLQESAPG LVKPSETLSL TCTVSGGSIR SYYWSWIRQP PGKGLEYIGY

16 187H 1 QVQLQESAPG LVKPSETLSL TCTVSGGSIR SYYWSWIRQP PGKGLEYIGY

17 187Hl 1 QVQLQESGPG LVKPSETLSL TCTVSGGSIS SYYWSWIRQP PGKGLEWIGY

SEQ

ID Clone

NO: No: UJJKZ Frame Region 3

2 3H 51 INHGGS-TNY NPSLKSRVTI SIDTSKNQVS LKLSSVTAAD TAVYYCAD—

4 7OH 51 IYSSGS-TNY NPSLKSRVTI SVDTSKNQFS LKLSSVTAAD TAVYYCARGL

6 87H 51 INHSGS-TNY NPSLKSRVTI SVDTSKNQCS LKLSSVTAAD TAVYYCAD—

8 97H 51 INTNTGNPAY AQGFTGRFVF SLDTSVSTAY LQISSLKAED TAVYYCAKDL

10 132H 51 ISSGSGNIYY ADSAKGRFTI SRDNAKNSLY LQMSSLRDED TAVYYCARVT

10a 132H 51 ISSGSGNIYY ADSAKGRFTI SRDNAKNSLY LQMSSLRDED TAVYYCARVT

12 170H 51 IYYTGSAI-Y NPSLQSRVTI SVDTSKNQFS LKLNSVTAAD TAVYYCAREG

14 184H 51 IYYTGSAI-Y NPSLQSRVTI SVDTSKNQFS LKLNSVTAAD TAVYYCAREG

16 187H 51 IYYTGSAI-Y NPSLQSRVTI SVDTSKNQFS LKLNSVTAAD TAVYYCAREG

17 187Hl 51 IYYTGSAI-Y NPSLQSRVTI SVDTSKNQFS LKLSSVTAAD TAVYYCAREG

Table 3 Amino Acid Sequence of TEMl Antibody Heavy Chains

SEQ

ID Clone

WO: No: CDR3 JH -CHl

2 3H 99 SGYDF- DY WGQGTLV TVSSASTKGP SVFPLAPSSK

4 7OH 100 WLHG— Y WGQGTLV TVSSASTKGP SVFPLAPSSK

6 87H 98 SGYVF- DY WGQGTLV TVSSASTKGP SVFPLAPSSK

8 97H 101 GEPPWVNW— FDP WGQGTLV TVSSASTKGP SVFPLAPSSK

10 132H 101 GDL FYYY GM -DVWGQGTTV TVSSASTKGP SVFPLAPSSK

10a 132H 101 GDL FYYY GM -DVWGQGTTV TVSSASTKGP SVFPLAPCSR

12 170H 100 VRGASGYYYY GM -DVWGQGTTV TVSSASTKGP SVFPLAPSSK

14 184H 100 VRGASGYYYY GM -DVWGQGTTV TVSSASTKGP SVFPLAPSSK

16 187H 100 VRGASGYYYY GM -DVWGQGTTV TVSSASTKGP SVFPLAPSSK

17 187Hl 101 VRGASGYYYY GM -DVWGQGTTV TVSSASTKGP SVFPLAPSSK

SEQ

ID Clone

NO: No:

2 3H 132 STSGGTAALG CLVK 4 7OH 132 STSGGTAALG CLVK 6 87H 132 STSGGTAALG CLVK 97H 139 STSGGTAALG CLVK

10 132H 139 STSΞSTAALG CLVK

10a 132H 139 STSESTAALG CLVK 12 170H 141 STSGGTAALG CLVK 14 184H 141 STSGGTAALG CLVK 16 187H 141 STSGGTAALG CLVK 17 187Hl 141 STSGGTAALG CLVK

Table 4 Subclass Identity of TEMl Antibody Heavy Chains

[0030] Any suitable framework regions (FR) with the CDRs disclosed herein. In some embodiments, consensus FR sequences may be employed. For example, the heavy chain FRl sequence can be used with the TEMl -#187 antibody may include one of the sequences listed below in Table 5.

Table 5

[0031] As used herein, the term "antigen binding domain" refers to the part of an antibody molecule which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. The antigen binding domain may be provided by one or more antibody variable domains (e.g., a Fd antibody fragment consisting of a V_H domain). Preferably, an antigen binding domain of an antibody comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (V_H)-

[0032] Also provided herewith is a hybridoma strain selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl ~#132(ATCC PTA-6065) and TEM 1-#187(ATCC PTA- 6066). Hybridoma TEM1-#87(ATCC PTA-6064), TEM1-#132(ATCC PTA-6065) and TEMl -#187(ATCC PTA-6066) were deposited to American Type Culture Collection (ATCC) (Manassas, VA 20108, USA) under the Budapest Treaty, on June 9, 2004.

[0033] The antibodies of the present invention are useful in any suitable form that retains at least one desirable biologic activity of the intact antibody. In one embodiment, the antibody is Fab fragment, F(ab')₂ fragment, a scFv, a diabody, a minibody, a nanobody, a multivalent single chain antibody, or an intact antibody molecule. "Single-chain Fv" or "scFv" antibody fragments comprise the V_H and V_L domains of the variable region of an antibody present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and V_L domains which enables the scFv to form the desired structure for antigen binding. See, e.g., Filipula et al, "Production of single chain Fv monomers or multimers" IN ANTIBODY ENGINEERING: A PRACTICAL APPROACH 253-68 (McCafferty et al., eds., Oxford University Press 1996). Fragments of intact molecules can be generated using methods well known in the art and include enzymatic digestion and recombinant means. See, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY (Cooligan et al, eds., most recent edition). Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the V_H and V_L domains. See, e.g., Reiter et al, Nature Biotech, 14:1239-45 (1996). Diabodies comprising multivalent or multispecific fragments constructed by gene fusion may also be used with the antibodies disclosed herewith. See, e.g., US 6,589,527; Holliger et al, Proc. Natl. Acad. ScL USA 90:6444-48 (1993). Diabodies and scFvmay be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Minibodies comprising a scFv joined to a CH3 domain may also be made using the antibody of the present invention. See, e.g., U.S. 5,837,821; Ba et al, Cancer Res. 56:3055-61 (1996). Nanobodies comprising single variable region (VHH) domain, originally characterized in camels and llamas can also be employed. See, e.g., Davies et al, BioTechnology 13:475-79 (1995); Cortez-Retamozo, et al, Cancer Res. 64:2853-57 (2004).

[0034] The fragments useful in the present methods are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that is capable of binding the desired, antigenic epitope and directly or indirectly exerting a biologic effect. Direct effects include, but are not limited to the inhibition of a growth signal, the inhibition of an anti-apoptotic signal, the elicitation of an apoptotic or necrotic signal, the initiation of the ADCC cascade, the elicitation of phagocytosis, and the initiation of the CDC cascade. Indirect effects include, but are not limited to toxicity due to conjugate delivery (e.g., radionuclide, toxin, drug, or other bioactive agent) or sensitization to secondary agents (e.g., delivery of agent that becomes toxic after exposure to additional agent, e.g., radiation).

[0035] Also provided herein is a monoclonal antibody, or a biologically active fragment thereof, comprising the same sequence of amino acids of the variable region as the TEMl antibodies originally produced or isolated from a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEM 1-#187(ATCC PTA-6066). It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the selective binding properties and/or specificity of the original antibody. Such techniques may involve joining DNA encoding an immunoglobulin variable region to a constant region, or introducing the complementarity determining regions (CDR' s) (CDR-grafting), of an antibody into the constant region plus framework regions, of a different immunoglobulin. For example, the selective binding of the antibody of the present invention may be artificially inserted and/or reconstructed by incorporating the amino acid sequence of the variable region or one or more of the complementarity determining regions (CDR' s) into the framework of another antibody or antibody-like molecule. See, e.g., Jones et al, Nature 321:522 (1996); EP-A-184187; GB 2188638A; EP-A-239400; CURRENT PROTOCOLS IN IMMUNOLOGY 2.12.1-12.15 (Cooligan et al., eds. current edition). The antibody of the present invention may also be "resurfaced" or "veneered", e.g., replacing the surface residues with the most common residues found in the acceptor, e.g., human, antibodies and those which use differing definitions of the extents of the CDR's (from the donor antibodies). A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the selective binding and/or specificity of antibodies produced.

[0036] The variable region and the associated CDR's of the present invention include completely identical sequences as well as those variants that retain the specificity of the source antibody. Thus, the variable region sequence and its associated CDR's may have one or more different amino acids from the source sequence. In one embodiment, the antibody is an IgG antibody, preferably an IgG₁ antibody. In a specific embodiment, the antibody is human, humanized, or chimeric. Alternatively, human TEMl antibodies can be murinized for greater cross-reactivity in murine disease models. Well known methods can be used to generate such antibodies. See, e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH (McCafferty et al, eds., Oxford University Press 1996).

[0037] The identification and employment of a CDR or a set of CDR's of the invention will generally be that of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDR's is located at a location corresponding to the CDR or set of CDR's of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat et al, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (4th Ed. 1987). Variable domains employed in the invention may be obtained from any germ-line or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. A CDR sequence of the invention may be introduced into a repertoire of variable domains lacking a CDR, using recombinant DNA technology.

[0038] For example, Marks et al. describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VK variable domains lacking a CDR2. Bio/Technology 10:779-83 (1992). Marks et al. further describe how this repertoire may be combined with a CDR2 of a particular antibody. Using analogous techniques, the CDR3- derived sequences of the present invention may be shuffled with repertoires of VH or V_L domains lacking a CDR3, and the shuffled complete VH or V_L domains combined with a cognate V_L or V_H domain to provide specific binding members of the invention. The repertoire may then be displayed in a suitable host system such as the phage display system of, e.g., W092/01047; Kay et al PHAGE DISPLAY OF PEPTIDES AND PROTEINS: A LABORATORY MANUAL (Academic Press 1996), so that suitable specific binding members may be selected. A repertoire may consist of from anything from 10⁴ individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰ members. Other suitable host systems include yeast display, bacterial display, T7 display, ribosome display and so on.

[0039] A further alternative is to generate novel VH or V_L regions carrying CDR-derived sequences of the invention using random mutagenesis of one or more selected V_H and/or V_L genes to generate mutations within the entire variable domain. One useful technique is error- prone PCR. See, e.g., Gram et al, Proc. Natl. Acad. ScL, USA 89:3576-80 (1992). In preferred embodiments, one or two amino acid substitutions are made within a set of heavy chain CDR's and/or light chain CDR's. Yet another method which may be used is to direct mutagenesis to CDR regions of V_H or V_L genes. Such techniques include, e.g., those disclosed in Barbas et al, Proc. Natl. Acad. Sd. USA, 91 :3809-13 (1994); Schier et al, J. MoI Biol. 263:551-67 (1996).

[0040] Bispecific antibodies may also be used with the antibodies and their associated variable regions and CDR's as disclosed herein. These may be conventional bispecific antibodies and can be manufactured in a variety of ways that include, but is limited to being prepared chemically or from hybrid hybridomas. See, e.g., Holliger et al, Current Opinion Biotechnol. 4:446-49 (1993). Examples of bispecific antibodies include those of the BiTE™ technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain. Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display from libraries. See, e.g., W094/13804. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against TEMl , then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. In one embodiment, bispecific whole antibodies may be made by knobs- into-holes engineering. See, e.g., Ridgeway et al., Protein Eng. 9:616-21, (1996).

[0041] All the above described techniques are known as such in the art and in themselves do not form part of the present invention. One of ordinary skill in the art will be able to use such techniques to provide specific binding members of the invention using routine methodology in the art.

[0042] The antibodies comprising the variable region amino acid sequence or one or more CDR' s of the TEMl antibodies disclosed herein can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, the antibody useful in the present methods is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one V_L and one V_H region in the host cell. In one embodiment, full length or variable regions of antibody gene can be cloned from hybridomas TEM1-#87(ATCC PTA- 6064), TEMl -#132(ATCC PTA-6065), or TEMl -#187(ATCC PTA-6066), followed by insertion into an antibody expression vector. Heavy and light chain variable region sequences can be joined to constant region sequence within an antibody expression vector. Such antibody expression vector is disclosed, for example, in US Patent 6,001,358. Heavy chain constant region of any class can be selected. The antibodies can be expressed in any suitable cell line. In one embodiment, the cell line is the CHO cell line. The preferred antibody class is IgG. The subclass of IgG can be selected from IgG₁, IgG₂, IgG₃ and IgG₄. The subclasses Of IgG₁, IgG₂ and IgG₃ are preferred if ADCC, CDC, and/or phagocytosis activity are necessary or expected for the desired therapeutic or biologic effect. When ADCC, phagocytosis, and/or CDC activity are necessary or expected for the desired therapeutic effect, IgG₁ is the most preferred embodiment.

[0043] Exemplary descriptions of recombinant means of antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et ah, MONOCLONAL ANTIBODIES (Oxford University Press, 2000); and Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993). Synthetic antibody molecules may be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et ah, J. MoI. Biol. 296:57-86 (2000); Krebs et ah, J. Immunol. Methods 254:67-84 (2001). Transgenic or genetically engineered mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies to human antigens.

[0044] A further aspect of the invention provides an antibody antigen binding domain specific for TEMl antigen, wherein the antibody antigen binding domain is one of the domains of the disclosed antibodies with an addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a V_H domain of an antibody of the invention, wherein the VH domain which is an amino acid sequence variant of the VH domain that retains the desired functional activity of the original TEMl antibody. In some embodiments at least one amino acid of the heavy chain is deleted, added, or substituted with an amino acid different from the original amino acid. Any suitable method can be used to modify the antibodies of the present invention. Typically, the substitutions will be conservative substitutions. See, e.g., U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and Angal, et ah, MoI. Immunol. 30:105-08 (1993). The modified amino acid residues in the amino acid sequence of the heavy chain are 40% or less, preferably 30% or less, more preferably 20% or less within the entire heavy chain. Modifications can facilitate or induce enhancement of desirable biologic activities, e.g., increased ADCC activity or serum half-life, or decrease an undesired biologic activity, e.g., immunogenicity. See, e.g., Sandlie et ah, "Choosing and manipulating effector functions" IN ANTIBODY ENGINEERING: A PRACTICAL APPROACH 187-202 (McCafferty et al., eds., IRL Press 2002). [0045] Provided herein is a monoclonal antibody, or a biologically active fragment thereof, wherein the antibody recognizes substantially the same epitope of TEMl recognized by the antibodies disclosed herewith. Such antibodies can be generated using well known techniques employing TEMl nucleic acid or protein or an immunogenic fragment thereof as the immunogen. To screen for antibodies which bind to a particular epitope on TEMl, a routine competition assay can be performed. See, e.g., ANTIBODIES: A LABORATORY MANUAL (Harlow et al, eds., Cold Spring Harbor Laboratory 1988). Alternatively, epitope mapping can be performed to determine whether the antibody binds an epitope of interest. See, e.g., Champe et al, J. Biol. Chem. 270: 1388-94 (1995); EPITOPE MAPPING: A PRACTICAL APPROACH (Olwyn et al, Oxford University Press 2001). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, single-cell clones may be subcloned by limiting dilution procedures and grown by methods well known in the art. See, e.g., ANTIBODIES: A LABORATORY MANUAL (Harlow et al., eds., Cold Spring Harbor Laboratory 1988). Such antibodies can be human, humanized, or chimeric antibodies generated by well known techniques in the art.

[0046] The TEMl antibodies of the present invention can also be employed in heteroconjugate antibodies. As used herein, the term "heteroconjugate antibody" refers to two co valently joined antibodies. Such antibodies can be prepared using known methods in synthetic protein chemistry, including using crosslinking agents. See, e.g., U.S. Patent No. 4,676,980.

[0047] It is also contemplated that the TEMl antibody of the present invention may be conjugated to a bioactive agent. As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that enhances or mediates a desired biological effect. In one embodiment, the desired biological effect is stasis or cell death (e.g., apoptosis). In another embodiment, the desired biological effect results from the antibody sensitizing the target cell to a secondary agent that induces stasis or cell death.

[0048] A fusion protein comprising the TEMl antibody of the present invention and a bioactive agent is also contemplated. Well known methods in recombinant DNA technology can be used to generate and produce such fusion proteins.

[0049] Bioactive agents include, for example, a pharmaceutical agent, such as a chemotherapeutic drug, a toxin, a cytokine, a ligand, another antibody, regulatory moieties such as zinc fingers and leucine zippers, or some combination thereof. In one embodiment, the agent in an antitumor agent. As used herein, the term "antitumor agent" refers to agent that inhibits tumor growth through the induction of an immune response, stasis, cell death, senescence, apoptosis, ankoisis or necrosis, hi one embodiment, the agent is an antiangiogenic agent. As used herein, the term "antiangiogenic agent" refers to an agent that inhibits endothelial cell and pericyte proliferation, tube formation, or some combination thereof, through the induction of an immune response, stasis, cell death, or senescence, apoptosis, ankoisis, necrosis.

[0050] Suitable agents for coupling to antibodies include cytokines, such as IL-2, IL- 12, interferon (IFN), Tumor Necrosis Factor (TNF); photosensitizers (for use in photodynamic therapy), including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as indium-Ill (¹¹¹In), iodine-131 (¹³¹I), yttrium-90 (⁹⁰Y), bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), technetium-99m (^99mTc), rhenium-186 (¹⁸⁶Re), and rhenium- 188 ( Re); antibiotics, such as doxorubicin, daunorubicin, methotrexate, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, mystatin, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-α toxin, cytotoxin from Chinese cobra (naja naja atra), and gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus), saporin (a ribosome inactivating protein from Saponaria officinalis^'), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing antitumor agents {e.g., antisense oligonucleotides, siRNA, plasmids encoding toxins, methotrexate, etc.); other antibodies or antibody fragments, such as F(ab); antiangiogenic agents including protamine, heparin, steroids, thalidomide, TNP -470, carboxyamidotriazole (CAI), interferon alpha (IFN-α), angiostatin, endostatin, and Avastin™ (ami- VEGF); enzymes (e.g., asparaginase); and catalytic nucleic acid, (e.g., hammerhead ribozymes). Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.

[0051] The antibody of the present invention may also be labeled for use in detection assays and methods. The antibody may be labeled with any suitable label, including but not limited to a detectable or functional label. Detectable labels include radiolabels such as ¹³¹I or

TC, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin, green fluorescence protein (GFP), red fluorescence protein (RFP), enhanced green fluorescence protein (EGFP), FITC, etc., which may be detected via binding to a specific cognate detectable moiety, e.g. labeled avidin or by excitation with a specific wavelength. Such antibodies are designed to be used in methods of diagnosis or treatment in human or animal subjects, preferably human.

[0052] Further provided herein is a monoclonal antibody that binds to TEMl , wherein the antibody inhibits endothelial cell proliferation in the range of 70-90% in vitro when 0.1 μg of the antibody is added to 5xlO⁵ endothelial cells and incubated at 37°C for 15 minutes.

[0053] In one embodiment, the antibody inhibits angiogenesis, particularly where the angiogenesis promotes or causes diseases such as cancer, polycystic kidney disease, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and psoriasis.

[0054] In one embodiment, the antibody inhibits tumor growth. The tumor can an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma. Additionally, the tumor may be a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, rectum, skin, spleen, testis, thymus, thyroid, or uterus.

[0055] In another embodiment, kits are provided that contain the necessary reagents to carry out the assays of the methods provided herein. Specifically provided herein is a compartment kit comprising one or more containers, wherein a first container comprises one or more antibodies selectively binding and/or specific for TEMl, and one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound antibody, diluent. The containers can be glass, plastic, or strips of plastic or paper. Types of detection agents include labeled secondary antibodies, other labeled secondary binding agents, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents that are capable of reacting with the labeled antibody.

Pharmaceutical Compositions of TEMl Antibodies

[0056] Also provided herein is a pharmaceutical composition comprising an amount of the monoclonal antibody, or a biologically active fragment thereof, of the TEMl antibody and a suitable excipient. hi one embodiment, the TEMl antibody, or biologically active fragment thereof, has a variable region sequence that comprises SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:15 and SEQ ID NO: 17. Further provided a pharmaceutical composition comprising a TEMl antibody originally produced by or isolated from the hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065, or TEMl -#187(ATCC PTA- 6066). The antibody of the pharmaceutical composition can also be one with the same variable region amino acid sequence as that of the antibody originally produced by or isolated from the hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl- #132(ATCC PTA-6065), or TEMl -#187(ATCC PTA-6066).

[0057] Pharmaceutical compositions are therapeutic agents that contain as an active ingredient the antibody of the present invention and also may contain pharmaceutically acceptable carriers, excipients, or additives depending on the route of administration. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The pharmaceutical composition of the invention may also contain other bioactive agents such as cytokines or chemotherapeutic agents.

[0058] Pharmaceutical compositions for use in accordance with the present methods thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.

[0059] When an effective amount of antibody of the methods herein is administered by intravenous, cutaneous, intramuscular, or subcutaneous injection, the antibody of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, intramuscular, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The antibodies may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0060] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0061] For administration by inhalation, the antibodies of the present methods are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. hi the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0062] Topical routes include administration in the form of salves, creams, jellies, ophthalmic drops or ointments, ear drops, suppositories, irrigation fluids for use in, e.g., the cleaning of wounds, or medicated shampoos. The antibodies of the present invention may also be administered transdermally using, e.g., a transdermal patch. In some embodiments, the antibody of the present invention can be administered in a topical administration in drop form with about 10 to 200 μL of the pharmaceutical composition being applied one or more times per day as determined by the treating physician.

Methods of Using TEMl Antibodies

[0063] The TEMl antibodies of the present invention are useful as therapeutic agents in methods to modulate tumor growth or unwanted angiogenesis. Provided herein is a method of inhibiting tumor growth, comprising: administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO.4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 15 and SEQ ID NO: 17, whereby the antibody inhibits tumor growth. In some embodiments, the antibody is an antibody originally produced by hybridomas TEM1-#87(ATCC PTA-6064), TEM1-#132(ATCC PTA-6065), or TEMl -#187(ATCC PTA-6066). It is contemplated that the subject expresses TEMl. In some embodiments, the antibody of the method is one with the same variable region sequence as that of the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEM1-#187(ATCC PTA- 6066). The TEMl antigen expressed by the subject include, but are not limited to allelic variants and other variants that are substantially similar to the known TEMl antigen. The antibody can be an intact antibody molecule, a scFv, a Fab fragment, or a F(ab')₂ fragment. In some embodiments, the antibody is conjugated to a bioactive agent, preferably an antitumor agent or an antiangiogenic agent. In some embodiments, the agent is a radionuclide. The tumor treated by this method can be an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma. hi some embodiments, the tumor is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus.

[0064] Also provided herein is a method of inhibiting angiogenesis, comprising: administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the variable region sequence of the antibody comprises SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:15 and SEQ ID NO:17, whereby the antibody inhibits angiogenesis. In some embodiments, the antibody is an antibody originally produced by or isolated from a hybridoma selected from the group consisting of TEMl -#87( ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), or TEMl -#187(ATCC PTA-6066). In some embodiments, the antibody of the method is one with the same variable region sequence as that of the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl -#187(ATCC PTA-6066). The subject treated by this method expresses the antigen bound by the antibody. The TEMl antigen expressed by the subject include, but are not limited to allelic variants and other variants that are substantially similar to the known TEMl antigen. In some embodiments, the angiogenesis targeted by this method is neoangiogenesis. The antibody can be an intact antibody molecule, a scFv, a Fab fragment, or a F(ab')₂ fragment. In some embodiments, the antibody is conjugated to a bioactive agent, preferably an antiangiogenic agent or an antitumor agent. In some specific embodiments, the subject treated by this method has a disease such as cancer, polycystic kidney disease, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, or psoriasis. Any subject can be treated with the methods and compositions provided herein. Such a subject is a mammal, preferably a human, with an angiogenesis associated disease or symptom. In one specific embodiment, the subject has cancer.

[0065] Veterinary uses of the disclosed methods and compositions are also contemplated. Such uses would include treatment of angiogenesis-related diseases and cancer, in domestic animals, livestock and thoroughbred horses. Thus, the TEMl antibodies of the instant invention are also those that are cross-reactive with xenogeneic TEMl homologues.

[0066] Subjects treated by these methods express TEMl . A screening or diagnostic analysis of patient samples can be performed prior to the initiation of treatment using TEMl antibody therapy. Such diagnosis analysis can be performed using any sample, including but not limited to cells, protein or membrane extracts of cells, biological fluids such as sputum, blood, serum, plasma, or urine, or biological samples such as formalin-fixed or frozen tissue sections employing the antibodies of the present invention. Any suitable method for detection and analysis of TEMl expression can be employed.

[0067] The reactivities of antibodies in a sample may be determined by any appropriate means. Radioimmunoassay (RIA) is one possibility. Radioactive labeled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.

[0068] Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are colored, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colors or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin, biotin/streptavidin, alkaline phosphatase, or horseradish peroxidase detection systems may be employed. The signals generated by individual antibody-reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant antibody binding in samples (normal and test).

[0069] The present invention also provides the use of a TEMl antibody as above for measuring antigen levels in a competition assay. This may be where the physical separation of bound from unbound antigen is not required. Linking a reporter molecule to the specific binding member so that a physical or optical change occurs on binding is one possibility. The reporter molecule may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g., a peptide bond, or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

[0070] The TEMl antibody is also useful in measuring levels of antigen directly, by employing a specific binding member according to the invention for example in a biosensor system.

[0071] The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

[0072] A kit comprising a pharmaceutical composition comprising a TEMl antibody according to any aspect or embodiment of the present invention is also provided. In a kit of the invention, the antibody may be labeled to allow its reactivity in a sample to be determined. Components of a kit are generally sterile and in sealed vials or other containers. Kits may be employed in diagnostic analysis or other methods for which antibody molecules are useful. A kit may contain instructions for use of the components in a method, e.g. a method in accordance with the present invention. Ancillary materials to assist in or to enable performing such a method may be included within a kit of the invention, hi one embodiment, the TEMl antibody is provided in a kit used to pre-screen subjects to determine suitability, i.e., presence of TEMl expression, for therapeutic administration of TEMl antibody.

[0073] As used herein, "inhibit" or "treat" or "treatment" includes a postponement of development of the symptoms associated with uncontrolled angiogenesis, tumor growth and/or a reduction in the severity of such symptoms that will or are expected to develop. The terms further include ameliorating existing uncontrolled or unwanted angiogenesis-related or tumor growth-related symptoms, preventing additional symptoms, and ameliorating or preventing the underlying metabolic causes of symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject with an angiogenesis-associated disease or symptom, particularly cancer, or with the potential to develop such a disease or symptom, hi particular, the terms "inhibit", "treat", or "treatment" include the successful diminution or eradication of established tumor growth.

[0074] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a TEMl antibody that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the angiogenic- and/or tumor-associated disease condition or the progression of the disease. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0075] The antibody of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be administered to a subject in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.

[0076] In practicing the methods of treatment or use provided herein, a therapeutically effective amount of antibody provided herein is administered to a mammal having a condition to be treated. The antibody may be administered in accordance with the methods herein either alone or in combination with other therapies such as treatments employing hematopoietic factors {e.g., cytokines), chemotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, and the like. When co-administered with one or more biologically active agents, the antibody provided herein may be administered either simultaneously with the biologically active agent(s), or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with the biologically active agent(s).

[0077] The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the area to be treated, the precise nature of the antibody {e.g., whole antibody, fragment or diabody), and the nature of any detectable label or other molecule attached to the antibody. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Goodman et ah, eds., McGraw-Hill Professionals, 9th Ed. 1996). Dosage amount and interval maybe adjusted individually to provide plasma levels of the active moiety sufficient to maintain the desired therapeutic effects, or minimal effective concentration (MEC). A typical antibody dose will be in the range 100 μg to 1 gm for systemic applications, and 1 μg to 1 mg for topical applications. Typically, the antibody will be a whole antibody, preferably the IgG₁ isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at, e.g., daily, twice- weekly, weekly, every 21 days, every 28 days, or monthly intervals, at the discretion of the physician. In preferred embodiments of the present invention, treatment is periodic, and the period between administrations is, e.g., weekly, sometimes about two weeks or more, preferably about three weeks or more, more preferably about four weeks or more, or about once a month.

[0078] The TEMl antibody of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.

[0079] The mode of administration is not particularly important. In one embodiment, the mode of administration is an LV. bolus. The prescribing physician will normally determine the dosage of the antibodies provided herein. It is to be expected that the dosage will vary according to the age, weight and response of the individual patient.

[0080] Techniques for formulation and administration of the antibodies of the instant methods may be found in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Easton, Pa., latest edition. Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of antibody used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection. Intravenous administration to the patient is preferred.

[0081] Alternately, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into an arthritic joint, fibrotic tissue, or a tumor, often in a depot or sustained release formulation. Furthermore, one may administer the antibody in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, arthritic or fibrotic tissue or a tumor. The liposomes will be targeted to and taken up selectively by the afflicted tissue.

[0082] The amount of antibody useful in the disclosed methods in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments that the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of antibodies of the present methods and observe the patient's response. Larger doses of antibodies of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the methods herein should contain about 0.01 μg to about 100 mg, preferably about 0.1 μg to about 15 mg, more preferably about 0.5 μg to about 1 mg, of antibody of the present invention per kg body weight. In one embodiment, the dose is about 10 to about 15 mg/kg. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than an antibody of the present methods that may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.

[0083] The antibody provided herein can be administered alone or in combination with other therapeutic modalities. For example, the treatment method can further comprise a step of delivering ionizing radiation to the cells contacted with the antibody. The ionizing radiation is delivered in a dose sufficient to induce a substantial degree of cell killing of the malignant cells, as judged by assays measuring viable malignant cells. The degree of cell killing induced is substantially greater than that induced by either the antibody alone or the ionizing radiation alone. Typical forms of ionizing radiation include beta rays, gamma rays, alpha particles, and X-rays. These can be delivered from an outside source, such as X-ray machine or a gamma camera, or delivered to the malignant tissue from radionuclides administered to the patient. The use of radionuclides is well understood in the art and need not be detailed further. The use of ionizing radiation in the treatment of malignancies is described, for example, in S. Hellman, "Principles of Radiation Therapy" IN CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY 248 (V. T. DeVita, Jr., et ah, eds., 4th ed., 1993). A range of dosages that can be used is between about 1 and 500 cGy (i.e., from about 1 to about 500 rads). [0084] The TEMl antibody also can be administered alone or in combination with other antibodies identified as inhibitors of endothelial cell and/or pericyte proliferation and/or tube formation using the methods disclosed herein. The co-administered antibodies can be specific for different epitopes of TEMl, a different TEM, or TEMl and another angiogenesis-inhibitory or antitumor target.

[0085] Any disease where angiogenesis is implicated can be treated with the present methods. Such diseases include, but are not limited to neoplasms of the central nervous system: glioblastoma multiforme, astrocytoma, oligodendroglial tumors, ependymal and choroids plexus tumors, pineal tumors, neuronal tumors, medulloblastoma, schwannoma, meningioma, meningeal sarcoma: neoplasm of the eye: basal cell carcinoma, squamous cell carcinoma, melanoma, rhabdomyosarcoma, retinoblastoma; neoplasm of the endocrine glands: pituitary neoplasms, neoplasms of the thyroid, neoplasms of the adrenal cortex, neoplasms of the neuroendocrine system, neoplasms of the gastroenteropancreatic endocrine system, neoplasms of the gonads; neoplasms of the head and neck: head and neck cancer, oral cavity, pharynx, larynx, odontogenic tumors: neoplasms of the thorax: large cell lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, neoplasms of the thorax, malignant mesothelioma, thymomas, primary germ cell tumors of the thorax; neoplasms of the alimentary canal: neoplasms of the esophagus, neoplasms of the stomach, neoplasms of the liver, neoplasms of the gallbladder, neoplasms of the exocrine pancreas, neoplasms of the small intestine, vermiform appendix and peritoneum, adenocarcinoma of the colon and rectum, neoplasms of the anus; neoplasms of the genitourinary tract: renal cell carcinoma, neoplasms of the renal pelvis and ureter, neoplasms of the bladder, neoplasms of the urethra, neoplasms of the prostate, neoplasms of the penis, neoplasms of the testis; neoplasms of the female reproductive organs: neoplasms of the vulva and vagina, neoplasms of the cervix, adenocarcinoma of the uterine corpus, ovarian cancer, gynecologic sarcomas; neoplasms of the breast; neoplasms of the skin: basal cell carcinoma, squamous carcinoma, dermatofibrosarcoma, Merkel cell tumor; malignant melanoma; neoplasms of the bone and soft tissue: osteogenic sarcoma, malignant fibrous histiocytoma, chrondrosarcoma, Ewing's sarcoma, primitive neuroectodermal tumor, angiosarcoma; neoplasms of the hematopoietic system: myelodysplastic syndromes, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, HTLV-I, and T-cell leukemia/lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, mast cell leukemia; neoplasms of children: acute lymphoblastic leukemia, acute myelocytic leukemias, neuroblastoma, bone tumors, rhabdomyosarcoma, lymphomas, renal and liver tumors. Other diseases include polycystic kidney disease; diabetic retinopathy; rheumatoid arthritis; psoriasis; osteoarthritis; adenocarcinoma; leukemia; lymphoma; melanoma; sarcoma; teratocarcinoma; acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas; macular degeneration; retinopathy of prematurity; corneal graft rejection; neo vascular glaucoma and retrolental fibroplasia. Other diseases associated with angiogenesis include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, corneal graph rejection, and chronic inflammatory diseases.

[0086] The antibody of the present invention also modulates the proliferation and activity of pericytes. Diseases associated with abnormal pericyte proliferation include hypertension, vascular disease, atherosclerosis (including formation of vascular calcifications and atherosclerotic plaques), restenosis, acute respiratory distress syndrome (ARDS), endometriosis or adenomyosis, asthma, asthmatic bronchitis, and normal aging. In particular, sequelae of hypertension, atherosclerosis and other vascular diseases include cerebrovascular ischemia or stroke, coronary artery disease and myocardial ischemia or infarction, peripheral vascular disease, Raynaud's syndrome, early occlusion of peripheral arteries or vascular remodeling associated with pulmonary hypertension.

Methods of Identifying Biologically Active TEMl Antibodies [0087] The present invention also provides methods of identifying an antibody as inhibitory for tumor growth or angiogenesis. More specifically, a method of identifying a TEMl antibody as inhibitory for tumor growth or angiogenesis comprises contacting a TEMl- expressing cell with a candidate antibody; assessing the inhibitory activity of the antibody in a tube formation assay in vitro, wherein the tubes are formed by endothelial cells, endothelial precursor cells or pericytes; assessing the inhibitory activity of the antibody in a proliferation assay in vitro, wherein the proliferating cell is an endothelial cell, endothelial precursor cells, or pericyte; and assessing the activity of the antibody in an ADCC, phagocytosis, and/or CDC assay, whereby the candidate antibody which inhibits the tube formation by endothelial cells, endothelial precursor cells, and/or pericytes, proliferation of endothelial cells, endothelial precursor cells, and/or pericytes, and mediates ADCC, phagocytosis, and/or CDC is identified as an antibody which inhibits tumor growth or angiogenesis. Preferably, the antibody is a human or humanized antibody.

[0088] Critical factors in selecting suitable TEMl antibodies include tight binding affinity, inhibitory activity in cell based assays including proliferation and tube formation (e.g., functional neutralization), activity in ADCC, CDC, and/or phagocytosis assays, reduction of tumor/disease site vasculature, and antitumor activity in tumor models in mice. In one embodiment, the binding affinity of the candidate antibody to the human TEMl extracellular domain is 1-5 x 10⁹ M. In some embodiments, the inhibitory activity in candidate antibodies in proliferation and tube formation assays is observed at < 120 μg/ml, preferably at < 20 μg/ml. In some embodiments, the candidate antibody demonstrates antitumor activity in vivo of greater than 5 days of tumor growth delay in syngeneic tumor models.

[0089] Any suitable method for assessing cellular proliferation may be used. In one embodiment, the method of inhibiting proliferation in a cell comprises the step of contacting target cells with a TEMl antibody, or biologically active fragment thereof, for a certain duration and then determining proliferation relative to target cells not treated with the antibody or treated with a non-binding antibody.

[0090] An antibody is inhibitory for proliferation if it inhibits the proliferation of cells relative to the proliferation of cells in the absence of the antibody or in the presence of a non- binding antibody. Proliferation may be quantified using any suitable methods. Typically, the proliferation is determined by assessing the incorporation of radioactive-labeled nucleotides into DNA (e.g., ³H-thymidine). In one embodiment, proliferation is determined by ATP luminescence. In a specific embodiment, proliferation is determined using the CellTiter-Glo™ Luminescent Cell Viability Assay (Promega).

[0091] In one embodiment, antibodies that block the function of TEMl associate with the enhanced proliferation of tumor endothelial cells and/or pericytes are most beneficial to subjects whose malignant disease generates measurable levels of endothelial precursor cells, thus indicating that the tumor vasculature of their disease originates, in some part, from proliferation of endothelial cells on co-opted existing vessels.

[0092] Any suitable method for assessing tube formation may be used for the method herein. In one embodiment, the method of inhibiting tube formation comprises the steps of contacting endothelial cells or pericytes with a TEMl antibody, or biologically active fragment thereof, in the presence of a suitable matrix, incubating the cells and antibody within the matrix, assessing the tube formation, whereby the antibody is inhibitory to tube formation when the number of tubes formed or the character of the tubes in the absence of the antibody is greater or significantly altered relative to the number of tubes or the character of the tubes in the presence of the antibody.

[0093] An antibody is inhibitory for tube formation if the number of tubes formed in the presence of the antibody is less relative to the number of tubes formed in the absence of the antibody or in the presence of a non-binding antibody. An antibody is inhibitory for tube formation if the character of tubes formed in the presence of the antibody is altered relative to the character of the tubes formed in the absence of the antibody or in the presence of a non- binding antibody. As used herein, the term "character of the tube" refers to the robustness and duration of the tube networks formed in the matrix. Tube formation may be quantified using any suitable methods. Typically, tube formation is assessed by microscopy. In one embodiment, the cells are pre-labeled with PKH67 green dye, according to manufacturer's instructions. In another embodiment, the cells are labeled with Calcein AM. Following incubation with or without antibody in the matrix, the tubes are examined using a fluorescent inverted phase microscope.

[0094] The activity of the TEMl antibody in ADCC is determined using suitable assays well known in the art. See, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY (Cooligan et al, eds., John Wiley & Sons, most recent edition) at Unit 7.27. Likewise, CDC activity can be assessed using well known methods. For example, target cells expressing TEMl are labeled with Cr, washed and incubated in triplicate with serial dilutions of the TEMl antibody or control antibody. Human complement (Pel-Freeze Biologicals, Roger, AR) is then diluted 1 :20 in complete media, added to the wells, and plates are incubated for at least 1 hr at 37°C. Following centrifugation, supernatants are removed and counted in a gamma counter. Controls and standards in triplicate include: background (cells only), maximum release (target cells plus 2% SDS), positive control (antibody known to mediate CDC and complement) and complement alone (cells plus complement only). K-562 cells (ATCC CCL 243) are used as the negative control.

[0095] Any suitable method can be employed to determine phagocytosis using TEMl antibodies of the present invention. One such assay is described in Example 6. [0096] The activity of a TEMl antibody in CDC is determined using suitable assays well known in the art. See, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY (Cooligan et ah, eds., John Wiley & Sons, most recent edition). One such assay is described in Example 6.

[0097] In the methods used to assess the inhibition of proliferation and tube formation and the ADCC, phagocytosis, and CDC activity, any cell that constitutively or inducibly expresses human TEMl, or expresses a suitable non-human TEMl homologue (e.g., murine, bovine, monkey and the like) may be used.

[0098] Any suitable source of endothelial cells may be employed in the present methods. In one embodiment, the cell is an endothelial cell line that endogenously expresses TEMl . In a specific embodiment, the cell line is the murine 2Hl 1 endothelial cell line (ATCC). In another embodiment, the cell is induced to express TEMl . In a specific embodiment, the cell is an AC133+/CD34+ human bone marrow cell cultured in the presence of basic FGF (bFGF), VEGF, and heparin to generate the endothelial precursor cell (EPCs) as described in Example 2. hi yet another embodiment, the cell is a transfected or transduced with TEMl . In a specific embodiment, the human umbilical vein endothelial cell (HUVEC) or the human microvascular endothelial cell (HMVEC) is transduced with an adenoviral vector encoding TEMl. hi another specific embodiment, the COS or HEK-293 cell is transfected with a plasmid encoding TEMl. hi some embodiments, the TEMl expressing cell may be contacted with growth factors or media conditioned by other cells. For example, colon carcinoma conditioned media can be prepared using confluent cultures of human HCTl 16 colon carcinoma cells or human HT29 colon carcinoma cells grown in serum free media for 3 days. Factors useful in supplementing media include VEGF and bFGF. In one embodiment, the cell is derived from or in a human or veterinary subject.

[0099] Any suitable source of pericytes may be employed in the present method. In one embodiment, human pericytes isolated from normal human brain vasculature are available from commercial sources. The pericytes are grown on poly-L-lysine coated flasks in pericyte basal media, 2% fetal bovine serum, and pericyte growth supplement containing bFGF, EGF, and IGF-I with transferrin, insulin, and hydrocortisone. Identity of the cells as pericytes can be confirmed using morphologic analysis or flow cytometric analysis of cell surface markers using reagents commercially available. Pericytes are characterized by the surface expression of NG2 chondroitin sulfate proteoglycan, alpha-smooth muscle actin, Thyl (CD90) and desmin. [0100] TEMl expressing cell can be contacted with the antibody in any suitable manner for any suitable length of time. The cells can be contacted with the antibody more than once during incubation or treatment. Typically, the concentration required is in the range of about 0.1 μg/ml to 1000 μg/ml, more typically in the range of 1 μg/ml to 100 μg/ml. The exact concentration can be readily determined from in vitro cultures of the cells and exposure of the cell to varying concentrations of the antibody. Typically, the length of time the cell is contacted with the antibody is 1 hour to 3 days, more typically for 24 hours. In tube formation assays, any suitable matrix may be used. In one embodiment, the matrix is reconstituted basement membrane Matrigel™ matrix (BD Sciences).

[0101] The following examples are offered to illustrate but not to limit the invention.

Example 1 Production of human anti-TEMl antibodies

[0102] Human antibodies to TEMl were raised in the KM mouse™ (WO 02/043478) by immunizing with TEMl -expressing FM3A cells (FM3A/TEM1) or TEMl -expressing L929 cells (L929/TEM1), both of which stably express high levels of human TEMl as determined by flow cytometric (FACS) analysis using reagent rabbit polyclonal antisera to human TEMl. For the establishment of FM3A/TEM1, murine FM3A mammary carcinoma cells (RIKEN BioResource Center (Japan): RCB0086) were transfected with a plasmid containing the human TEMl cDNA by electroporation. The plasmid was constructed by subcloning TEMl cDNA from pcDNA3.1 human TEMl into pTracerEF-Bsd (Invitrogen) using the BstXI site. For the establishment of L929/TEM1, murine L929 fibroblasts (ATCC CCL-I) were transfected with the same plasmid by lipofection using TransIT-LTl (Mirus). TEMl -expressing cells were, then, collected from the transfectants stained with reagent rabbit polyclonal antisera, and separated based on expression using a cell sorter, FACSVantage (BD Biosciences). FM3A/TEM1 were maintained in modified Eagle's medium supplemented with 10% Fetal Bovine Serum (FBS) and 10 μg/ml of Blasticidin S, and L929/TEM1 were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and 10 μg/ml of Blasticidin S. The mice were immunized with 5xlO⁶ cells of FM3A/TEM1 or IxIO⁶ cells of L929/TEM1 intraperitoneally 3 times at 2 weeks intervals. At the last immunization, 3 days before cell fusion, 5 μg of human interleukin-6 was injected intraperitoneally into the immunized KM mice. Hybridomas were prepared from the spleens of the immunized animals using SP2/0-Agl4 myeloma cells (ATCC) as a fusion partner. Hybridomas which produce antibody against human TEMl were screened by FACS analysis of their supernatants using TEMl -expressing FM3A cells. In brief, TEMl -expressing FM3A cells were incubated with the supernatants at O⁰C for one hour. After washing, the cells were incubated with RPE- conjugated anti-human kappa chain specific antibody or RPE-conjugated anti-human gamma chain specific antibody at O⁰C for 1 hour. After washing, the cells were subjected to FACS analysis. Selected hybridomas were cloned by limiting dilution method.

[0103] Purification of Antibodies: For purification of antibodies, hybridomas were adapted to eRDF medium (Kyokuto Pharmacy) supplemented with 1% Low IgG FBS (HyClone), 5 μg/ml of insulin, 5 μg/ml of transferrin, 10 μM of ethanolamine, and 25 nM of sodium selenite. The purification of the IgG from the fermentation broth was performed using a combination of conventional techniques commonly used for antibody production. The culture harvest was clarified to remove cells and cellular debris prior to starting the purification scheme. This was achieved using filtration of the harvest. Following clarification, the antibody was captured and significantly purified using affinity chromatography on Protein A matrix (MabSelect; Amersham Biosciences). The antibody was bound to Protein A matrix and, following washing of the matrix, was eluted by a reduction of the pH. Further purification of the antibody was then achieved by anion exchange chromatography (Q Sepharose Fast Flow; Amersham Biosciences) and cation exchange chromatography (SP Sepharose Fast Flow; Amersham Biosciences). As well as removing impurities, this step can also be used to buffer exchange into PBS.

Example 2

Endothelial Precursor Cells TEPCs)

[0104] Human bone marrow cell expressing the markers ACl 33 and CD34 were stimulated with VEGF, bFGF and heparin in heparin or collagen monolayer culture to differentiate into a phenotype described as endothelial precursor cells (EPC). Briefly, to differentiate bone marrow progenitor cells into EPC, the AC133+/CD34+ bone marrow subpopulation was isolated using conventional means. The CD34+/AC133+ progenitors cells (1-2 x 10⁵ cells/ml) were grown in IMDM medium (Cambrex Inc.) supplemented with 15% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA), 50 ng/ml VEGF (R&D Systems, Minneapolis, MN), 50 ng/ml rhbFGF (R&D Systems), and 5 U/ml heparin (Sigma Chemical Co., St. Louis MO) on fibronectin coated flasks (BD Biosciences, Franklin Lakes, NJ) at 37⁰C with humidified 95% air/ 5% CO₂ to generate endothelial precursor cells (EPC). See, e.g., Bagley et al, Cancer Res. 63: 5866-73 (2003); Gerwins et al, CritRev Oncol Hematol 34: 185-94 (2000); Quirici et al, Brit J Haematol 115: 186-94 (2001); Bastaki et al, Aterioscler Thromb Vase Biol 17: 454-64 (1997). Fresh media was added to the cultures every three to five days. The adherent cells that were generated from the original population of mixed non¬ adherent and adherent cells were designated EPC. The EPC were grown to confluency and could be passaged up to a dozen times. After the second passage, the EPC were maintained in IMDM media supplemented with 15% FBS without additional growth factors. The EPC were divided 2-3 fold at each passage. The EPC were employed in the in vitro assays examining TEM function. The EPC appeared to be an intermediary between progenitor cells from bone marrow and fully differentiated endothelial cells.

[0105] Characterization of the EPC indicated that they possessed many of the same properties as mature endothelial cells such as HUVEC and HMVEC. In vitro, human EPC formed tubes/networks on Matrigel™ matrix (BD Sciences), migrated and invaded, important properties in the formation of new vessels with in a tumor microenvironment. SAGE analysis of EPC was performed comparing gene expression under stimulatory (+ VEGF) and non- stimulatory (- VEGF) conditions with notable differences being observed. In vivo human EPC formed tubes when implanted in Matrigel™ plugs in immunodeficient mice, and thus allowed modeling of angiogenesis with human endothelium in mice. EPC expressed many endothelial cell surface markers including CD31, P IHl 2 and CD 105 at levels similar to those of HUVEC and HMVEC as determined by flow cytometry. In addition, human EPC expressed TEMl. TEMl mRNA was identified as being expressed at high levels in endothelial cells derived from a fresh surgical specimen of colon tumor compared to the endothelium of normal colon mucosa. The levels of TEMl mRNA expressed in human EPC were markedly higher than was observed in HUVEC and HMVEC. Bagley, et al, Cancer Res. 63: 5866-73 (2003).

Example 3 Pericytes

[0106] Human pericytes isolated from normal brain by conventional methods were purchased from ScienCell Research Laboratories (San Diego, CA). Pericytes were cultured in pericyte media with supplements including bFGF, EGF, IGF-I, transferrin, insulin and hydrocortisone and 2% fetal bovine serum (Sciencell). Pericytes were cultured on flasks coated with a Ix solution of poly-L-lysine (Sciencell) in sterile dH₂O for 30 minutes at 37⁰C. Pericytes were found on vessels, arteries, veins, capillaries, and post-capillary venules though the relative frequency and distribution of pericytes varies depending upon tissue, type of vessel, and stage of development. See, e.g., Majno et al., J. Biophys. Biochem. Cytol. 11:571- 605 (1961); Majno, Circulation (Section 2) IN HANDBOOK OF PHYSIOLOGY 2293-375 (W.F. Hamilton and P. Dow, eds. Amer. Physio. Soc, Washington, D.C. 1965); Diaz-Flores et al, Histol. Histopath. 6:269-86 (1991); Hirschi et al, Cardiov. Res. 32:687-98 (1996); Hirshi, IN REGULATION OF ANGIOGENESIS 419-28 (LD. Goldberg and E.M. Rosen, eds., 1997); Rouget et al, Arch. Physiol. Norm. Pathol. 5:603-63 (1973); and Zimmermann et al, Z. Anat. Entwicklungsgesch, 68: 3-109 (1923). Pericytes were polymorphic, elongated cells with a distinctive morphology comprised of an irregularly shaped plasma membrane with extended cytoplasmic processes. These extensions of the pericytes interdigitated with endothelial cells to form contact. Pericytes regulated capillary and venule blood flow through contractile activity and also controlled vascular permeability. Markers characteristic of pericytes were confirmed by flow cytometry analysis using alpha-smooth muscle actin, NG2, Thy-1, desmin, CD54/ICAM, CD106/VCAM, P1H12, LFA-I, CD105/endoglin and fibronectin. The pericytes expressed TEMl and were employed in the in vitro assays examining TEM function. The pericytes formed tubes/networks on Matrigel™ matrix (BD Sciences) that were stable for several days.

[0107] Expression of TEMl : Purified TEMl antibodies were analyzed for binding to EPC and pericytes by fluorescence activated cell cytometry (FACs). TEMl antibodies were tested at final concentrations of 0.1 and 1.0 μg/ml using 100,000 EPC or pericytes. TEMl antibodies were compared with DNP or OMP isotype control antibody. There were no significant differences between DNP and OMP in FACs analysis. TEMl antibodies were incubated with EPC or pericytes for 1 hr on ice, washed twice and then a secondary goat anti-human PE- labeled antibody was added. EPC or pericytes were incubated with the secondary antibody for 30 minutes, washed twice, and subjected to fluorescence activated cell cytometry immediately. Approximately 42 TEMl antibodies were assayed for binding to EPC and pericytes. Overall, the antibody originally produced by ATCC-6064 gave the highest mean fluorescence with the EPC and pericytes. Example 4 Antiproliferative activity in EPCs and pericytes by TEMl antibodies

[0108] Proliferation Assay: Proliferation was assayed by measurement of adenosine triphosphate (ATP) bioluminescence (Crouch et al, J. Immunol. Methods 160:81-88 (1993); Kangas et al., Med. Biol. 62:338-43 (1984)) using human endothelial precursor cells (EPC) and pericytes prepared as described in Examples 2 and 3. TEMl antibodies were diluted in phosphate-buffered saline (PBS) to 2x concentrations ranging from 10 μg/ml to 1600 μg/ml. TEMl antibodies were pipetted in triplicate into 96-well plate format and serially diluted two¬ fold or ten-fold such that 50 μls remain in the well. EPC or pericytes were plated in 96-well plate format at 2,000 cells per well in 50 μls of media with 6% fetal bovine serum. The final concentration of TEMl antibodies ranged from 0.01 - 800 μg/ml. EPC are cultured in IMDM (Cambrex) and pericytes were cultured in pericyte basal media (Sciencell Research Labs) both with final fetal bovine serum concentration of 3%. Cells were assayed for proliferation after 72 hours using Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) that measured ATP levels by bioluminescence. The bioluminescence units readout was converted to EPC and pericyte cell number using a standard curve set up with untreated cells ranging from 1000 to 10,000 cells per well. When the cells were at a resting state but not proliferating, 5 hours post transfer, the luminescence reagent was added and a standard curve of luminescence units versus cell number was constructed for EPC and for pericytes. The results were expressed as numbers of cells per well +/- standard deviation (SD).

[0109] Multiple clones were shown to inhibit EPC and pericytes proliferation. See Table 6 below.

Table 6 Effective concentrations to achieve 50% activity (EC ) for

TEMl antibodies to inhibit endothelial precursor cells (EPC) and eric te roliferation and tube formation in cell-based assays

Example 5 Inhibition of EPC and pericyte tube/network formation in vitro by TEMl antibodies

[0110] Endothelial Tube Formation Assay: Reduced Growth Factor Matrigel™ (BD Biosciences, Bedford, MA) was added to the wells of a 48-well plate in a volume of 150 μls and allowed to solidify at 37⁰C for 30 minutes. TEMl antibodies were diluted in phosphate- buffered saline (PBS) to a final volume of 700 μls to which 70,000 pericytes or 100,000 EPC in 175 μls of media with 10% fetal bovine serum was added. Basal media for EPC was IMDM (Cambrex, Walkersville, MD), and for pericytes the basal media was Pericyte Media (Sciencell Research Labs, San Diego, CA). EPC or pericytes were preincubated with TEMl antibodies at final concentrations ranging from 150 ng/ml to 100 μg/ml for 30 minutes at 37⁰C. After the Matrigel solidified, 30,000 EPC or 20,000 pericytes that were preincubated with TEMl antibodies were added in 250 μl of media in triplicate. Cells were incubated at 37⁰C with humidified 95% air/5% CO₂ for 16 hours (Pelletier et al, Lab Invest. 80:501-11 (2000)). The tube networks were stained with 8 μg/ml calcein for 30 minutes and quantified by image analysis using Scion image or Metamorph software with data expressed as fluorescent pixel area +/- standard deviation (SD).

[0111] The clones of the present invention effectively inhibited both EPC and pericyte tube formation. See Table 6 above. Example 6 ADCC, Phagocytosis, and CDC activity of TEMl antibodies

[0112] ADCC Assay: Antibody-dependent cellular cytotoxicity (ADCC) was described as a potentially important mode of action for antibody therapeutics in vivo (Velders et al, Brit J Cancer 74:478-83 (1998); Flieger et al, Hybridoma 18:63-68 (1999). Human SKOV3 ovarian carcinoma cells expressed very high levels of Her2/neu. SKO V3 cells were infected with adeno-TEMl to develop a TEMl expressing target cell that also allowed the use of Herceptin binding to Her2/neu, as a control antibody in ADCC assay. SKO V3 /TEMl expressing cells or stably expressing HEK 293/TEM1 target cells were labeled overnight with ⁵¹Cr, and then washed in DMEM to remove unincorporated ⁵¹Cr. The chromium-labeled adeno-TEMl - infected SKO V3 cells (7 x 10⁴) were mixed with 1 μg to 5 μg of TEMl antibodies or Herceptin in 1.4 ml of DMEM (Barnes et al., Lancet 355: 160-61 (2000); Plowman et al., Proc Natl Acad Sd USA 90: 1746-50 (1993)). The reaction mixture was divided into 12 wells of a 96-well plate (5 xlO³ cells per well in 100 μl DMEM). Human peripheral blood mononuclear cells (PBMC) or monocyte effector cells were added to the wells at an E:T ratio of 200:1 and 100:1 in 100 μl DMEM bringing the total volume per well to 200 μl. Therefore the final TEMl antibody concentrations were 0.036 μg to 0.18 μg per well. The plate was centrifuged at 900 rpm for 3 minutes and then incubated at 37⁰C for 5 hours to overnight (20 hours). The ⁵¹Cr released was measured using beta counter. All samples were in triplicate with two E:T ratios. Percent specific lysis was calculated as: % Target cell lysis = 100 X (Experimental cpm — spontaneous cpm/Total cpm — spontaneous cpm).

[0113] The ADCC activity of the TEMl antibodies at 0.18 μg/ml is shown in Table 7.

Table 7 ADCC Activity of TEMl Antibodies

[0114] CDC Assay: Complement-dependent cytotoxicity (CDC) assays were carried out using Chinese hamster ovary cells transformed with ras and stably transfected to express human TEMl as the complement sensitive target cells. Purified TEMl antibodies were used at concentrations ranging from 0.05 μg/ml to 5 μg/ml. Baby rabbit complement (Cedarlane Labs, Cat #3441) or human complement was used at a final concentration of 10% in the assay mixture. The target CHO/TEM1 cells were labeled with ⁵¹Cr for 1 hour. The reaction with complement was allowed to proceed for 4 hours. The endpoint for the assay was ⁵¹Cr released into the medium, which was then used to calculate specific cellular lysis as described above. The results are shown in Table 8 below using TEMl antibodies at 5 μg/ml.

Table 8 CDC Activity of TEMl Antibodies

[0115] Phagocytosis Assay: For the phagocytosis assay, macrophages were prepared starting from a Leukopak. The PBMC were isolated from a Leukopak by passage over Ficoll. The monocytes were then isolated from the PBC by passage over Percoll. The monocytes were cultured for 6 days in media containing GM-CSF (200U/ml) and, thus were induced to differentiate into macrophages. The day-6 macrophages (1-2 x 10⁵ /well) were arrayed in a round bottom 96- well plate 24 hr prior to the assay. The target cells were prepared by infecting human MDA-MB-231 breast cancer cells or human SKO V-3 ovarian cancer cells with an adeno-virus containing the gene for TEMl . The infected cells were allowed to grow for 72 hrs. The target cells were labeled with the lipophilic dye PKH67-GL, 24 hrs prior to the assay and were plated to decrease the number of dead or dying cells. On the day of the assay, the labeled target cells (10⁵/well) were arrayed directly over the macrophages. Purified TEMl antibodies were added to the wells at concentration over the range from 0.01 to 100 μg/well. The total reaction volume was 200 μl. The reactions were allowed to incubate at 37⁰C for 4 hrs. AU of the cells were removed from the plate with Versene. The suspended cells were stained with CD14-PE and CDl Ib-PE macrophage markers. The cells were then rinsed, and images were collected. Cell that stained with both red and green represent macrophages that have phagocytosed TEMl expressing cells. An exemplary experiment is shown in Table 9 below. In this experiment, 1-2 x 10⁵ day-6 macrophages were plated with TEMl -expressing MDA-MB-231 cells (targets) at 10⁵ cells per well in the presence of 10 ng/well of anti-TEMl antibody.

Table 9 Pha oc tosis activit of TEMl antibodies

Example 7 Binding Affinity of TEMl antibodies

[0116] BIAcore binding assay was used to determine the binding constants for TEMl antibodies association and dissociation from the extracellular domain of human TEMl. First, HPC4-tagged TEMl extracellular domain protein was bound covalently to the BIAcore CM5 chip (2500 RU). Second, the TEMl antibody (at concentration ranging from 1.2 nM to 100 nM) was injected into the flow through over the surface in binding buffer (HBS-EP:10 mM HEPES, pH 7.4, 150 niM NaCl, 0.005% surfactant P-20, 3 niM EDTA) for 3 min at 120 ul/min followed by 2.5 min washout with buffer (PBS-P: PBS, pH 7.2 (Gibco), 0.005% surfactant P-20). The surface was regenerated with 2 pulses of 10 mM EGTA in binding buffer. Multiple TEMl antibodies were characterized in BIAcore kinetic binding experiments, which result in distinct K_0n, K₀^ and Ka. The Ka ranges for the TEMl antibodies range from sub-nanomolar to tens of nanomolar as shown in Table 10.

Table 10 BIAcore Analysis of TEMl Antibodies

Example 8 In vivo efficacy of TEMl antibodies

[0117] The in vivo efficacy of the TEMl antibodies was tested in several tumor model systems including the fully syngeneic mouse CT26 colon carcinoma grown in Balb/C mice, the murine L929 fibrosarcoma and the subline L929/TEM1 grown in SCID mice. The L929/TEM1 subline was stably transfected to express human TEMl . The tumors were implanted subcutaneously on the flank of the animals as a suspension of 10⁶ viable tumor cells derived from donor tumors. The day of tumor cell implantation was day 0. For the in vivo studies, TEMl antibodies were administered by intraperitoneal injection at a dose of 5 mg/kg on days 4, 7, 10, 13, 16, and 19 or on days 15, 18, 21, 24, 27, and 30 after tumor cell implantation. All studies used the following control groups: untreated animals, vehicle-treated animals, and animals treated with an isotype control human antibody. Body weights and tumor measurements were determined twice per week. Tumors were measured in two diameters using calipers, and tumor volumes were calculated using the formula: short diameter x short diameter x long diameter x 0.5. The progress in individual animals was followed over time. Two animals from each group of seven were sacrificed for serum and tumor collection 24-48 hours after the final dose of TEMl antibody. The remaining five animals per group were followed until tumors reached 4000 mm³. The efficacy of TEMl antibodies was determined by the difference in days for tumors in the TEMl antibody groups to reach 2000 mm³ compared with the tumors in animals treated with an isotype control antibody to reach the same volume. The calculation provides a determination of tumor growth delay (TGD) in days. There was little effect on body weights by any of the treatments (data not shown). The tumor growth delays for TEMl antibodies ranged from 3 days to 17 days. See Tables 11-13 below.

Table 11

Growth delay of subcutaneously implanted murine CT-26 colon carcinoma treated by intraperitoneal injection with TEMl monoclonal antibodies

Table 12

Growth delay of subcutaneously implanted murine L929/TEM1 fibrosarcoma treated by intraperitoneal injection with TEMl monoclonal antibodies

Table 13

Growth delay of subcutaneously implanted murine CT-26 colon carcinoma treated by intraperitoneal injection with TEMl monoclonal antibodies

Example 9

Epitope binding of TEMl antibodies

[0118] Purified TEMl antibodies were analyzed with a competitive ELISA format. The competitive ELISA with the purified TEMl antibodies was performed to determine a preliminary epitope map. For the experiment, an ELISA plate was coated with 1 μg/ml of extracellular TEM1-HPC4 protein and then a biotinylated purified TEMl antibody was added in the presence or absence of excess non-labeled (cold) purified TEMl antibody. The concentration of biotinylated purified TEMl antibody was <50 ng/ml and the concentration of the non-labeled purified TEMl antibody was either 1 μg/ml or 10 μg/ml. By this competition assay, the purified TEMl antibodies were found to represent seven different groups. See, e.g., Table 14 below. Table 14 Competition ELISA of TEMl Antibodies

BlOTINYLATED ANTIBODIES

Example 10

Intratumoral vessel counts after treatment with TEMl antibodies

[0119] Tumors were collected and frozen after treatment of the tumor-bearing host with purified TEMl antibodies. The frozen tumors were sectioned and stained lmmuno- histochemically for CD31 (Pharmingen, clone MEC13.3) with DAB visualization. The tissue was not counterstained with hematoxylin to allow greater discrimination of the vessels during imaging. The threshold for the slides was at 2Ox magnification for the CD31 DAB immunohistochemical staining, and the vessel number, average perimeter, average length and total average area were collected for 10 fields per tissue slice. The data were filtered to eliminate single objects or cells so that data accurately reflected vessel numbers. Using the L929/TEM1 tumor, there was a decrease in the number of intratumoral vessels in animals treated with purified TEMl antibodies to one-half to one-third of the number of vessels found in the tumors of vehicle-treated animals as shown in Table 15.

Table 15

Intratumoral vessel counts in subcutaneously implanted L929/TEM1 fibrosarcoma after treatment with TEMl antibody

Example 11 In vivo efficacy of TEM-I # 187Pro in the mouse CT-26 colon carcinoma

[0120] The in vivo efficacy of anti-TEM 1 #187Pro is tested in the fully syngeneic mouse CT26 colon carcinoma grown in Balb/C mice over a range of antibody doses. The tumors are implanted subcutaneously on the flank of the animals as a suspension of 10 viable tumor cells derived from donor tumors. The day of tumor cell implantation is day 0. For the in vivo studies, anti-TEM 1 #187Pro is administered by intraperitoneal injection at doses of 2.5, 5, 10, 15 or 25 mg/kg on days 4, 7, 10, 13, 16, and 19 after tumor cell implantation. The standard agent, 5-fluorouracil, is administered by intraperitoneal injection at a dose 25 mg/kg once per day on days 4 through 8 after tumor cell implantation. The study has as control groups animals that remain untreated, and vehicle-treated animals. Body weights and tumor measurements are determined twice per week. Tumors are measured in two diameters using calipers and tumor volumes are calculated using the formula: short diameter x short diameter x long diameter x 0.5.

[0121] The progress in individual animals is followed over time. The animals are followed until tumors reached 4000 mm³. The efficacy of anti-TEM 1 #187Pro and 5-fluorouracil is determined by the difference in days for tumors in the anti-TEM 1 #187Pro-treated or the 5- fluorouracil-treated groups to reach 2000 mm³ compared with the tumors in animals in the control groups to reach the same volume. The calculation provides a determination of tumor growth delay (TGD) in days. There is little effect on body weights by any of the treatments.

[0122] The tumor growth delays for anti-TEM 1 #187Pro and 5-fluorouracil in the mouse CT26 colon carcinoma shown in Table 16 below.

Table 16

Example 12

Whole body autoradiography of iodine-125 (¹²⁵I) labeled TEMl antibodies [0123] TEMl-#33 that highly cross-reacts with mouse TEMl, and TEM1-#184 that has the same sequence as TEMl -#170 and TEMl -#187 (ATCC PTA-6066) were labeled with ¹²⁵I using iodogen method. Relative radioactivities were 656 kBq/μg and 585 kBq/μg, respectively, and radiochemical activities were 93.7% and 98.7%, respectively. By mixing the labeled antibodies with non-labeled ones, 1 mg/0.206 MBq/mL of TEMl-#33 and 1 mg/0.250 MBq/mL of TEMl -#184 for injections were prepared. Those samples were injected intravenously into female mice at 5 mg/1.03 MBq/5 mL/kg for TEMl -#33 and 5 mg/1.25 MBq/5 mL/kg for TEMl -#184, respectively. Mice used here were C57BL/6 for both samples, and, for TEMl -#184, human TEMl transgenic (Tg) mice (WO 2004/078942) which was established using RPCIl 1-867G23 BAC clone as a transgene, were also used. Non-tumor bearing mice and tumor bearing mice were used in each group. Tumors used here were MCA 207 fibrosarcoma and Lewis lung carcinoma (LLC) (ATCC). MCA207 fibrosarcoma cells were implanted subcutaneously on right side of back and LLC cells were on left side of back of the same mice. Time points of sacrifice after injection were 24-hour and 168-hour. After sacrifice by anesthesia, mice were frozen in dry ice/hexane bath and embedded in 2% CMC- Na. The frozen samples were sectioned with cryo-microtome at 20 μm thickness. The sections were dried at -2O⁰C, and then exposed to imaging plate (Fuji Film, Tokyo, Japan) for 16-hour, and analyzed with bioimaging analyzer (Fuji Film). Standard radiation source was put on each image, and used for quantitative analysis.

[0124] The results suggest that TEMl antibodies are selectively retained in some kind of tumors and that there is no serious retention of TEM antibodies in normal tissues. It was also demonstrated that tumor-bearing and TEMl transgene clearly accelerated the decrease of the injected TEMl Abs. See Tables 17 and 18 below.

Table 17

Concentration of radioactivity (μg equiv. /g) after a single intravenous administration of ^12SI-#33 at a dose of 5 mg/kg to female C57BL/6 mice

LLOQ : Below the lower limit of quantitation. ULOQ : Above the upper limit of quantitation. Table 18

Concentration of radioactivity (ιιg equiv./g) after a single intravenous administration of ^• 12_5τ I- #184 at a dose of 5 mg/kg to female C57BL/6 mice and the TEMl Tg mice

C57BL/6 mice TEMl Fg mice

Non-tumor-bearing Tumor-bearing Non-tumor-bearing Tumor-bearing lVCglOIl

Time (h) Time (h) Time (h) Time (h)

24 168 24 168 24 168 24 168

Blood 15.8 15.6 12.9 1.9 15.1 1.1 11.3 LLOQ

Cerebrum 1.1 LLOQ LLOQ LLOQ LLOQ LLOQ LLOQ LLOQ

Cerebellum LLOQ LLOQ LLOQ LLOQ LLOQ LLOQ 1.0 LLOQ

Hypophysis 1.6 1.8 1.5 LLOQ 2.8 LLOQ 0.9 1.5

Spinal cord 1.4 LLOQ 0.9 LLOQ LLOQ LLOQ 0.7 LLOQ

Eyes 1.2 LLOQ LLOQ LLOQ LLOQ LLOQ LLOQ LLOQ

Submaxillary glands 5.6 5.4 7.0 1.2 5.6 1.4 7.0 1.3

Thyroid gland ULOQ ULOQ ULOQ ULOQ ULOQ ULOQ ULOQ ULOQ

Thymus 4.2 2.8 3.1 1.0 3.8 LLOQ 2.5 LLOQ

Lungs 8.3 8.4 4.7 1.3 7.9 1.1 5.5 LLOQ

Heart 5.9 4.9 4.6 0.9 4.9 LLOQ 3.8 LLOQ

Liver 7.8 6.3 5.5 0.9 7.9 LLOQ 5.9 LLOQ

Stomach 3.7 3.9 5.0 1.2 5.0 2.5 5.7 LLOQ

Adrenals 9.5 8.0 5.4 1.1 6.9 1.6 7.3 LLOQ

Kidneys 10.0 8.9 7.1 1.5 8.4 LLOQ 7.8 LLOQ

Pancreas 2.7 2.7 2.4 LLOQ 3.1 LLOQ 1.6 LLOQ

Spleen 6.2 5.6 3.0 LLOQ 6.0 1.4 2.7 LLOQ

Mesenteric lymph nodes 3.7 2.2 LLOQ LLOQ 5.7 0.9 3.5 LLOQ

Skeletal muscle 1.5 1.4 1.1 LLOQ 1.1 LLOQ 0.8 LLOQ

Bone marrow 4.9 3.4 2.8 LLOQ 5.9 2.1 3.3 LLOQ

Urinary bladder 3.1 9.1 4.4 3.4 6.5 1.6 1.8 LLOQ

Fat 2.6 1.4 2.4 LLOQ 2.4 1.2 2.8 LLOQ

Brown fat 4.6 3.8 4.5 LLOQ 4.7 1.2 3.9 LLOQ

Intestine 5.6 5.1 3.0 LLOQ 4.9 LLOQ 3.4 LLOQ

Skin 3.7 3.5 3.6 LLOQ 3.5 LLOQ 2.7 LLOQ

Ovaries 8.3 9.0 8.0 LLOQ 9.1 1.7 5.1 1.4

Uterus 5.2 2.2 3.9 2.6 8.4 1.4 8.3 1.0

Gastric contents 0.9 LLOQ 11.1 LLOQ 2.6 LLOQ 9.6 1.0

Intestinal contents 1.4 1.0 0.8 LLOQ 2.2 LLOQ 0.7 LLOQ

Tumor (left side) 7.9 2.2 8.0 LLOQ

Tumor (right side) 1.9 LLOQ 3.7 LLOQ

LLOQ : Below the lower limit of quantitation. ULOQ : Above the upper limit of quantitation.

[0125] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow.

[0126] Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. U.S. patents and other publications referenced herein are hereby incorporated by reference.

Claims

Claims

1. A monoclonal antibody, or a biologically active fragment thereof that binds to TEMl, wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO: 8, SEQ ID NO:9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:15 and SEQ ID NO:17.

2. A monoclonal antibody, or a biologically active fragment thereof that binds to TEMl, wherein the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl-

#187(ATCC PTA-6066).

3. The monoclonal antibody of claim 1 or 2, wherein the antibody is produced by a hybridoma.

4. The monoclonal antibody of claim 1 or 2, wherein the antibody is produced recombinantly.

5. The monoclonal antibody of claim 4, wherein the antibody is produced in CHO cells.

6. A monoclonal antibody, or a biologically active fragment thereof that binds to TEMl, wherein the antibody comprises a variable region with the same amino acid sequence as the TEMl antibody originally isolated from a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEM 1-#187(ATCC PTA-6066).

7. The antibody of claim 1 or 6, wherein the antibody is an IgG antibody.

8. The antibody of claim 7, wherein the antibody is an IgG₁, IgG₂ or IgG₃ antibody.

9. The antibody of claim 8, wherein the IgG antibody is an IgG₁ antibody.

10. The antibody of claim 1 , 2, or 6, wherein the antibody is conjugated to an antitumor agent or an antiangiogenic agent.

11. The antibody of claim 1 , 2, or 6, wherein the antibody inhibits angiogenesis.

12. The antibody of claim 11, wherein the angiogenesis promotes or causes cancer.

13. The antibody of claim 11 , wherein the angiogenesis promotes or causes polycystic kidney disease.

14. The antibody of claim 11 , wherein the angiogenesis promotes or causes diabetic retinopathy.

15. The antibody of claim 11 , wherein the angiogenesis promotes or causes rheumatoid arthritis.

16. The antibody of claim 11 , wherein the angiogenesis promotes or causes psoriasis.

17. The antibody of claim 1 , 2, or 6, wherein the antibody inhibits tumor growth.

18. The antibody of claim 17, wherein the tumor is an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma.

19. The antibody of claim 17, wherein the tumor is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus.

20. A pharmaceutical composition comprising an amount of the monoclonal antibody, or a biologically active fragment thereof, of claim 1, 2, or 6 and a suitable excipient.

21. A monoclonal antibody, or a biologically active fragment thereof that binds to TEMl, wherein the antibody recognizes substantially the same epitope recognized by the antibody of claim 1, 2 or 6.

22. A hybridoma strain selected from the group consisting of TEMl -#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl -#187(ATCC PTA-6066).

23. A method of inhibiting tumor growth, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 15 and SEQ ID NO: 17, whereby the antibody inhibits tumor growth.

24. A method of inhibiting tumor growth, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the antibody was originally produced by a hybridoma selected from the group consisting of TEMl -#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl-

#187(ATCC PTA-6066), whereby the antibody inhibits tumor growth.

25. A method of inhibiting tumor growth, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the antibody comprises a variable region with the same amino acid sequence as the TEMl antibody originally isolated from a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEM1-#187(ATCC PTA-6066), whereby the antibody inhibits tumor growth.

26. The method of claim 23, 24, or 25, wherein the subject expresses the antigen bound by the antibody.

27. The method of claim 23, 24, or 25, wherein the antibody is an intact antibody molecule, a scFv, a Fab fragment, or a F(ab') fragment.

28. The method of claim 23, 24, or 25, wherein the antibody is conjugated to an antitumor agent or an antiangiogenic agent.

29. The method of claim 23, 24, or 25, wherein the tumor is an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma.

30. The method of claim 23, 24, or 25, wherein the tumor is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus.

31. A method of inhibiting angiogenesis, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or SEQ ID NO:15 and SEQ ID NO:17, whereby the antibody inhibits angiogenesis.

32. A method of inhibiting angiogenesis, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the antibody was originally produced by a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEMl -#132(ATCC PTA-6065), and TEMl- #187(ATCC PTA-6066), whereby the antibody inhibits angiogenesis.

33. A method of inhibiting angiogenesis, comprising administering to a subject in need thereof, an effective amount of an antibody, or a biologically active fragment thereof, wherein the antibody comprises a variable region with the same amino acid sequence as the TEMl antibody originally isolated from a hybridoma selected from the group consisting of TEM1-#87(ATCC PTA-6064), TEM1-#132(ATCC PTA-6065), and TEM1-#187(ATCC PTA-6066), whereby the antibody inhibits angiogenesis.

34. The method of claim 31, 32, or 33, wherein the subject expresses the antigen bound by the antibody, or an allelic variant thereof.

35. The method of claim 31, 32, or 33, wherein the angiogenesis is neoangiogenesis.

36. The method of claim 31 , 32, or 33, wherein the antibody is an intact antibody molecule, a scFv, a Fab fragment, or a F(ab') fragment.

37. The method of claim 31, 32, or 33, wherein the antibody is conjugated to an antiangiogenic agent or an antitumor agent.

38. The method of claim 31, 32, or 33, wherein the subject has cancer.

39. The method of claim 31 , 32, or 33, wherein the subject has polycystic kidney disease.

40. The method of claim 31 , 32, or 33, wherein the subject has diabetic retinopathy.

41. The method of claim 31, 32, or 33, wherein the subject has rheumatoid arthritis.

42. The method of claim 31 , 32, or 33, wherein the subject has psoriasis.

43. A method to identify an antibody that inhibits tumor growth comprising: contacting a TEMl -expressing cell with a candidate antibody; assessing the inhibitory activity of the antibody in a tube formation assay in vitro at concentrations <20 μg/ml, wherein the tubes are formed by endothelial cells, endothelial precursor cells, and/or pericytes; assessing the inhibitory activity of the antibody in a proliferation assay in vitro at concentrations <20 μg/ml, wherein the proliferating cell is an endothelial cell, endothelial precursor cell, and/or a pericyte; and assessing the activity of the antibody in an antibody-dependent cell cytotoxicity assay, phagocytosis assay, and/or a complement-mediated cytotoxicity assay at concentrations <5 μg/ml; whereby the candidate antibody which inhibits the tube formation by endothelial cells, endothelial precursor cells, and/or pericytes, inhibits the proliferation of endothelial cells, endothelial precursor cells, and/or pericytes, and mediates ADCC, phagocytosis, and/or CDC activity is identified as an antibody which inhibits tumor growth at doses <5 mg/kg.

44. The method of claim 43, wherein the antibody is human.

45. A method to identify an antibody that inhibits angiogenesis comprising: contacting a TEMl -expressing cell with a candidate antibody; assessing the inhibitory activity of the antibody in a tube formation assay in vitro at concentrations <20 μg/ml, wherein the tubes are formed by endothelial cells, endothelial precursor cells, and/or pericytes; assessing the inhibitory activity of the antibody in a proliferation assay in vitro at concentrations <20 μg/ml, wherein the proliferating cell is an endothelial cell, endothelial precursor cell and/or a pericyte; and assessing the activity of the antibody in an antibody-dependent cell cytotoxicity assay, phagocytosis, and/or a complement-mediated cytotoxicity assay at concentrations <5 μg/ml, whereby the candidate antibody which inhibits the tube formation by endothelial cells, endothelial precursor cells, and/or pericytes, inhibits the proliferation of endothelial cells, endothelial precursor cells, and/or pericytes, and mediates ADCC, phagocytosis, and/or CDC activity is identified as an antibody which inhibits angiogenesis.

46. The method of claim 45, wherein the antibody is human.

47. The monoclonal antibody of claim 1, wherein SEQ ID NO:16 or SEQ ID NO:17 has a FRl region selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

48. The method of claim 23 or 31 , wherein the variable region of the antibody comprises SEQ ID NO: 16 or SEQ ID NO: 17, wherein the FRl is selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.