RU2465282C2 - Combined therapy with using alpha5beta1 antagonists - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there are offered: versions of alpha5beta1 class IgG2 antibodies of hybridomas deposited under access number in ATCC No. PTA-7421, No. PTA-7420; as well as a conjugate of the antibody and the therapeutic agent, and the marked antibody for diagnosing a disease related to alpha5beta1 expression. There are also disclosed: a coding nucleic acid; an expression vector; a recombinant cell; a method for producing the antibody; a method for detecting alpha5beta1 protein; an antibody-based pharmaceutical composition; versions for applying the antibody for treating pathological angiogenesis.

EFFECT: use of the invention provides the antibody affine to Kd 0,1 nM which can find application in treating pathology associated with angiogenesis.

37 cl, 16 dwg, 6 tbl, 17 ex

Description

Field of Invention

The present invention relates to the use of VEGF antagonists and alpha5beta1 antagonists for treating cancer and suppressing angiogenesis and / or suppressing vascular permeability, including abnormal angiogenesis in certain diseases. The presented invention also relates to the use of VEGFR agonists and alpha5beta1 agonists to increase angiogenesis and vascular permeability. The presented invention also relates to antibodies against alpha5beta1, including their compositions and kits and methods for their manufacture and use.

BACKGROUND OF THE INVENTION

The important role of VEGF-A in pathological and non-pathological angiogenesis is recognized. The introduction of VEGF in in vivo models elicits a strong angiogenic response (Plouet, J et al., (1989) EMBO J. 8: 3801-3808; Leung, D.W., et al., (1989) Science 246: 1306-1309). The loss of one VEGF-A allelic gene leads to increased embryonic mortality in mice (Carmeliet, P., et al., (1996) Nature 380: 435-439; Ferrara, N et al., (1996) Nature 380: 439-442 ) VEGF is also known as a vascular permeability factor due to its ability to cause hemorrhage (Senger, DR et al., (1995) Science 219: 983-985; Dvorak, HF, et al., (1995) Am. J. Pathol. 146: 1029-1039). Thus, VEGF-A is involved in angiogenesis at the developmental stage, reproduction and bone angiogenesis, in addition to other non-pathological types of angiogenesis.

VEGF-A binds to two receptor tyrosine kinases (PTKs), VEGFR-1 (Flt-1) and VEGFR-2 (KDR, Flk-1). It is generally believed that VEGFR-2 is the main mediator of mitogenic, angiogenic and permeability-enhancing actions of VEGF-A. In February 2004, the US Food and Drug Administration (FDA) approved bevacizumab, a humanized monoclonal antibody against VEGF (vascular endothelial growth factor) -A, for the treatment of metastatic colon cancer in combination with 5-fluorouracil (FU)-based chemotherapy regimens. Subsequently, the FDA approved pegaptinib, an aptamer that blocks 165 amino acids of VEGF-A isoform, for treating the wet (neovascular) form of age-related macular degeneration (AMD).

Despite these advances, many patients receiving treatment with VEGF antagonists will ultimately become victims of their illness. Therefore, there is a need to develop new drugs and treatments for treating diseases that are no longer or only partially amenable to therapy with VEGF antagonists. There is also a need for the development of alternative and / or better treatments for the treatment of cancer and complicated diseases caused or affected by abnormal angiogenesis.

SUMMARY OF THE INVENTION

The presented invention relates to medicines and methods for treating patients who may benefit from a reduction in angiogenesis, suffering from abnormal angiogenesis and / or suffering from neoplasia. In accordance with one embodiment, the present invention provides a method of inhibiting angiogenesis and / or vascular permeability in a subject, comprising the step of administering to the subject a therapeutically effective amount of a VEGF antagonist and an alpha5beta antagonist simultaneously or sequentially. In accordance with another embodiment of the invention, the present invention provides a method of treating a subject suffering from a disease, the subject either previously responding to the treatment of the disease with a VEGF antagonist, but only partially or no longer responding to a VEGF antagonist, comprising the step of administering to the subject a therapeutically effective amount of the antagonist alpha5beta1. According to another embodiment, the present invention provides a method of treating a subject suffering from a disease that is persistent or difficult to treat by treatment with an alpha5beta antagonist, alone or in combination with chemotherapy, comprising the step of administering to the subject a therapeutically effective amount of a VEGF antagonist.

The presented invention also relates to new antibodies against alpha5beta1, kits and compositions including them, and methods for their manufacture or use. In one embodiment, the novel anti-alpha5beta1 antibodies are the 7H5 or 7H12 antibodies described herein, or a humanized or chimeric form thereof. According to another particular embodiment, the 7H5 or 7H12 antibodies or their chimeric or humanized forms can be in the form of Fab, Fab ', F (ab)' ₂ fragments, Fv single chain (scFv), Fv fragment, diabody, multispecific antibody and linear antibody . In accordance with another embodiment, the new anti-alpha5beta1 antibodies can be conjugated to another object, but not limited to, such as a therapeutic drug, or a fluorescent dye, or other marker for detecting alpha5beta1 in patients or in patient samples. Similar new antibodies against alpha5beta1 can be used in various therapeutic and diagnostic methods. For example, such antibodies against alpha5beta1 can be used in the treatment of abnormal angiogenesis, neoplasia, eye diseases and autoimmune diseases. Such antibodies can be used to detect the alpha5beta1 protein in patients or in patient samples by contacting such antibodies with the alpha5beta1 protein in patients or in patient samples and to quantitatively or qualitatively determine the anti-alpha5beta1 antibodies associated with the alpha5beta1 protein.

In accordance with yet another embodiment, the present invention provides a method for treating cancer in a subject, comprising the step of administering a VEGF antagonist and an alpha5beta1 antagonist simultaneously or sequentially. In accordance with one preferred embodiment, the cancer responds to therapy with a VEGF antagonist. Another embodiment of the invention is a method of treating age-related macular degeneration in a subject suffering from AMD, comprising the step of administering a therapeutically effective amount of a VEGF antagonist and an alpha5beta1 antagonist simultaneously or sequentially. In yet another embodiment, a method for treating autoimmune diseases in a subject is provided, comprising the step of administering a therapeutically effective amount of a VEGF antagonist and an alpha5beta antagonist simultaneously or sequentially.

In one embodiment, a VEGF antagonist may be administered first to a subject to treatment and then treated with an alpha5beta1 antagonist. In another embodiment, the subject is treated with a VEGF antagonist and an alpha5beta1 antagonist simultaneously. According to another embodiment, the subject is treated with a VEGF antagonist until the subject no longer responds to treatment with a VEGF antagonist, after which the subject is treated with an alpha5beta1 antagonist. In one particular embodiment, the subject was treated with a VEGF antagonist if the cancer was non-invasive or at an early stage and was treated with an alpha5beta 1 antagonist when the cancer was invasive. In another embodiment, a subject treated with an alpha5beta1 antagonist has an increased level of alpha5beta1 in the diseased tissue compared to tissue from a non-diseased subject. In this case, the method may further include the step of detecting alpha5beta1 in a subject, for example in diseased tissue, after treatment with a VEGF antagonist. In accordance with one embodiment, the invasive cancer is metastatic cancer. In accordance with another embodiment, the early stage cancer is cancer that can be treated with adjuvant therapy (e.g., chemotherapy or surgical removal).

In one preferred embodiment, the subject suffering from the disease has a pathology of angiogenesis. In accordance with another embodiment, the disease is selected from the group consisting of cancer, immune disease, or eye disease. In accordance with one preferred embodiment, the disease is selected from the group consisting of a solid tumor, a metastatic tumor, a soft tissue tumor, a disease associated with neovascularization of the eye, an inflammatory disease associated with abnormal angiogenesis, a disease that occurs after transplantation to a subject, and a disease, associated with abnormal development of vascular fibrous tissue. In another preferred embodiment, the disease is selected from the group consisting of breast cancer (including metastatic breast cancer), cervical cancer, colon cancer (including metastatic cancer of the intestine), lung cancer (including non-small cell lung cancer), non-Hodgkin’s lymphoma ( NHL), chronic lymphocytic leukemia, renal cell carcinoma, prostate cancer, including hormone-resistant prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, soft tissue cancer, gastrointestinal stroma tumor, gliobiform multiforme fins and multiple myeloma. According to another preferred embodiment, the disease is selected from the group consisting of retinopathy caused by age-related macular degeneration (e.g., wet AMD), diabetic retinal edema, redness of the iris, psoriasis, inflammatory kidney disease, hemolytic uremic syndrome, diabetic retinopathy (e.g. , proliferative diabetic retinopathy), arthritis (e.g., psoriatic arthritis, osteoarthritis, rheumatoid arthritis), inflammatory bowel disease, chronic pricking, chronic exfoliation of the cornea, chronic uveitis, chronic vitritis, corneal rejection of transplanted tissue, corneal neovascularization, corneal neovascularization of transplanted tissue, Crohn’s disease, myopia, ocular neovascular disease, Paget’s disease, pemphigoid, polyarthritis, post-laser neurosurgery, Sjogren’s syndrome, ulcerative colitis, implant rejection, pneumonia, nephrotic syndrome, edema, ascites associated with malignant tumors, ud Macaw, angiofibroma and neovascular glaucoma. In one embodiment, the subject is additionally administered a therapeutic drug selected from the group consisting of an anti-neoplastic drug, a chemotherapeutic drug, and a cytotoxic drug.

According to a preferred embodiment of the present invention, the subject being treated with an alpha5beta1 antagonist suffers from a relapse after treatment with a VEGF antagonist or becomes immune to treatment with a VEGF antagonist. According to another embodiment, a subject being treated with an alpha5beta1 antagonist and a VEGF antagonist is suffering from metastatic cancer or has previously undergone adjuvant therapy. In one embodiment, the disease in the candidate patient is relapse, refractory, or resistant to chemotherapeutic drugs, such as irinotecan. Examples of such diseases include, but are not limited to, metastatic colon cancer, relapse of metastatic colon cancer, metastatic breast cancer, relapse of metastatic breast cancer, metastatic breast cancer HER2 +, adjuvant breast cancer, adjuvant breast cancer HER2 +, metastatic cancer of the pancreas, adjuvant colon cancer , adjuvant non-small cell lung cancer, adjuvant rectal cancer, adjuvant non-small cell lung cancer, metastatic non-small cell lung cancer, metastatic ovarian cancer, metastat cal adjuvant renal cell carcinoma and renal cell carcinoma.

In accordance with one embodiment, a subject suffering from the disease described herein is provided with maintenance therapy after treatment with a VEGF antagonist, where maintenance therapy involves using an alpha5beta1 antagonist alone or after, or simultaneously with, a VEGF antagonist.

In accordance with one preferred embodiment, the VEGF antagonist can be selected from the group consisting of an antibody, immunoadhesin, peptibody, small molecule and nucleic acid that hybridizes with a nucleic acid molecule encoding VEGF under stringent conditions (e.g., ribozyme, siRNA or aptamer) . In accordance with one preferred embodiment, the VEGF antagonist is an antibody. In accordance with another embodiment, the antibody is a monoclonal antibody. In accordance with one preferred embodiment of the anti-VEGF antibody binding to human VEGF can be completely inhibited by antibodies Avastin ^®. In accordance with one preferred embodiment, the anti-VEGF antibodies are human, humanized, or chimeric. In accordance with one specific embodiment of the anti-VEGF antibody is the Avastin ^®. In accordance with another embodiment, anti-VEGF antibodies are selected from the group consisting of Fab, Fab ', F (ab)' ₂ , single-chain Fv (scFv), Fv fragment; diabodies and linear antibodies. According to another embodiment, the VEGF antagonist is a bispecific antibody that binds to VEGF and alpha5beta1 and is an alpha5beta1 antagonist.

In accordance with one preferred embodiment, the alpha5beta1 antagonist may be selected from the group consisting of an antibody, immunoadhesin, peptibody, small molecule, and nucleic acid that hybridizes to a nucleic acid molecule encoding alpha5beta1 under stringent conditions.

In accordance with one preferred embodiment, the alpha5beta1 antagonist is an antibody. In accordance with another embodiment, the antibody is a monoclonal antibody. According to a further embodiment, the monoclonal antibody is a chimeric antibody, such as an anti-human alpha5beta1 antibody, known as M200 or F200. In accordance with one embodiment, antibodies against alpha5beta1 include the sequence of VH SEQ ID NO: 1 and the sequence of VL SEQ ID NO: 2. In accordance with another embodiment, antibodies against alpha5beta1 include the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. In accordance with another embodiment, antibodies against alpha5beta1 include the sequence of SEQ ID NO: 4 and the sequence of SEQ ID NO: 5. According to a preferred embodiment of the invention, the binding of anti-alpha5beta1 antibodies to human alpha5beta1 can be completely inhibited by 7H5 antibodies or 7H12 antibodies. In accordance with one preferred embodiment, antibodies against alpha5beta1 are human, humanized or chimeric. In accordance with one specific embodiment, the anti-human alpha5beta1 antibody is a 7H5 antibody, a 7H12 antibody, or a chimeric or humanized antibody thereof. According to another embodiment, anti-alpha5beta1 antibodies are selected from the group consisting of Fab, Fab ', F (ab)' ₂ , single chain Fv (scFv), Fv fragment; diabodies and linear antibodies. According to another embodiment, the alpha5beta1 antagonist is a bispecific antibody that binds VEGF and alpha5beta1 and is a VEGF antagonist. According to yet another embodiment, the anti-alpha5beta1 antagonist has altered effector function. In one embodiment, anti-alpha5beta1 antibodies are modified to reduce or suppress antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) (for example, by changing the nucleic acid sequence encoding an antibody's Fc protein). According to yet another embodiment, anti-alpha5beta1 antibodies have been modified to increase the half-life in the human body (for example, by changing the nucleic acid sequence encoding the Fc region of an antibody).

In accordance with one embodiment, the VEGF antagonist or alpha5beta1 antagonist is conjugated to a cytotoxic drug or chemotherapeutic drug. In accordance with another embodiment, the cytotoxic drug is a radioisotope or toxin.

The present invention provides compositions comprising a VEGF antagonist, an alpha5beta1 antagonist and a pharmaceutically acceptable carrier. The present invention also provides articles comprising instructions for detecting alpha5beta1 in a subject treated with a VEGF antagonist.

The present invention also relates to the use of VEGFR agonists and alpha5beta1 agonists to stimulate angiogenesis and vascular permeability and compositions comprising VEGFR agonists and alpha5beta1 agonists and a pharmaceutically acceptable carrier. Combination therapy with VEGFR agonists and alpha5beta1 agonists can be used in the treatment of various diseases in which improvement can occur with increased angiogenesis and vascular permeability, including, for example, healing of wounds, such as treatment of chronic wounds, acute wounds and conventional wounds.

Brief Description of the Figures

Figure 1 shows an increase in the population of alpha5beta1 expressing stromal cells after treatment of xenograft HT29 tumors with anti-VEGF antibodies, B20-4.1.

Figure 2 is a diagram showing the binding of 7H5 and 7Hl2 antibodies to HUVEC cells in a direct binding assay.

Figure 3 shows the binding of 7H5 and 7H12 antibodies to HUVEC cells, but not to RAJI cells according to FACS analysis.

Figure 4 is a diagram showing the adhesion of HUVEC to fibronectin in the presence of purified monoclonal antibodies 7H5 and 7H12.

Figure 5 is a (A) bar graph showing the effect of 7H5 and 7H12 on HUVEC cell development by the total number of cells; and (B) a bar graph showing the effect of 7H5 and 7H12 on HUVEC cell development by Alamar blue stain in another study.

Figure 6 is a photograph of the migration of HUVEC cells after treatment with 7H5 for 0 h and 30 h compared with the negative control (IgG).

Figure 7 is a bar graph quantitatively showing the migration of HUVEC cells after treatment with 7H5 and 7H12.

Figure 8 is a bar graph showing the percentage of HUVEC cells expressing activated caspase-3 in apoptosis studies after treatment with 7H5 and 7H12.

Figure 9 is a bar graph showing caspase 3/7 activity in HUVEC cells after treatment with 7H5 and 7H12.

Figure 10 is a graph showing the activity of 7H12 and / or bevacizumab in a rabbit ear wound healing model.

Figure 11 shows the results of treatment with anti-VEGF antibodies with or without anti-alpha5beta1 antibodies on a breast cancer model in the form of (A) a chart showing the average tumor volume in the group of treated mice, or (B) a Kaplan-Meier graph showing the percentage of animals remaining in the study as a function of time. Animals were withdrawn from the study when their tumors reached or exceeded 1,500 mm ³ .

Figure 12 shows the results of treatment with anti-VEGF antibodies with or without anti-alpha5beta1 antibodies on a colon cancer model in the form of (A) a chart showing the average tumor volume in the group of treated mice, or (B) a Kaplan-Meier graph showing the percentage animals remaining in the study as a function of time. Animals were withdrawn from the study when their tumors reached or exceeded 1,500 mm ³ .

Figure 13 shows the results of treatment with anti-alpha5beta1 antibodies or a chemotherapeutic drug in a colon cancer model in the form of (A) a chart showing the average tumor volume in the group of treated mice, or (B) a Kaplan-Meier graph showing the percentage of animals remaining in the study as a function of time. Animals were withdrawn from the study when their tumors reached or exceeded 1,500 mm ³ .

Figure 14 shows a Scatchard plot of the binding of ¹²⁵ I-7H5 to alpha5beta1 on the rabbit R9ab rabbit fibroblast cell line.

Figure 15 shows a Scatchard plot of the binding of ¹²⁵ I-7H12 to alpha5beta1 on the rabbit R9ab rabbit fibroblast cell line.

Figure 16 shows the results of mapping IgG epitopes against integrin alpha5beta1 and studies of the competitive binding of various antibodies against alpha5beta1.

DETAILED DESCRIPTION OF THE INVENTION

Without being bound by theory, the authors hypothesized that increased recovery of the stromal cell population may deliver other vascular growth factors to the diseased areas, which may compensate for the loss of VEGF activity in patients treated with VEGF antagonists. Exposure of alpha5beta1 expressing stromal cells to anti-alpha5beta1 antibodies can lead to a decrease in the number of stromal cells, thereby reducing the production of potentially compensating vascular growth factors. Alternatively or additionally, the authors suggested that inhibition of the interactions between the endothelium and the extracellular matrix and partial inhibition of alpha5beta1 binding interactions will increase the effectiveness of treatment with VEGF antagonists by inhibiting the recurrence of angiogenesis along the traces in the extracellular matrix left by the regressing vessels resulting from treatment with the VEGF antagonist. Thus, treatment with alpha5beta1 antagonists, simultaneously with any treatment with or after VEGF antagonists, can inhibit vascular repair after treatment with a VEGF antagonist and, consequently, the resumption of neovascular growth.

“Alpha5beta1” or “α5β1” or “a5b1” is an integrin comprising two different proteins (ie, alpha5 and beta1 subunits). For alpha5beta1, it has been shown to bind to fibronectin, L1-CAM, and fibrinogen. Integrin alpha5beta1 is also called very late activation protein-5, VLA-5, alpha5beta1, CD49e / CD29, fibronectin receptor, FNR and GPIc-IIa. In a preferred embodiment, alpha5beta1 is human alpha5beta1.

"Alpha5" is also known as CD49e, alpha5, integrin alpha5 subunit, VLA-5 alpha subunit, GPIc-IIa IC subunit and FNR alpha chain has four isoforms resulting from alternative splicing (A-D). Variations undergo the cytoplasmic domains of proteins. Amino acid sequences of isoforms of human alpha5 can be found, for example, under access numbers Genbank X07979, U33879, U33882 and U33880, respectively.

“Beta1” is also called CD29, beta1, GPIIa platelet; VLA beta chain; beta-1 integrin chain, CD29; FNRB; MDF2; VLAB; GPIIA; MSK12 and VLA5B. Amino acid sequences of human beta1 can be found, for example, under Genbank accession number X06256.

The term “VEGF” or “VEGF-A,” as used herein, refers to 165 amino acid human vascular endothelial cell growth factor and related 121, 189, and 206 amino acid human vascular endothelial cell growth factors, as described in Leung et al. Science, 246: 1306 (1989), and Houck et al. Mol. Endocrin., 5: 1806 (1991), along with naturally occurring allelic and altered forms thereof. The term “VEGF” also refers to VEGF of non-human species, such as a mouse, rat or primate. Sometimes VEGF from a particular species is indicated by terms such as hVEGF for human VEGF, mVEGF for mouse VEGF, and the like. The term "VEGF" is also used to refer to shortened forms of a polypeptide comprising amino acids from 8 to 109 or from 1 to 109 of 165 165 amino acid human vascular endothelial cell growth factor. References to any such forms may be referred to herein, for example, as “VEGF (8-109)”, “VEGF (1-109)” or “VEGF ₁₆₅ ”. The amino acid positions in the “shortened” natural VEGF are numbered as indicated in the natural VEGF sequence. For example, the amino acid at position 17 (methionine) in truncated natural VEGF corresponds to amino acid 17 (methionine) in natural VEGF. Shortened natural VEGF has a binding affinity for the KDR and FIt-1 receptors, comparable to natural VEGF. According to a preferred embodiment, VEGF is human VEGF.

The term “VEGF antagonist” refers to a molecule capable of neutralizing, blocking, inhibiting, inactive, decreasing or interfering with the action of VEGF, in a manner comprising binding to VEGF or one or more VEGF receptors or nucleic acids encoding them. Preferably, the VEGF antagonist binds to VEGF or a VEGF receptor. VEGF antagonists include anti-VEGF antibodies and antigen binding sites thereof, polypeptides that bind to VEGF or VEGF receptors and block ligand-receptor interaction (e.g., immunoadhesins, peptibodies), anti-VEGF receptor antibodies and VEGF receptor antagonists such as low molecular weight inhibitors VEGFR tyrosine kinases, aptamers that bind VEGF and nucleic acids that hybridize under certain conditions with nucleic acid sequences that encode VEGF or a VEGF receptor (e.g. mRNA). In accordance with one preferred embodiment, the VEGF antagonist binds to VEGF and inhibits VEGF-induced proliferation of endothelial cells. According to one preferred embodiment, the VEGF antagonist binds to VEGF or the VEGF receptor with greater affinity than not to VEGF or not to VEGF receptor. According to one preferred embodiment, the VEGF antagonist binds to a VEGF or VEGF receptor with a Kd of between 1 μM and 1 pM. According to another preferred embodiment, the VEGF antagonist binds to a VEGF or VEGF receptor with a Kd between 500 nM and 1 pM.

According to a preferred embodiment, the VEGF antagonist is selected from the group consisting of a polypeptide, such as an antibody, peptibody, immunoadhesin, a low molecular weight compound or an aptamer. In a preferred embodiment, the antibody is an anti-VEGF antibody such as AVASTIN ^® antibody or an anti-VEGF receptor antibody such as an anti-VEGFR2 or against VEGFR3. Other examples of VEGF antagonists include VEGF-Trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258 , AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975 / CT6758 and KRN-633.

The term “anti-VEGF antibody” means an antibody capable of binding to VEGF with sufficient affinity and specificity. Preferably, the anti-VEGF antibody of the invention can be used as a therapeutic drug that targets and prevents diseases and conditions in which the action of VEGF is involved. Antibodies against VEGF usually do not bind to either other VEGF homologues, such as VEGF-B or VEGF-C, or to other growth factors such as P1GF, PDGF or bFGF. Preferred anti-VEGF antibodies are monoclonal antibodies that bind to the same epitope as the anti-VEGF monoclonal antibodies A.4.6.1 produced by the ATCC HB 10709 hybridoma. More preferably, the anti-VEGF antibodies are recombinant humanized anti-VEGF monoclonal antibodies generated in accordance with Presta et al. (1997) Cancer Res. 57: 4593-4599, including but not limited to the antibody known as bevacizumab (BV; Avastin ^®). In accordance with another embodiment, anti-VEGF antibodies that may be used include, but are not limited to, the antibodies disclosed in WO 2005/012359. In accordance with one embodiment, antibodies against VEGF include variable heavy and variable light regions of any of the antibodies disclosed in figures 24, 25, 26, 27 and 29 of application WO 2005/012359 (for example, G6, G6-23, G6-31 , G6-23.1, G6-23.2, B20, B20-4 and B20.4.1). In another preferred embodiment, the anti-VEGF antibody known as ranibizumab is a VEGF antagonist prescribed for eye diseases such as diabetic neuropathy and AMD.

An anti-VEGF antibody "bevacizumab (BV)", also known as "rhuMAb VEGF" or "Avastin ^® ", is a recombinant humanized monoclonal anti-VEGF antibody prepared according to Presta et al. (1997) Cancer Res. 57: 4593-4599. It includes mutant structural regions of human IgG1 and antigen-binding complementarity determining regions of a murine monoclonal antibody against hVEGF A.4.6.1, which blocks the binding of human VEGF to its receptors. About 93% of the amino acid sequence of bevacizumab, including most of the structural sites, is a derivative of human IgG1, and about 7% of the sequence is a derivative of mouse antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated. Other anti-VEGF antibodies include those described in United States Patent No. 6884879 and WO 2005/044853.

Antibodies against VEGF ranibizumab or antibodies LUCENTIS ^® or rhuFab V2 are humanized antibodies with a formed affinity against the human fragment of VEGF Fab. Ranibizumab is produced by conventional recombinant techniques using an expression vector for Escherichia coli and bacterial fermentation. Ranibizumab is not glycosylated and has a molecular weight of approximately 48,000 daltons. See WO98 / 45331 and US20030190317.

The term "alpha5beta1 antagonist" refers to any molecule that inhibits the biological effect of alpha5beta1. In accordance with one preferred embodiment, the antagonist molecule specifically binds alpha5beta1. In accordance with one preferred embodiment, the antagonist molecule binds to alpha5. According to one preferred embodiment, the alpha5beta1 antagonist preferably binds to alpha5beta1 with greater affinity than integrins other than alpha5beta1. In accordance with one preferred embodiment, the antagonist is selected from the group consisting of a polypeptide, such as an antibody, peptibody, or immunoadhesin, a low molecular weight compound or aptamer that inhibits the binding of alpha5beta1 to its ligands (in particular fibronectin), or a nucleic acid that hybridizes under stringent conditions with a nucleic acid molecule encoding alpha5beta1 (for example, mRNA that inhibits alpha5 expression). The biological effect of alpha5beta1 can be any of the effects, their combination, or all effects selected from the group consisting of (1) binding to fibronectin, (2) increased cell migration on fibronectin, (3) increased survival of cells containing alpha5beta1 in the presence of fibronectin, (4) increasing the proliferation of cells containing alpha5beta1 in the presence of fibronectin; and (5) increasing the formation of tubes from cells containing alpha5beta1 in the presence of fibronectin.

Examples of anti-alpha5beta1 antibody antagonists include M200 and F200 (WO 2004 / 089988A2), the 7H5 antibodies and 7H12 antibodies described herein, and chimeric, fully human and humanized antibodies based on them. For example, antibodies M200 and F200 can be obtained from the variable heavy and variable light chains of murine antibodies against human alpha5beta1, IIA1 (Pharmingen, San Diego, Ca). Examples of low molecular weight alpha5beta1 inhibitors include Ac-PHSCN-NH2 (WO-9822617A1) and (S) -2 - [(2,4,6-trimethylphenyl) sulfonyl] amino-3- [7-benzyloxycarbonyl-8- (2-pyridinylaminoethyl ) -1-oxa-2,7-diazaspiro (4,4) non-2-en-3-yl] carbonylamino] propionic acid. According to one preferred embodiment of the invention, an alpha5beta1 antagonist binds to alpha5beta1 but does not bind to alphaVbeta3, alphaVbeta5 or alphaVbeta1. According to one preferred embodiment of the invention, an alpha5beta1 antagonist binds to alpha5beta1 with a Kd of between 1 μM and 1 pM. According to another preferred embodiment of the invention, the alpha5beta1 antagonist binds to alpha5beta1 with a Kd of between 500 nM and 1 pM. In accordance with one preferred embodiment, the anti-alpha5beta1 antibody is an antibody that can compete with 7H5 or 7H12 when binding to alpha5beta1 in a competitive binding assay. According to another preferred embodiment, the antibody is an antibody whose binding to alpha5beta1 can be completely inhibited by antibodies produced by a hybridoma deposited as alpha5 / beta1 7H5.4.2.8 (ATCC No. PTA-7421) or a hybridoma deposited as alpha5 / beta1 7H12.5.1.4 (ATCC No. PTA-7420) March 7, 2006

The term "VEGFR agonist" refers to a molecule that can activate the VEGF receptor or increase its expression. VEGFR agonists include, but are not limited to, VEGFR ligand agonists, VEGF variants, antibodies, and active fragments.

The term "alpha5beta1 agonist" refers to a molecule that can activate alpha5beta1 or increase its expression. Alpha5beta1 agonists include, but are not limited to, for example, alpha5beta1 ligand agonists.

Molecules, such as antibodies, characterized by binding to overlapping or similar sites of the target, can be identified by conducting studies of competitive inhibition / binding.

In one embodiment, HUVEC cells or other cells expressing alpha5beta1 were used in competitive inhibition studies, FACS was used to evaluate the location of the binding sites of the two anti-alpha5beta1 antibodies relative to each other. For example, HUVEC cells can be washed in a conical tube and pelleted by centrifugation at 100 rpm for 5 minutes. The precipitate was usually washed twice. Cells can then be resuspended, counted and stored on ice until use. 100 μl of the first anti-alpha5 beta1 antibody can be added to the well (for example, starting at a concentration of 1 μg / ml or lower concentration). Then, 100 μl of cells (e.g., 20 × 10 ⁵ cells) can be added to each well, followed by incubation on ice for 30 minutes. Then, 100 μl of biotinylated anti-alpha5beta1 antibodies (stock solution 5 μg / ml) can be added to each well, followed by incubation on ice for 30 minutes. The cells were then washed and pelleted by centrifugation for 5 min at 1000 rpm. The supernatant has been removed. R-picoerythrin-conjugated streptavidin (Jackson 016-1 10-084) was added (100 μl, 1: 1000) to the well. The plate was then wrapped in foil and incubated on ice for 30 minutes. After incubation, the pellet could be washed and pelleted by centrifugation for 5 minutes at 1000 rpm. The pellet was resuspended and transferred to microtubes for FACS analysis.

The term "angiogenic factor or agent" refers to a growth factor that stimulates the development of blood vessels, for example, promotes angiogenesis, endothelial cell growth, blood vessel stability, and / or vasculogenesis, etc. Angiogenic factors include, but are not limited to, for example, VEGF and members of the VEGF family, P1GF, the PDGF family, the family of fibroblast growth factor (FGF), TIE ligands (angiopoietins), etherins, Del-1, fibroblast growth factors: acidic (aFGF) and major (bFGF), follistatin, granulocyte colony stimulation factor (G-CSF), hepatocyte growth factor (HGF) / scattering factor (SF), interleukin 8 (IL-8), leptin, midkine, placental growth factor, platelet growth factor endothelial cells (PD-ECGF), platelet-derived growth factor, in particular PDGF-BB or PD GFR-beta, pleiotrophin (PTN), progranulin, proliferin, transforming growth factor alpha (TGF-alpha), proliferin, transforming growth factor beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF) / vascular permeability factor (VPF), etc. They also include factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), CTGF and its family members, and TGF-alpha and TGF-beta . See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol 53: 217-39 (1991); Streit and Detmar, Oncogene, 22: 3172-3179 (2003); Ferrara & Alitalo, Nature Medicine 5 (12): 1359-1364 (1999); Tonini et al., Oncogene, 22: 6549-6556 (2003) (e.g., Table 1 listing known oncogenic factors); and, Sato Int. J. Clin. Oncol., 8: 200-206 (2003).

The term “Kd” or “Kd value” for an anti-VEGF antibody of the invention in one preferred embodiment is measured by examining the binding of a radiolabeled VEGF (RIA) between the Fab version of an antibody and a VEGF molecule, as described in the following measurement, in wherein the affinity of binding in Fab solution to VEGF is determined by balancing Fab with a minimum concentration of labeled ( ¹²⁵ I) VEGF (109) in the presence of a series of dilutions of unlabeled VEGF, followed by capture of bound VEGF on coated anti bodies against Fab plate (Chen, et al., (1999) J. Mol Biol 293: 865-881). To establish the conditions for the analysis, microtiter plates (Dynex) were coated with exciting anti-Fab antibodies (Cappel Labs) during incubation overnight with 5 μg / ml antibodies in 50 mM sodium carbonate (pH 9.6), after which 2% were blocked ( by weight) bovine serum albumin in FSB for two to five hours at room temperature (approximately 23 ° C). In a non-absorbent plate (Nunc No. 269620), 100 pM or 26 pM [ ¹²⁵ I] VEGF (109) was mixed with a series of dilutions of the Fab of interest, for example Fab-12 (Presta et al., (1997) Cancer Res. 57: 4593-4599 ) Then the Fab of interest was incubated overnight; however, incubation could continue for up to 65 hours in order to ensure that equilibrium is achieved. The mixtures were then transferred onto an exciting plate and incubated at room temperature for one hour. The solution was then removed, after which the plate was washed eight times with 0.1% Tween-20 in PBS. When the plates were dried, 150 μl of scintillation liquid (MicroScint-20; Packard) was added per well, after which the plates were counted on a Topcount gamma counter (Packard) for ten minutes. The concentration of each of the Fab, which gave binding less than or equal to 20% of the maximum, were selected for use in studies on competitive binding. In another embodiment, Kd or Kd value was measured using surface plasmon resonance assays using BIAcore ^™ -2000 or BIAcore ^™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C with immobilized hVEGF (8 -109), CM5 chips, and ~ 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Human VEGF was diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml (~ 0.2 μM) before administration at a flow rate of 5 μl per minute to obtain approximately 10 response units (RU) of the bound protein. Following the administration of human VEGF, 1M ethanolamine was introduced to block unreacted groups. For kinetic measurements, a series of two-fold dilutions of Fab (from 0.78 nM to 500 nM) was introduced into the FSB with the addition of 0.05% Tween 20 (PBST) at 25 ° C at a flow rate of approximately 25 μl / min. Association rates (k _on ) and dissociation rates (k _off ) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously substituting association and dissociation sensograms. The equilibrium dissociation constant (Kd) was calculated as the ratio k _off / k _on . See, for example, Chen, Y., et al., (1999) J. Mol Biol 293: 865-881. If the association rate exceeds 10 ⁶ M ⁻¹ S ⁻¹ according to the surface plasmon resonance studies described earlier, the association rate can be determined using the fluorescence quenching technique, which measures the increase or decrease in the fluorescence intensity (excitation 295 nm, radiation 340 nm , 16 nm bandwidth) at 25 ° C 20 nM anti-VEGF antibody (in the form of Fab) in FSB, pH 7.2 in the presence of increasing concentrations of short-form human VEGF (8-109) or mouse VEGF according to measurements using pektrometra, such as a spectrometer, equipped with a stop-flow (Aviv Instruments) or SLM-Aminco spectrophotometer series 8000 (ThermoSpectronic) to shake the cuvette. Similar binding studies can be carried out to determine Kd Fab or antibodies against alpha5beta1 using alpha5beta1 as a target.

As used herein, the subject to be treated is a mammal (e.g., a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). The subject may be a clinical patient, a clinical trial volunteer, an experimental animal, etc. The subject may be suspected of having or at risk of developing cancer, an immune disease, or any other disease characterized by pathological angiogenesis. Many methods for diagnosing cancer, immune diseases, or other diseases exhibiting abnormal angiogenesis and clinical presentation of these diseases are well known in the art. In accordance with one preferred embodiment, the subject to be treated is a human.

The term "abnormal angiogenesis" is used when new blood vessels grow either excessively or inappropriately (for example, the site, time or onset of angiogenesis is undesirable from a medical point of view) in a disease state or in such a way that it causes a disease state. Excessive, inappropriate, or uncontrolled angiogenesis occurs when the growth of a new blood vessel contributes to a worsening condition of the disease or causes a condition of the disease, such as cancer, especially vacuolated solid tumors and metastatic tumors (including cancer of the colon, lung (especially small cell lung cancer ), or prostate cancer), diseases caused by neovascularization of the eye, in particular diabetic blindness, retinopathy, mainly diabetic retinopathy or related with age of macular degeneration, choroidal neovascularization (CNV), diabetic macular edema, pathological myopia, Van Hippel-Lindau disease, histoplasmosis of the eye, retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization and rubeosis; psoriasis, psoriatic arthritis, hemangioblastoma, such as hemangioma; inflammatory kidney diseases such as glomerulonephritis, in particular mesangioproliferative glomerulonephritis, hemolytic uremic syndrome, diabetic nephropathy or hypertensive nephrosclerosis; various inflammatory diseases such as arthritis, in particular rheumatoid arthritis, inflammatory bowel disease, psoriasis, sarcoidosis, arteriosclerosis and diseases occurring after transplantation, endometrosis or chronic asthma and more than 70 other conditions. New blood vessels can nourish affected tissues, destroy normal tissues, and, in the case of cancer, new vessels can allow tumor cells to go into circulation and settle in other organs (tumor metastases). The present invention contemplates the treatment of patients at risk of the aforementioned diseases.

Other patients who are candidates for antibodies or polypeptides of this invention have or are at risk of developing, abnormal proliferation of vascular fibrous tissue, red acne, acquired immunodeficiency syndrome, clogged arteries, atopic keratitis, bacterial ulcers, Behcet's disease, blood-borne tumors , obstructive carotid artery disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitritis, excessive contact lens wear, corneal transplant rejection, corneal neovascularization, corneal neovascularization, Crohn’s disease, Ailes disease, epidemic keratoconjunctivitis, fungal ulcers, herpes simplex infection, herpes zoster infections, high viscosity syndromes, lipoma leukemia, sarcoma leukemia, sarcoma leukemia , regional keratolysis, Muren's ulcer, mycobacterial infections with the exception of leprosy, myopia, ocular neovascular disease, congenital pits of the optic nerve, syn Osler-Weber rum (Osler-Weber-Randu), osteoarthritis, Paget’s disease, ciliary circle inflammation, pemphigoid, phylectenulosis, polyarteritis, complications after laser radiation, infections of protozoa, elastic pseudoxanthoma, pterygium, dry keratitis, pterygitomyceratitis neovascularization of the retina, retrolental fibroplasia, sarcoid, scleritis, sickle cell anemia, Sjogren's syndrome, solid tumors, Starthart's disease, Steven Johnson's disease, higher limbic keratitis, syphilis, systemic lupus erythematosus, marginal degeneration of the cornea, toxoplasmosis, trauma, Ewing's sarcoma tumors, neuroblastoma tumors, osteosarcoma tumors, retinoblastoma tumors, rhabdomyosarcoma tumors, ulcerative colitis, vein occlusion, Vitamin A deficiency and Wegner sarcoidosis, parasitic disease, anemia wound healing, post-surgical hypertrophy, wounds or injuries, suppression of hair growth, suppression of ovulation and corpus luteum formation, suppression of implantation and suppression m development of the embryo in uterus.

Anti-angiogenesis therapy is useful in the general treatment of transplant rejection, pneumonia, nephrotic syndrome, preeclampsia, effusion into the pericardial region, such as associated with pericarditis, and pleural effusion, diseases and disorders characterized by undesirable vascular permeability, for example, edema associated with brain tumors , ascites associated with malignant tumors, Meigs syndrome, pneumonia, nephrotic syndrome, pericardial effusion, pleural effusion, permeable Tew associated with cardiovascular diseases, such as postinfarction state, shocks and the like.

Other angiogenesis-dependent diseases in accordance with this invention include angiofibroma (abnormal bleeding from bleeding vessels), neovascular glaucoma (growth of blood vessels in the eye), arteriovenous malformations (abnormal communication between arteries and veins), non-healing fractures (fractures that cannot be cured), atherosclerotic plaques (hardening of the arteries), pyogenic granuloma (a frequent pathological change in the skin consisting of blood vessels), scleroderma (a form of disease connective tissue), hemangioma (a tumor consisting of blood vessels), trachoma (the main cause of blindness in the third world), hemophilic arthropathy, vascular adhesion and hypertrophic scars (improper scar formation).

The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include those who already have the disorder and those in whom the disorder should be prevented.

The terms “recurrence,” “relapse” or “relapse” refer to the return of a cancer or disease after the clinical definition of the disappearance of the disease. Diagnosis of distant metastases or local recurrence may be considered a relapse.

The terms “immune” or “resistant” refer to a cancer or disease that does not respond to treatment.

The term "adjuvant therapy" refers to treatment carried out after the main treatment, usually surgical. Supportive therapy for cancer or disease may include immune therapy, chemotherapy, radiotherapy or hormone therapy.

The term “supportive therapy” refers to a planned re-treatment that is performed to help maintain the effects of prior therapy. Maintenance therapy is often done to maintain cancer in remission or to lengthen the response to a particular therapy, regardless of the progress of the disease.

The term “invasive cancer” refers to cancer that has spread beyond the layer of tissue in which it started into normal surrounding tissue. Invasive cancer may or may not be metastatic.

The term "non-invasive cancer" refers to a very early stage of cancer or to cancer that does not extend beyond the tissue from which it originates.

The term "progression-free" in oncology refers to a period of time during and after treatment during which the cancer does not grow. Progression-free survival includes the amount of time that the patient has experienced a complete or partial response, as well as the period of time that the patient has experienced a stable illness.

The term "progressive disease" in oncology may refer to tumor growth of more than 20 percent since the start of treatment, either due to an increase in the mass of the tumor, or due to the spread of the tumor.

The term “disorder” refers to any condition in which treatment with antibodies will benefit. For example, mammals suffering from abnormal angiogenesis (excessive, inappropriate or uncontrolled angiogenesis) or vascular permeability or in need of their prevention. This includes chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question. Non-limiting examples of treatable disorders include malignant and benign tumors, non-leukemic and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocele disorders; and inflammatory, angiogenic and immunological disorders.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals, which is usually characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Specific examples of such cancers include squamous cell cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, intestinal cancer, colon cancer, endometrial sarcoma, salivary gland sarcoma, kidney cancer, renal tubular cancer , prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, head and neck cancer, colorectal cancer, colon cancer, lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma, squamous cell carcinoma etiology (e.g., epithelial squamous cell cancer), prostate cancer, peritoneal cancer, hepatic cell carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, retinoblastoma, astrocytoma, tekoma, arrenoblastoma, hepatoma, hematologic malignancies, including non-Hodgkin’s lymphoma (NHL), multiple myeloma and acute hematologic malignant conditions, endometrial or intrauterine carcinoma, endometriosis, fibrosarcoma, choriocarcinoma, salivary carcinoma, vulvar cancer, cancer thyroid gland, carcinoma of the esophagus, carcinoma of the liver, carcinoma of the rectum, carcinoma of the penis, nasopharyngeal carcinoma, carcinoma of the larynx, Kaposi’s sarcoma, melanoma, carcinoma of the skin, schwannomas, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, cariomyoma, cariomyoma, leukomyocytoma, leiomyocytoma Wilm's tumor, as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; medium differentiated / follicular NHL; diffuse NHL moderate; highly differentiated immunoblastic NHL; highly differentiated lymphoblastic NHL; highly differentiated small cell NHL with undigested nuclei; NHL with massive lesion, mantle cell lymphoma; AIDS-associated lymphoma, Wandelstrom disease (macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL), nappy cell leukemia; chronic leukemia of myeloblasts; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatoses and Meigs syndrome.

The term “tumor,” as used herein, refers to neoplastic cell growth and proliferation, malignant and benign, and all precancerous and cancerous cells and tissues.

The term “anti-neoplastic composition” or “anti-neoplastic preparation” refers to a composition useful in the treatment of cancer, including at least one active therapeutic factor, for example, “anti-cancer drug”. Examples of therapeutic agents (anti-cancer drugs) include, but are not limited to, for example, chemotherapeutic drugs, growth inhibitory drugs, cytotoxic drugs, drugs used in radiotherapy, anti-angiogenesis drugs, apoptotic drugs, anti-tubulin drugs, and other cancer drugs, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor antagonist (EGFR) (e.g. tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g. er lotinib (Tarceva ^TM ), platelet growth factor inhibitors (e.g. Gleevec ^TM (imatinib mesylate)), a COX-2 inhibitor (e.g. celecoxib), interferons, cytokines, antagonists (e.g. neutralizing antibodies) that bind to one or more of the following goals - with the receptor (s) ErbB2, ErbB3, ErbB4, PDGFR-beta, BAFF, BR3, APRIL, BCMA or VEGF, TRAIL / Apo2 and other biologically active and organic chemical factors, etc. Their combinations are also included in this invention.

As used herein, the term “growth inhibitory factor” refers to a compound or composition that inhibits cell growth in vitro and / or in vivo. Thus, a factor that significantly reduces the percentage of cells in the S phase can be a factor for suppressing growth. Examples of growth inhibitory factors include factors that block the cell cycle (at a point other than the S phase), such as factors that cause G1 to stop and M phase to stop. Classical M phase blockers include vinca alkaloids (vincristine and vinblastine), TAXOL ^® and topoII inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. G1-blocking factors (agents) also extend to S-phase blocking, for example, DNA-alkylating drugs such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.

The term "cytotoxic drug (agent)", as used here, refers to a substance that inhibits or prevents the functioning of cells and / or causes the destruction of cells. The term is intended to include radioactive isotopes (e.g., I ¹³¹ , I ¹²⁵ , Y ^90, and Re ¹⁸⁶ ), chemotherapeutic drugs, and toxins, such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof.

The term “chemotherapeutic drug” means a chemical compound used in the treatment of cancer. Examples of chemotherapeutic drugs include chemical compounds that can be used to treat cancer. Examples of chemotherapeutic drugs include alkylating agents such as thiotepa and CYTOXAN ^® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and pyrosulfan; aziridines such as benzodopa, carboxwon, meturedopa, and uredopa; ethyleneimines and methyleneimines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl lomelamine; acetogenins (mainly bullatacin and bullatacinone); camptothecin (including the synthetic analogue of topotecan); bryostatin; callistatin; CC-1065 (including synthetic analogues of adoselesin, carzelesin and biselezin); cryptophycins (namely cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodictin a; spongistatin; nitrogen mustards, such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydroxide oxide, melphalan, novembicin, fenesterin, prednimustine, trophosphamide, uramustine; nitrosourea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediin antibiotics (for example, calichimycin, in particular calichemicin gamma-II and calichimycin omega-II (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynamin, including dinemycin A; bisphosphonates, such as chlodronate; esperamycin; as well as the neocarcinostatin chromophore and associated chromoprotein and endiotic antibiotic chromophores), aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, chrominomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carcinomycin, carinomycin 5-oxo-L-norleucine , ADRIAMYCIN ^® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycin, pomymnomycin, oligomycin, pomymnomycin, oligomycin, pomymnomycin, oligomycin, pomenomycin quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimerexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluuridine, enocytabine, phloxuridine; androgens such as calusterone, dromostanolone propionate, epitostanol, mepostostan, testolactone; adrenal inhibitors such as aminoglutethimide, mitotan, trilostane; a folic acid replenisher such as frolinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabucil; bisanthrene; edatrexate; defofamine; demecolcin; diaziquon; elfornithine; elliptin acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maitanzinoids such as maitanzine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethyl hydrazide; procarbazine; PSK ^® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2,2 ', 2 "trichlorotriethylamine; trichotecenes (in particular T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobromane; gacitosin; arabinoside (Ara"");cyclophosphamide;thiotepa; taxoids such as paclitaxel TAXOL ^® (Bristol-Myers Squibb Oncology, Princeton, NJ.), Cremophor ABRAXANETM, an albumin-based nanoparticle form of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and doxetaxel TAXOTERE ^® (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine GEMZAR ^® ; 6-thioguanine; mercaptopurine; methotrexate; analogues platinum, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine NAVELBINE ^® ; novantrone; teniposide; edatrexate; daunomycin; aminopterin; CP-xeloda; ibandronate ) (including treatment regimen with irinotecan with 5-FU and leucovorin); RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimen (FOLFOX); PKC-alpha, Raf, H-Ras and EGFR inhibitors (e.g., erlotinib (TarcevaTM)) that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of all of the above drugs.

Also included in this specification are anti-hormonal drugs that regulate or suppress the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor regulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX ^® ), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifen , keoxifene, LY117018, onapriston, and FARESTON-toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate MEGASE ^®, ekzemestan AROMASIN ^®, formestane, fadrozole, vorozole RIVISOR ^®, letrozole FEMARA ^® and Anastrozole ARIMIDEX ^® ; antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (1,3-dioxolane, a cytosine nucleoside analogue); antisense oligonucleotides, in particular inhibiting the expression of signaling pathway genes involved in the propagation of aberrant cells, such as, for example, PKC-alpha, Raf and H-Ras; ribozymes such as an expression inhibitor of VEGF (e.g., ribozyme ANGIOZYME ^®) and HER2 expression inhibitor; vaccines such as genetic therapy vaccines, for example ALLOVECTIN ^® vaccine, LEUVECTIN ^® vaccine, and VAXID ^® vaccine; PROLEUKIN ^® rIL-2; LURTOTECAN ^® topoisomerase 1 inhibitor; ABARELIX ^® rmRH; vinorelbine and esperamycins (see US patent No. 4675187), and pharmaceutically acceptable salts, acidic forms or derivatives of the above drugs.

The term “prodrug”, as used herein, refers to a precursor or derivative of a pharmaceutically active substance (eg, a low molecular weight compound) that is less cytotoxic to diseased cells than the parent drug and can be enzymatically activated or converted to a more active parent form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al, "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (Ed.), Pp. 247-267, Humana Press (1985). Prodrugs in accordance with this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, possibly containing substituted phenoxyacetamide prodrugs or optionally substituted phenylacetamide prodrugs containing 5-fluorocytosine and other 5-fluoruridine prodrugs which They can be converted to a more cytotoxic free drug. Examples of cytotoxic drugs that can be converted to prodrugs for use in this invention include, but are not limited to the chemotherapeutic drugs described above.

The term "isolated" when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and isolated and / or isolated from the cell or cell culture in which it is expressed. Contaminating components in its natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to produce at least 15 residues of the N-terminal or internal amino acid sequence using a rotary beaker, or (2) to homogeneity according to SDS-PAGE under non-reducing and reducing conditions using blue Coomassie or, preferably, silver. An isolated polypeptide includes an in situ polypeptide in recombinant cells since at least one component of the natural environment of the polypeptide is not present. However, usually an isolated polypeptide can be made using at least one purification step.

The term "isolated" nucleic acid encoding a polypeptide or other nucleic acid encoding a polypeptide refers to a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule to which it is normally associated in a natural source of a nucleic acid encoding a polypeptide. An isolated polypeptide encoding a nucleic acid molecule is in a different form or environment than those in which it is found in nature. An isolated polypeptide encoding a nucleic acid molecule is thus different from a polypeptide encoding a nucleic acid molecule in natural cells. However, isolated polypeptide-encoding nucleic acid molecules include a polypeptide-encoding nucleic acid molecule, which typically express the polypeptide in the case, for example, when the nucleic acid molecule is in a position on the chromosome other than the position in natural cells.

The term "control sequences" refers to DNA sequences necessary for the expression of operably linked coding sequences in a particular host organism. Control sequences suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is in functional relationship with another nucleic acid sequence. For example, a DNA presequence or secretory leader sequence is operatively linked to a DNA encoding a polypeptide if it expresses a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned in a manner that facilitates translation. In general, “operably linked” means that the DNA sequences are connected and continuous, and, in the case of a secretory leader sequence, continuous and are in the same reading phase. However, enhancers do not have to be adjacent. Binding occurs by ligation at suitable restriction sites. If such sites do not exist, synthetic linkers and adapters are used in accordance with normal practice.

“Stringent conditions” or “high stringency conditions” as described herein can be defined as (1) including low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% dodecyl sulfate sodium at 50 ° C; (2) comprising a denaturing factor, such as formamide, during hybridization, for example, 50% (volume) of formamide with 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer with pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) overnight hybridization in a solution that contains 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution sonicated with salmon sperm (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C with washing for 10 minutes at 42 ° C in 0.2x SSC (sodium chloride / sodium citrate) s followed by a 10 minute rinse under high stringency conditions consisting of 0.1 x SSC containing EDTA at 55 ° C.

“Percentage (%) of amino acid sequence identity” with respect to the polypeptide sequences given here is defined as the percentage in the sequence of amino acid residues discussed that are identical to the amino acid residues in the compared polypeptide after alignment and, if necessary, to achieve the maximum percentage of sequence identity, introducing gaps, conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining the identity of amino acid sequences can be achieved in various ways that are within the knowledge of a person skilled in the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Specialists in the art can determine the appropriate parameters for measuring homology, including the algorithms required to achieve maximum homology within the full length of the compared sequences. However, for the tasks to be solved here,% amino acid sequence identity was obtained using the ALIGN-2 computer program for comparing sequences. A computer program for comparing sequences of ALIGN-2 was developed by Genentech, Inc, the source code (table 1) was entered into the registry along with user documentation in U.S. Copyright Office, Washington D.C., 20559, where it was registered under US registration number TXU510087. ALIGN-2 is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program must be compiled for use on a UNIX operating system, preferably a digital UNIX V4.0D system. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

The amino acid sequences described herein are contiguous amino acid sequences unless otherwise indicated.

As used herein, the term “immunoadhesin” refers to antibody-like molecules that combine the binding specificity of a heterologous protein (“adhesin”) with the effector functions of the immunoglobulin constant domains. Structurally, immunoadhesins include a fusion sequence consisting of an amino acid sequence with a desired binding specificity different from that of the antibody antigen recognition and binding site (ie, being “heterologous”), and immunoglobulin constant domain sequences. The adhesion portion of an immunoadhesin molecule is typically a continuous amino acid sequence comprising at least a receptor or ligand binding site, such as a VEGFR or fibronectin ligand. The constant portion of the immunoglobulin sequence in the immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4, IgA subtypes (including IgA-1 and IgA-2), IgE, IgD or IgM . Peptibodies, often containing the sequence obtained by selection from phage display sequences that specifically bind to the target, and their fusion with the Fc portion of the immunoglobulin can also be considered immunoadhesins here.

The term “antibody” is used in the most general sense and, in particular, refers to, for example, single monoclonal antibodies (including agonists, antagonists and neutralizing antibodies), antibody compositions with multi-epitope specificity, polyclonal antibodies, single chain antibodies and antibody fragments (see below) , as long as they specifically bind to the native polypeptide and / or exhibit biological or immunological activity according to this invention. In accordance with one embodiment, the antibody binds to an oligomeric form of the target protein, for example a trimeric form. In accordance with another embodiment, the antibody specifically binds to a protein, this binding can be inhibited by monoclonal antibodies of the invention (e.g., a deposited antibody of the invention, etc.). The phrase “functional fragment or analogue” of an antibody refers to a compound having qualitative biological activity common with the antibody with which it is compared. For example, a functional fragment or analog of an antibody of the invention may specifically bind to VEGF or alpha5beta1. In one embodiment, the antibody can prevent or significantly reduce the ability of VEGF to cause cell proliferation.

An “isolated antibody” is an antibody that has been identified and separated and / or isolated from components of its natural environment. The polluting components of its natural environment are materials that can interfere with the diagnostic or therapeutic use of antibodies, including enzymes, hormones and other protein or non-protein soluble substances. In preferred embodiments, the antibodies will be purified (1) to more than 95% by weight of antibodies, as determined by the Lowry method, and, most preferably, to more than 99% by weight, (2) to a degree sufficient to produce at least 15 amino acid residues of the N-terminal or internal amino acid sequence using a rotary beaker, or (3) homogeneity according to SDS-PAGE under reducing or non-reducing conditions using Coomassie blue staining or, preferably, sulfur Br. Isolated antibodies include in situ antibodies in recombinant cells since at least one component of the natural environment of the antibodies will not be present. However, usually isolated antibodies are prepared using at least one purification step.

The basic 4-chain unit of an antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 conventional heterotetrameric units, along with an additional polypeptide called chain J, and thus contains 10 antigen binding sites, and secreted IgA antibodies can polymerize to form polyvalent structures, including 2-5 basic 4-chain units together with the J-chain). In the case of IgG, the 4-chain unit usually has a mass of about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain. Each of the H and L chains also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (V _H ) at the N-terminus, followed by three constant domains (C _H ) for each of the chains α and γ, and four constant C _H domains for isotypes μ and ε. Each L chain has a variable domain (V _L ) at the N-terminus, followed by a constant domain (C _L ) at the other end. V _L is opposite V _H and C _L is opposite the first constant domain of the heavy chain C _H ¹ . It is believed that certain amino acid residues form an interaction between the variable regions of the heavy and light chains. Paired together, V _H and V _L form the antigen binding site. The structure and properties of various classes of antibodies are described, for example, in Basic and Clinical Immunology. 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinguishable types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C _H ), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, which have heavy chains designated respectively by α, δ, γ and μ. The classes γ and α are further subdivided on the basis of relatively small differences in the C _H sequence and function, for example, the following subclasses are expressed in humans: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term "variable" refers to the fact that some segments of the variable domains have significant sequence differences among antibodies. Domain V provides antigen binding and determines the specificity of a particular antibody to a particular antigen. However, the variability is not evenly distributed within the 110 amino acids of the variable domains. Instead, sections V are composed of relatively constant gaps called framework sections (FR) of 15-30 amino acids, separated by shorter sections of exceptional variability, called "hypervariable sections", each of which is 9-12 amino acids in length. Each of the variable domains of natural heavy and light chains includes four FRs, mainly taking the beta-sheet configuration, associated with three hypervariable regions that form loops, connecting and in some cases forming part of the beta-sheet structure. In each chain, the hypervariable regions are held together in close proximity by FR and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding region of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit a variety of effector functions, such as the participation of an antibody in antibody dependent cell cytotoxicity (ADCC).

The term “hypervariable region”, as used herein, refers to the amino acid residues in an antibody responsible for antigen binding. A hypervariable region typically includes amino acid residues from a “complementarity determining region” or “CDR” (eg, residues of about 24-34 (L1), 50-56 (L2) and 89-97 (L3) in V _L , and about 31- 35B (H1), 50-65 (H2), and 95-102 (H3) in V _H (in one embodiment, H1 is about 31-35); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or “hypervariable loop” residues (eg, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in V _L , and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in V _H ; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

As used herein, the term “monoclonal antibody” refers to antibodies obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies contained in the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single site of antigen. Moreover, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized uncontaminated by other antibodies. The “monoclonal” modifier should not be construed as requiring the production of antibodies in any particular way. For example, monoclonal antibodies useful in the present invention can be obtained using the hybridoma technology first described by Kohler et al., Nature, 256: 495 (1975), or can be obtained using recombinant DNA techniques in bacterial, eukaryotic animals or plants cells (see, for example, US patent No. 4816567). "Monoclonal antibodies" can also be isolated from phage libraries of antibodies using the techniques described, for example, in Clackson et al., Nature. 352: 624-628 (1991), Marks et al., J. Mol. Biol., 222: 581-597 (1991), and the examples below.

Here, monoclonal antibodies also include “chimeric” antibodies in which a part of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from certain species or belonging to a particular class or subclass, while the remaining part of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of these antibodies, until they demonstrate biological activity in accordance with this invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA. 81: 6851-6855 (1984)). Chimeric antibodies of interest in this invention include primatized antibodies comprising variable-domain antigen binding sequences derived from a non-human primate (eg, Old World monkeys, anthropoid apes, etc.), and human sequences constant sections.

An “intact” antibody is an antibody containing an antigen binding site, as well as C _L and at least constant heavy chain domains C _H 1, C _H 2 and C _H 3. The constant domains can be constant domains of the natural sequence (eg, human constant domains of the original sequence) or a variant of their amino acid sequence. Preferably, the intact antibody has one or more effector functions.

“Antibody fragments” include parts of whole antibodies, preferably an antigen binding or variable region of a whole antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ , and Fv fragments; diabodies, linear antibodies (see US Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); single chain antibody molecules; and multispecific antibodies assembled from antibody fragments.

The expression "linear antibodies" usually refers to the antibodies described in Zapata et al., Protein Eng., 8 (10): 1057-1062 (1995). In short, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding sites. Linear antibodies may be bispecific or monospecific.

Papain digestion of antibodies gives two identical antigen-binding fragments, called "Fab" fragments, and the residual "Fc" fragment, the designation reflects the ability to crystallize easily. The Fab fragment consists of an entire L chain together with the variable domain H of the chain (V _H ), and the first constant domain of one heavy chain (C _H ¹ ). Each Fab fragment is monovalent with respect to antigen binding, i.e. has a single antigen binding site. Pepsin treatment of the antibody yields one large F (ab ') ₂ fragment, which roughly corresponds to two Fab disulfide-linked bridged fragments having divalent antigen-binding activity and still capable of cross-linking the antigen. Fab 'fragments differ from Fab fragments in that they have several additional amino acid residues at the C-terminus of the C _H 1 domain, including one or more cysteine residues from the hinge region of the antibody. Fab'-SH is the designation used here for Fab ', in which one or more cysteine residues of the constant domain carry a free thiol group. Antibody fragments F (ab ') ₂ were initially prepared as pairs of Fab' fragments having hinged cysteine residues between them. Other chemical compounds of antibody fragments are also known.

The Fc fragment includes the C-terminal regions of both H chains linked together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which is also a part recognized by the Fc receptors (FcR) found on certain types of cells.

"Fv" is the smallest fragment of an antibody containing the entire antigen recognition and binding site. This fragment is a dimer consisting of one variable region of the heavy and one light chain in a close, non-covalent bond. As a result of the binding of these two domains, six hypervariable loops are formed (3 loops from H and L chains), which contain amino acid residues involved in antigen binding and impart specificity for antigen binding to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity compared to the whole binding site.

“Single chain Fvs”, also abbreviated as “sFvs” or “scFvs,” are antibody fragments that include V _H and V _L antibody domains joined to the same polypeptide chain. Preferably, the sFv polypeptide also comprises a polypeptide linker between the V _H and V _L domains, allowing sFv to form the structure required for antigen binding. For a description of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, below.

The term “diabodies” refers to small antibody fragments obtained by constructing sFv fragments (see the previous paragraph) with short linkers (approximately 5-10 residues) between the V _H and V _L domains, thus pairing the V domains between the chains, and not inside chain, which leads to the formation of a bivalent fragment, for example, a fragment having two antigen binding sites. Bispecific diabodies are heterodimers of two “crossed” sFv fragments in which the domains of two antibodies V _H and V _L are present in different polypeptide chains. Diabodies are described more fully, for example, in EP 404097, WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

“Humanized” forms of non-human antibodies (eg, rodent antibodies) are chimeric antibodies that contain a minimal sequence derived from non-human antibodies. Mostly, humanized antibodies are human immunoglobulins (recipient antibodies) in which the remnants of the recipient’s hypervariable region are replaced by the remnants of the hypervariable region of the antibody (donor antibody) from a non-human species, such as a mouse, rat, rabbit or non-human primate, having desired specificity, affinity and capabilities. In some cases, residues of the structural region (FR) of a human immunoglobulin are replaced by corresponding non-human residues. Moreover, humanized antibodies may contain residues not found in either the recipient or the donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody includes essentially all of at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those found in non-human immunoglobulin and all or substantially all FRs are FRs from the human immunoglobulin sequence. Humanitariannet antibodies can also include at least a portion of the constant region of immunoglobulin (Fc), usually from human immunoglobulin. For more details see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

A "species-dependent antibody" is an antibody that has a greater affinity for binding to an antigen from one mammalian species than to its homologue from another mammalian species. Typically, species-dependent antibodies “specifically bind” to the human antigen (ie, have a binding affinity (Kd) of not more than 1 × 10 ^-7 M, preferably not more than 1 × 10 ^-8 , and most preferably not more than 1 × 10 ^-9 M), but have an affinity for binding to a homologue of an antigen from another non-human mammalian species, which is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than the affinity of its binding to the human antigen. Species-dependent antibodies can belong to any of the various types of antibodies defined above, but are preferably humanized or human antibodies.

In such embodiments, the degree of binding of the polypeptide, antibody, antagonist or composition to a “non-target” protein will be less than about 10% of the binding of the polypeptide, antibody, antagonist or composition to a protein that is its direct objective according to analysis by activated fluorescence sorting of cells ( FACS) or radioimmune precipitation (RIA). With regard to the binding of a polypeptide, antibody, antagonist or composition to a target molecule, the term “specific binding” or “specifically binds to” or “specific” for a particular polypeptide or epitope on a particular polypeptide target means a binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is usually a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, with an excess of unlabeled target. In this case, specific binding will be shown if the binding of the labeled target to the probe is competitively suppressed by an excess of unlabeled target. As used herein, the term “specific binding” or “specifically binds to” or “specific for” a particular polypeptide or epitope on a particular polypeptide target can be illustrated, for example, by a molecule having a Kd relative to the target of at least about 10 ^-4 M, alternatively not less than about 10 ^-5 M, alternatively not less than about 10 ^-6 M, alternatively not less than about 10 ^-7 M, alternatively not less than about 10 ^-8 M, alternatively not less than about 10 ^-9 M alternatively not less than about 10 ^-10 M, alternatively not less than about 10 ^-11 M, alternatively not less than about 10 ^-12 M or better. In one embodiment, the term “specific binding” refers to a binding in which a molecule binds to a particular polypeptide or epitope of a particular polypeptide in the absence of significant binding to any other polypeptide or epitope of the polypeptide.

The term "effector functions" of an antibody refers to the biological activities attributed to the Fc region (Fc region of the natural sequence or amino acid sequence variant of the Fc region) of the antibody and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors; and B cell activation. The term “natural sequence of an Fc region” means an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Examples of Fc sequences are described, for example, but not limited to Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The term “Fc region variant” means an amino acid sequence that differs from the natural sequence of the Fc region by the advantage of having at least one “amino acid modification” described in this invention. Preferably, the Fc region variant has at least one amino acid substitution as compared to the natural sequence of the Fc region or the Fc region of the parent polypeptide, for example from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the natural sequence of the Fc region or in the Fc region of the parent polypeptide. In one embodiment, an embodiment of the Fc region described herein will have at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, or at least about 99% homology to the Fc region of the natural sequence. In accordance with another embodiment, an embodiment of the Fc region described herein will have at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, or not less than approximately 99% homology with the Fc region of the parent polypeptide.

“Percentage (%) of amino acid sequence identity” or “amino acid sequence homology” with respect to the polypeptide sequences described herein is defined as the percentage in the sequence of amino acid residues discussed that are identical to the amino acid residues in the compared polypeptide after alignment and, if necessary, to achieve the maximum percentage of sequence identity , introducing gaps, and conservative substitutions are not considered part of the identity of subsequent flaxes. Alignment for the purposes of determining the% identity of amino acid sequences can be achieved in various ways that are within the skill of a person skilled in the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Specialists in the art can determine the appropriate parameters for measuring homology, including the algorithms required to achieve maximum homology within the full length of the compared sequences. However, for the tasks to be solved here,% amino acid sequence identity was obtained using the ALIGN-2 computer program for comparing sequences. ALIGN-2 sequence comparison software was developed by Genentech, Inc, the source code was entered into the registry along with user documentation in U.S. Copyright Office, Washington D.C., 20559, where it was registered under US registration number TXU510087. ALIGN-2 is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program must be compiled for use on a UNIX operating system, preferably a digital UNIX V4.0D system. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

The term "polypeptide comprising an Fc region" refers to a polypeptide, such as an antibody or immunoadhesin (see definitions above), comprising an Fc region. The lysine located at the C-terminus (residue 477 according to the EU numbering system) of the Fc region can be removed, for example, by purification of the polypeptide or by recombinant construction of a nucleic acid encoding the polypeptide. Accordingly, a composition comprising polypeptides, including antibodies having an Fc region of the invention, may comprise populations of polypeptides in which all K447 residues are deleted, polypeptide populations in which not a single K447 residue is deleted, or polypeptide populations having a mixture of polypeptides with and without a K447 residue.

Within the scope of this specification and claims, with respect to residues in the variable domain (approximately 1-107 light chain residues and 1-113 heavy chain residues), a Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest) is commonly used. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). An “EU numbering system” or “EU index” is typically used when referring to a residue in a constant region of an immunoglobulin heavy chain (for example, the EU index is given in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), summarized here by reference). Unless otherwise indicated, references to residue numbers in the variable domain of antibodies mean the numbering of residues according to the Kabat numbering system. Unless otherwise indicated, references to residue numbers in the constant domain of antibodies mean the numbering of residues according to the EU numbering system.

The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to an Fc region of an antibody. In one embodiment, the FcR of the present invention binds an IgG antibody (gamma receptor) and includes receptors of the subclasses FcγRI, FcγRII, and FcγRIII, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains a tyrosine-based activation site (ITAM) in the cytoplasmic domain. The inhibitory receptor FcγRIIB contains in the cytoplasmic domain a tyrosine-based inhibitory site (ITIM) (see review publication M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). The term includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those that will be discovered in the future, are also covered herein by the term “FcR”. The term also includes the neonatal FcRn receptor, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).

The term “FcRn” refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of a цепи chain non-covalently linked to ∃2-microglobulin. Numerous functions of the neonatal Fc FcRn receptor are discussed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays a role in the passive delivery of IgG immunoglobulins from mother to calf and the regulation of serum IgG levels. FcRn can act as a reutilization receptor by binding and transporting pinocytosed IgG intact into and through cells, allowing them to avoid the usual metabolic pathway leading to their degradation.

WO00 / 42072 (Presta) and Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001) describes antibody variants with increased or decreased binding to FcR. The contents of these publications are incorporated herein by reference.

The “CH1 domain” of the human IgG Fc region (also called the “C1” or “H1” domain) typically ranges from about 118 amino acids to about 215 amino acids (according to the EU numbering system).

The term “hinge region” is usually defined as the length from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22: 161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine bases forming S-S bonds between the heavy chains in one position.

The “lower hinge portion” of the Fc portion is usually defined as the length of the bases directly at the C-end of the hinge portion, i.e. residues from 233 to 239 sites Fc. In previous studies, binding to FcR was usually attributed to amino acid residues in the lower hinge region of the IgG Fc region.

The "CH2 domain" of the human IgG Fc region (also referred to as the "C2" or "H2" domain) is usually in the region of 231 amino acids to 340 amino acids. The CH2 domain is unique in that it is not closely paired with another domain. Instead, between the two domains of the CH2 intact natural IgG molecule, there are two N-linked branched carbohydrate chains. It has been suggested that carbohydrates can replace the pairing of a domain with a domain and help stabilize the CH2 domain. Burton, Molec. Immunol. 22: 161-206 (1985).

The “CH3 domain” (also referred to as the “C2” or “H3” domain) includes the extent of residues at the C-terminus of the CH2 domain in the Fc region (i.e., approximately from amino acid residue 341 to the C-terminus of the antibody sequence, usually at the amino acid residue 446 or 447 IgG).

A “functional Fc region” has “effector functions” of the natural sequence of the Fc region. Examples of “effector function” include C1q binding, complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis; negative regulation of cell surface receptors (i.e., B cell receptors, BCR), etc. Such effector functions typically require a combination of the Fc region with a binding domain (e.g., the variable domain of an antibody) and can be evaluated using various assays, for example, as disclosed herein.

“C1q” is a polypeptide that includes the binding site of an Fc region to an immunoglobulin. C1q, together with two serine proteases, C1r and C1s, form the C1 complex, the first component of the complement dependent cytotoxicity (CDC) pathway. Human C1q can be purchased commercially, for example from Quidel, San Diego, CA.

The term “binding domain” refers to a portion of a polypeptide that binds to another molecule. In the case of FcR, the binding domain may include a part of its polypeptide chain (for example, its alpha chain), which is responsible for the binding of the Fc region. One useful binding domain is the extracellular domain of the FcR alpha chain.

An antibody or peptibody with an IgG Fc variant with an “altered” FcR binding affinity or ADCC activity has an increased or decreased ability to bind FcR (eg, FcγR or FcRn) and / or ADCC activity compared to the parent polypeptide or to a polypeptide comprising the parent sequence of the Fc region . An Fc variant that "exhibits improved binding" to FcR binds to at least one FcR with a higher affinity (i.e., lower Kd or IC50) than the native sequence Fc polypeptide or IgG. In accordance with some variants of implementation, the increase in binding compared with the original polypeptide can be approximately 3-fold, preferably approximately 5, 10, 25, 50, 60, 100, 150, 200, or 500 times, or from about 25% to 1000 % improvement in binding. A variant of a polypeptide that "shows reduced binding" to FcR binds to at least one FcR with a lower affinity (i.e., with a higher Kd or IC50) than the parent polypeptide. In accordance with some variants of implementation, the decrease in binding compared with the original polypeptide can be approximately 40% or more.

The term “antibody-dependent cell mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig binds to FcR receptors present on some cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages), allowing these cytotoxic cells -effects to specifically bind to antigen-carrying target cells and subsequently kill target cells using cytotoxins. Antibodies “cock” cytotoxic cells and are absolutely necessary for such destruction. The main cells that are ADCC mediators, NK cells express FcγRIII only, while monocytes express FcγRI, FcγRII and FcγRIII. The expression of FcR in hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). In order to evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed as described in US Pat. No. 5,500,362 or 5,821,337 or in the Examples below. Effector cells used for such studies include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively or additionally, the ADCC activity of the molecule of interest can be investigated in vivo, for example in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

A polypeptide comprising an Fc region variant that "exhibits increased ADCC" or is a mediator of antibody-dependent cell mediated cytotoxicity (ADCC) in the presence of human effector cells is more effective than a polypeptide having wild-type IgG Fc or the parent polypeptide is significantly more effective in vitro or in vivo as an ADCC mediator, if the amounts of the polypeptide with the variant Fc region and the polypeptide with the wild-type Fc region (or the parent polypeptide) in the study are almost the same vym. Typically, such variants are determined using in vitro ADCC studies, such as those described herein, but other assays or methods for determining ADCC activity are implied, for example in an animal model, etc. In one embodiment, the preferred embodiment is more effective as an ADCC mediator compared to wild-type Fc (or parent polypeptide) about 5 to about 100 times, for example, about 25 to about 50 times.

The term “complement dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to antibodies (corresponding subclass) that are associated with an antigen that they recognize. To evaluate complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

Variants of the polypeptide with altered amino acid sequences of the Fc region and a reduced or increased ability to bind C1q are described in US patent No. 6194551B1 and WO99 / 51642. The contents of these publications are hereby incorporated by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term "human effector cells" refers to white blood cells expressing one or more FcRs and performing effector functions. In one embodiment, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer cells (NKs), monocytes, cytotoxic T cells, and neutrophils; PBMC and NK cells are preferred. Effector cells can be isolated from their natural source, for example from blood or PBMC, as described herein.

Methods for measuring binding to FcRn are known (see, for example, Ghetie 1997, Hinton 2004), and are also described in the examples below. In vivo binding to human FcRn and the life span of high affinity human FcRn binding polypeptides can be studied, for example, on transgenic mice or transfected human cell lines expressing human FcRn, or on primates that have been introduced with an Fc polypeptide variant. In one embodiment, anti-alpha5beta1 antibodies having altered Fc IgG in accordance with the invention showed increased binding affinity for human FcRn compared to a polypeptide having wild-type Fc IgG of at least 2 times, not less than 5 times, not less than 10 times, not less than 50 times, not less than 60 times, not less than 70 times, not less than 80 times, not less than 100 times, not less than 125 times, not less than than 150 times. In a specific embodiment, the binding affinity for human FcRn increased by approximately 170 times.

In one embodiment, the EC50 or apparent Kd (at pH 6.0) of the FcRn binding affinity polypeptide was less than 1 μM, more preferably less than or equal to 100 nM, more preferably less than or equal to 10 nM. In one embodiment, for an increased binding affinity for FcγRIII (F158; i.e., a low affinity isotype), the EC50 or apparent Kd was less than or equal to 10 nM, and for FcγRIII (V158; a high affinity isotype), the EC50 or apparent Kd was less than or equal to 3 nM. According to another embodiment, a decrease in the binding of the antibody to the Fc receptor relative to the control antibody (e.g., Herceptin ^® antibodies) can be considered significant relative to the control antibody if the ratio of the absorption values at the midpoint of the binding curves of the test antibody and the control antibody (e.g., A _{450nm (antibody)} / A _{450nm (control antibody)} ) less than or equal to 40%. According to another embodiment, an increase in the binding of the antibody to the Fc receptor relative to the control antibody (e.g., Herceptin ^® antibodies) can be considered significant relative to the control antibody if the ratio of the absorption values at the midpoint of the binding curves of the test antibody and the control antibody (e.g., A _{450nm (antibody)} / A _{450nm (control antibody)} ) is greater than or equal to 125%. See, for example, Example 16.

A “parent polypeptide” or “parent antibody” is a polypeptide or antibody comprising an amino acid sequence from which an altered polypeptide or antibody is generated and with which a modified polypeptide or antibody is compared. Typically, the parent polypeptide or parent antibody does not have one or more modifications of the Fc region described herein, and differs from the altered polypeptide described herein in effector functions. The parent polypeptide may include the natural sequence of the Fc region or the Fc region with existing modifications of the amino acid sequence (such as additions, deletions and / or substitutions).

Antibodies in accordance with this invention can be obtained using phage display. As used herein, the term “library” refers to a plurality of antibody sequences, or antibody fragments, or nucleic acids that encode these sequences, the sequences have differences in the combination of variable amino acids that have been introduced into these sequences in accordance with the methods of the invention.

A “phage display” is a technique by which variants of a polypeptide are demonstrated as fusion proteins as at least a portion of a membrane protein of phage particles, such as filamentous phage. The usefulness of phage display is that large libraries of random protein variants can be quickly and efficiently sorted by the presence of sequences that bind the target antigen with high affinity. Mapping peptide and protein libraries on phages is used to test millions of polypeptides for specific binding features. Methods of polyvalent phage displays are used to demonstrate small random peptides and small proteins by fusing them with either gene III or gene VIII of the filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3: 355-362 (1992) and references cited therein. In a monovalent phage display, a protein or peptide library is fused to gene III or part thereof and is expressed at low levels in the presence of wild-type gene III, so that phage particles show one copy or do not show fusion proteins. Compared to polyvalent phage, avidity is reduced, so that sorting based on a significant affinity for ligands, and phagemid vectors that facilitate DNA manipulation are used. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3: 205-0216 (1991).

A “phagmid” is a plasmid vector having a bacterial origin of replication, such as ColE1, and a copy of the intergenic region of the bacteriophage. The phagemid can be used with any known bacteriophage, including filamentous bacteriophages and lambdoid bacteriophages. The plasmid also usually contains a selection marker for antibiotic resistance. DNA segments cloned into these vectors can be transmitted as plasmids. When the cells containing these vectors are supplied with all the genes necessary for the production of phage particles, the method of plasmid replication is changed to roll ring replication to obtain copies of one strand of plasmid DNA and package phage particles. Phagemids can form infectious and non-infectious phage particles. This term refers to phagmids that contain the gene for the phage coat protein or its fragment associated with the gene of the heterologous polypeptide so that, as a result of gene fusion, the heterologous polypeptide is displayed on the surface of the phage particle.

The term “phage vector” means a double-stranded replication form of a bacteriophage containing a heterologous gene and capable of replicating. The phage vector has a phage replication initiation point, allowing phage replication and the formation of a phage particle. The phage is preferably a filamentous bacteriophage such as M13, fl, fd, Pf3 phage or a derivative thereof, or a lambdoid phage such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative thereof.

Covalent modifications of the polypeptides, such as peptibodies, immunoadhesins, antibodies and short peptides, are included within the scope of this invention. One covalent modification method involves reacting the desired amino acid residues of the polypeptide with an organic modifying reagent that is capable of reacting with selected side chains or with the N- or C-terminal residues of the polypeptide. Modification with bifunctional reagents is useful, for example, to create cross-linking of a polypeptide with a water-insoluble support matrix or surface for use in the method of purification of antibodies, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, 4-azidosalicylic acid esters, homobifunctional imido esters, including disuccinimidyl esters, such as 3.3 ' -dithiobis (succinimidyl propionate), bifunctional maleides, such as bis-N-maleimido-1,8-octane, and reagents such as methyl-3 - [(p-azidophenyl) dithio] propioimidate.

Other modifications include the deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, of hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains [T. E. Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of an N-terminal amine, amidation of any C-terminal carboxyl group.

Other modifications include conjugation with toxin antagonists such as maytansine and maytansinoids, calicheamicin and other cytotoxic substances.

Another type of covalent modification of the polypeptides includes the binding of the polypeptide to one or a variety of non-protein polymers such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene, according to the method described in US patent No. 4640835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The polypeptide in accordance with the present invention can also be modified, if this is beneficial, so as to form a chimeric molecule comprising a polypeptide fused to another, heterologous polypeptide or amino acid sequence (for example, immunoadhesins or peptibodies).

In one embodiment, such chimeric molecules include a polypeptide fused to a protein transduction domain that targets the polypeptide to be delivered to various tissues, and more particularly through the blood-brain barrier, using, for example, the transduction domain of the human immunodeficiency virus TAT protein (Schwarze et al. 1999, Science 285: 1569-72).

In another embodiment, such a chimeric molecule comprises a polypeptide fused to a tag polypeptide that provides an epitope to which anti-tag antibodies can selectively bind. The epitope tag is usually located at the amino or carboxyl end of the polypeptide. The presence of such epitope-labeled forms of the polypeptide can be detected using antibodies against the tag polypeptide. Also, the presence of an epitope tag allows the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds the tag-epitope. Various label polypeptides and their corresponding antibodies are known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) labels; the flu HA polypeptide label and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988)]; the c-myc label and its antibodies 8F9, 3C7, 6E10, G4, B7, and 9E10 [Evan et al., Molecular and Cellular Biology. 5: 3610-3616 (1985)]; herpes simplex virus glycoprotein D (gD) label and antibodies thereto [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include the Flag peptide [Hopp et al., BioTechnology. 6: 1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)]; α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and peptide tag of the 10 T7 protein gene [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA 87: 6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise a polypeptide fused to an immunoglobulin or a specific portion of an immunoglobulin. For a bivalent form of a chimeric molecule (for example, “immunoadhesin”), a similar fusion can be performed with the Fc region of an IgG molecule. Fusion Igs of the present invention include polypeptides that comprise approximately or only residues 94-243, residues 33-53, or residues 33-52 from a human, instead of at least one variable region in the Ig molecule. In a particularly preferred embodiment, the fusion immunoglobulin comprises a hinge region and CH2 and CH3 regions, or a hinge region and CH1, CH2, and CH3 regions of an IgG1 molecule. The preparation of fusion immunoglobulins is also described in US Pat. No. 5,428,130, June 27, 1995.

The invention provides methods and compositions for inhibiting or preventing relapse of tumor growth or relapse of cancer cell growth. In various embodiments, the cancer is a relapse of the growth of tumors or a relapse of the growth of cancer cells if the number of cancer cells has not markedly decreased, or increased, or the size of the tumor has not significantly decreased, or increased, or attempts to further reduce the size or number of cancer are unsuccessful. cells. The determination of whether the cancer cells are a relapse of the tumor growth or a relapse of the growth of the cancer cells is carried out in vivo or in vitro by any method for evaluating the effectiveness of the effect on cancer cells known in the art. A tumor resistant to treatment against VEGF is an example of a recurrence of tumor growth.

An “effective amount” of a polypeptide, antibody, antagonist, or composition described herein corresponds to an amount sufficient to fulfill a specific stated purpose. An “effective amount” can be determined empirically and using known methods related to the declared purpose.

The term “therapeutically effective amount” refers to an amount of an antibody, polypeptide or antagonist according to this invention, effective for “treating” a disease or disorder in a mammal (ie, patient). In the case of cancer, a therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size or weight of the tumor; suppress (i.e. slow down to some extent or, preferably, stop) cancer infiltration into peripheral organs; suppress (i.e. slow down to some extent, or preferably stop) tumor metastases; suppress, to some extent, tumor growth; and / or alleviate to some extent one or more symptoms associated with cancer. In order for the drug to prevent the growth and / or kill existing cancer cells, it must be cytostatic and / or cytotoxic. In one embodiment, the therapeutically effective amount is an amount that inhibits growth. In another embodiment, the therapeutically effective amount is an amount that improves progression-free patient survival. In another embodiment, the therapeutically effective amount is an amount that extends the life of the patient.

In the case of treatment of wounds, the term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective to accelerate or improve wound healing in a subject. A therapeutic dose is a dose that demonstrates a therapeutic effect on a patient, and a subtherapeutic dose is a dose that does not demonstrate a therapeutic effect on a patient being treated.

The term "chronic wound" refers to a wound that does not heal. See, for example, Lazarus et al., Definitions and guidelines for assessment of wounds and evaluation of healing, Arch. Dermatol. 130: 489-93 (1994). Chronic wounds include, but are not limited to, for example, arterial ulcers, diabetic ulcers, pressure sores, trophic ulcers, etc. An acute wound can develop into a chronic wound. Acute wounds include, but are not limited to, wounds caused, for example, by thermal damage, trauma, surgery, removal of extensive skin cancer, deep fungal and bacterial infections, vasculitis, scleroderma, pemphigoid, toxic epithelial necrosis, etc. See, for example, Buford, Wound Healing and Pressure Sores, HealingWell.com, published: October 24, 2001. The term “conventional wound” refers to a wound that undergoes conventional wound healing.

The term "growth inhibitory amount" of a polypeptide, antibody, antagonist or composition according to this invention refers to an amount capable of inhibiting the growth of cells, especially tumor, for example cancer cells, both in vitro and in vivo. A growth inhibitory amount of a polypeptide, antibody, antagonist, or composition of this invention for inhibiting the growth of neoplastic cells can be determined empirically or by known methods, or as in the examples given here.

The term "cytotoxic amount" of a polypeptide, antibody, antagonist or composition according to this invention refers to an amount capable of causing destruction of cells, especially tumor, for example cancer cells, both in vitro and in vivo. The cytotoxic amount of a polypeptide, antibody, antagonist or composition according to this invention for inhibiting the growth of neoplastic cells can be determined empirically or by methods known in the art.

The term "autoimmune disease" here means a disease or disorder originating from the individual tissues of the individual and directed against them or their joint manifestation or symptoms, as well as the resulting condition. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, psoriatic arthritis spinal arthritis, and juvenile rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, deforming arthritis, primary chronic polyarthritis, reactive arthritis, and ankylosing pondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as spotted psoriasis, teardrop psoriasis, pustular psoriasis and nail psoriasis, dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis, associated dermatitis, associated dermatitis, dermatitis and associated dermatitis X chromosome syndrome of increased IgM, urticaria, such as chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, youth Oske dermatomyositis, toxic epidermal necrosis, scleroderma (including systemic scleroderma), sclerosis, such as systemic sclerosis, multiple sclerosis (MS), such as spinoptic MS, primary progressive MS, and remitting MS, progressive systemic sclerosis, atherosclerosis, atherosclerosis , Charcot-Vulpian disease, and atactic sclerosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, colitis such as ulcerative colitis, microscopic colitis, collagen colitis, polyposis colitis, necr tyrosing enterocolitis, and transmural colitis and autoimmune inflammatory bowel diseases), gangrenous pyoderma, erythema nodosum, primary sclerosing cholangitis, episiscleritis, respiratory distress syndrome, respiratory distress syndrome in adults or acute respiratory distress syndrome (ARDS), meningitis, inflammation eyeball, iritis, choroiditis, and autoimmune hematologic disorder, rheumatoid spondylitis, sudden hearing loss, mediation IgE diseases such as anaphylactic, allergic and atopic rhinitis, encephalitis, such as Ramussen's encephalitis and limbic encephalitis and / or brain stem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, uveitis, or autoimmune uveitis, with glomerulonephritis (GN) with or without nephrotic syndrome, such as chronic or acute glomerulonephritis, such as primary GN, immuno-mediated GN, membrane GN (non-membrane phropathy), idiopathic membrane GN, membrane proliferative GN (MPGN), including type I and type II, and fast progressive GN, allergic conditions, allergic reactions, eczema, including allergic or atopic eczema, asthma such as bronchial asthma, and autoimmune asthma , conditions involving T-cell infiltration and chronic inflammatory responses, chronic pulmonary inflammatory diseases, autoimmune myocarditis, lymphocyte adhesion failure, systemic lupus erythematosus (SLE) or syst many lupus erythematosus, such as cutaneous SLE, subacute systemic lupus erythematosus, newborn lupus erythematosus (NLE), distributed lupus erythematosus, lupus erythematosus (including nephritis, encephalitis, pediatric, nonrenal, discoid, alopecia), juvenile diabetes mellitus (type I), and diabetes mellitus diabetes, including pediatric insulin-dependent diabetes mellitus (IDDM), adult diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus; immune responses associated with immediate and delayed hypersensitivity, mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, including lymphomatoid granulomatosis, Wegner’s granulomatosis, agranulocytosis, vasculitides, including vasculitis (rheumatic arteriomyocytes and arthritis) medium vessels (including Kawasaki disease and nodular polyarthritis), microscopic polyarthritis, central nervous system vasculitis, necrotizing, skin or allergic mi vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as vasculitis or Churg-Strauss syndrome (CSS), transient arthritis, aplastic anemia, autoimmune aplastic anemia, positive Kumba anemia, autoimmune hemolytic anemia, and incomplete anemia hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia, Addison's disease, true red blood cell anemia, or apl Asia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, inflammatory CNS disorders, multiple organ damage syndromes, such as secondary after sepsis, trauma or bleeding, antigen-mediated diseases antibody, glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Becet and Behcet's disease, Castleman’s syndrome, Goodpaste’s syndrome, Rey’s syndrome o, Sjögren’s syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and pemphigoid skin, pemphigus (including pemphigus foliaceae, pemphigus pemphigus, mucous membrane pemphigoid and erythematous pemphigus fibrosis, nephroimmutociomas, neurologic fibroids, and nephritis, chronic neuropathy, such as IgM neuropathies or IgM-mediated neuropathies, thrombocytopenia (developing, for example, in patients with myocardial infarction), including thrombocytopenic thrombohemolytic purpura (TTP) and autoimmune or immuno-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP, autoimmune disease of testes and ovaries, hypothyroid syndromes, and hypothyroid syndromes, including including thyroiditis, such as autoimmune thyroiditis, Hashimoto’s disease, chronic thyroiditis (Hashimoto’s thyroiditis), or subacute thyroiditis, autoim moon thyroid diseases, idiopathic hypothyroidism, Grave’s disease, polygranular syndromes such as autoimmune polygranular syndromes (or polygranular endocrinopathic syndromes), paraneoplastic syndromes, including neurological neuroplastic syndromes, such as Lambert-Eaton’s myasthenic syndrome, or Itast syndrome, human "or" harsh person ", encephalomyelitis, such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergies RP G encephalomyelitis (EAE), myasthenia gravis, a degeneration of the cerebellum, neyromiotoniey, opsoclonus or opsoklonicheskim myoclonic syndrome (OMS), and sensory neuropathy syndrome Sheehan, autoimmune hepatitis, chronic hepatitis, lyupoznym hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis , lymphoid intermediate pneumonitis, obliterating bronchioitis (non-transplant) or NVP (non-specific intermediate pneumonia), Guillain-B syndrome arre, Berger’s disease (IgA nephropathy), IgA idiopathic neuropathy, IgA linear dermatosis, primary biliary cirrhosis, pneumocirrhosis, autoimmune enteropathic syndrome, celiac disease, celiac disease, abdominal sprue (celiac enteropathy), refractory cirrhosis, cirrhosis, refractory, cystic fibrosis, refractory, cystic fibrosis, refractory, cirrhosis, refractory, (ALS, Lou Gehrig's disease), coronary heart disease, autoimmune disease of the inner ear (AIED), or autoimmune hearing loss, opsoclonic myoclonic syndrome (OMS), polychondritis and, such as refractory or recurrent polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis, including monoclonal B-cell lymphocytosis (for example, benign monoclonal gammopathy and monoclonal gamma neuropathy neuropathy, neoplastic neuropathy, neuroplasty, canalopathies such as epilepsy, migraine, arrhythmia, muscle disorders, deafness, blindness, periodic paralysis and CNS canalopathies, autism, inflammation myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatologic disorder, fibromyalgia, multiple endocrine insufficiency, such as nephrotic disease, adrenal gland disease, and diabetic disease Dressler’s syndrome, focal alopecia, CREST syndrome (calcification, Reynald’s phenomenon, violation of motility of the esophagus, sclerodact lia and telangiectasia), male and female autoimmune infertility, mixed collagenosis, Chagas disease, rheumatic fever, recurring miscarriage, "farmer's lungs", exudative erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, pulmonary pulmonary allergic allergy, benign pulmonary allergy, angiitis, Alport syndrome, alveolitis, such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, lei shmaniozom, kipanosomiazom, schistosomiasis, askaridiazom, aspergillosis syndrome Sumpter, syndrome Kaplan, dengue, endocarditis, endomyocardial fibrosis, pulmonary fibrosing alveolitis interstrtsialnym pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, congenital anemia of newborn, eosinophilic fasciitis, Schulman's syndrome, Felty's syndrome, filariasis, cyclitis, such as chronic cyclitis, heterologous cyclitis, iridocyclitis or Fuchs cyclitis, purpura Genoch a-Scheinen, infection with human immunodeficiency virus (HIV), infection with echovirus, cardiomyopathy, Alzheimer's disease, infection with parvovirus, infection with rubella virus, post-vaccination syndromes, congenital infection with rubella, infection with Epstein-Barr virus, mumps, and Emanoma syndrome glands, Sydenham chorea, post-streptococcal nephritis, thromboangitis obliterans, thyrotoxicosis, spinal cord dryness, chorioretinitis, giant cell polymyaglia, endocrine ophthalmos Opatija, chronic hypersensitivity pneumonitis, dry keratokonyuktivitom, epidemic keratokonyuktivitom, idiopathic nephritic syndrome, minimal-change nephropathy, benign familial and ischemia-reperfusion injury, autoimmune lesions of the retina, inflammation of joints, bronchitis, chronic obstructive airway diseases, silicosis, thrush, aphthous stomatitis, arteriosclerotic disorders, aspergimiogenesis, autoimmune hemolysis, Beck's disease, cryoglobulin ile, Dupuytren's contracture, phacoanaphylactic endoophthalmia, allergic enteritis, type II lepromatous reaction, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hammen-Rich disease, sensorineural hemorrhage, paraxinemia pulmonary gonorrhea, hypoxic leukemia, hypoxic leukemia, hypoxic leukemia, hypoxic leukemia, hypoxic leukemia myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyrad iculoneuritis, gangrenous pyoderma, subacute thyroiditis, acquired spenic atrophy, sterility due to the presence of antibodies against sperm, non-malignant thymoma, vitiligo, diseases associated with SCID and Epstein-Barr viruses, acquired immunodeficiency syndrome (AIDS), para Leishmania, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses associated with hypersensitivity an immediate and delayed type, mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ trauma syndrome, diseases mediated by the antigen-antibody complex, diseases of the main antglomerular membrane, allergic neuritis, autoimmune polyendocrinopathic, mycophoriomatous, mycophoriomatous, myoforimouropory, gastritis, sympathetic ophthalmia, rheumatic diseases, mixed collagenosis, nephrotic syndrome, ins lithi, polyendocrine insufficiency, peripheral neuropathy, autoimmune polygranular type I syndrome, ideopathic hypoparathyroidism of adults (AOIH), generalized allopecia, dilated cardiomyopathy, acquired bullous epidermolysis, hemochromatosis, nephritic fibrosis, myocardiomyositis, myocardiomyositis, myocarditis, myocarditis acute or chronic sinusitis, epimoid, frontal, maxillary and ofenoid sinusitis associated with eosinophilic disorders and, such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Leffler’s syndrome, chronic eosinophilia pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma or Kimura’s syndrome, anaphyloxia, seronegative spondylorromatosis, scleral dermatitis, polyenditis, polyenditis, and chronic mucocutaneous candidiasis, Bruton's syndrome, temporary infant hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia, autoimmu disorders associated with collagenosis, rheumatism, neurological diseases, ischemic reperfusion disorder, a response to lowering blood pressure, vascular dysfunction, angiectasia, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and concomitant vascular hypertrophy, vascular hyperflammation injuries, reperfusion injuries of the myocardium or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other inflammatory disorders of the central nervous system, syndromes associated with transfusion of granulocytes, toxicity induced by cytokines, severe acute inflammations, chronic intractable inflammations, pyelitis, pneumocirrhosis, diabetic retinopathy of the large intestine arteriosis, hypertension , valvulitis and endometriosis.

Methods of treating cancer can be evaluated by, for example, tumor regression, reduction in tumor weight or size, progression time, survival time, progression-free survival, overall response rate, response duration, quality of life, expression and / or protein activity, but not limited to these indicators. Since the anti-angiogenic drugs described here are aimed at the vessels of the tumor, and not necessarily at the neoplastic cells themselves, they represent a unique class of anticancer drugs and, therefore, require unique criteria and definitions of clinical responses to the drugs. For example, tumor reduction of more than 50% in a 2-dimensional analysis is the standard boundary value for confirming the response. However, alpha5beta1 antagonists and the VEGF antagonists of the invention can cause inhibition of the spread of metastases without reducing the primary tumor, or can simply cause a tumorigenic effect. Accordingly, approaches can be applied to determine the effectiveness of therapy, including, for example, measuring markers of angiogenesis in plasma or urine and measuring response by radiological imaging.

Depending on the indications for treatment and the factors relevant to the dosage that a medical professional in this field should be familiar with, the antibodies of the invention will be administered in a dosage that is effective in treating symptoms while minimizing toxicity and side effects. For the treatment of cancer, an autoimmune disease or immunodeficiency disease, a therapeutically effective dose may be, for example, in the range of 50 mg per dose to 2.5 g / m ² . In one embodiment, dosages of from about 250 mg / m ² to about 400 mg / m ² or 500 mg / m ² are administered. In another embodiment, the dosage is approximately 250-375 mg / m ² . In yet another embodiment, the dosage range is 275-375 mg / m ² .

Therapies for age-related macular degeneration (AMD) can be evaluated, but not limited to, reducing speed or preventing further loss of vision. For the treatment of AMD, in vivo efficacy can be measured, for example, by one of the following methods: estimating the average change in the best corrected visual activity (BCVA) from the baseline over a given time, estimating the relative number of subjects who have lost less than 15 characters of visual activity compared to the main line for a certain time, the assessment of the relative number of subjects who acquired the number of characters of visual activity, more than or equal to 15 compared with the main line for definitely time, by assessing the relative number of subjects with the equivalent of Snellen's visual activity of 20/2000 after a certain time, evaluating with the help of a questionnaire the visual functioning of NEI, estimating the size of CNV and the amount of CNV leaking for a certain time according to fluorescein angiography, etc.

The term “detection” is intended to include determining the presence or absence of a substance or quantifying the amount of a substance. Thus, the term refers to the use of materials, compositions and methods in accordance with the invention for qualitative and quantitative determinations. In general, the particular technique used to determine is not critical to the practice of the invention.

For example, “detection” in accordance with the invention may include: observing the presence or absence of an alpha5 gene product, mRNA molecules, or alpha5 polypeptide; a change in alpha5 polypeptide levels or the amount associated with the target; changes in the biological function / activity of the alpha 5 polypeptide. In some embodiments, “detection” may include detection of wild-type alpha5 levels (eg, mRNA or polypeptide levels). Detection may include quantifying the change (increase or decrease) of any value between 10% and 90%, or any value between 30% and 60%, or more than 100% compared to the control. The definition may include quantifying the change in any value from 2-fold to 10-fold, inclusive, or more, for example, 100-fold.

The term “label,” as used herein, refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody. The label can be detectable on its own (for example, radioisotope tags or fluorescent tags) or, in the case of an enzymatic tag, can catalyze a detectable chemical change in the substrate, compound or composition.

New antibodies against alpha5beta1

New antibodies are proposed here that can bind human alpha5beta1 and competitively inhibit the binding of anti-alpha5beta1 antibodies to human alpha5beta1. In accordance with one embodiment, antibodies against alpha5beta1 are produced by a hybridoma selected from the group consisting of a hybridoma deposited as Alpha5 / beta1 7H5.4.2.8 (ATCC No. PTA-7421) and a hybridoma deposited as Alpha5 / beta1 7H12.5.1. 4 (ATCC No. PTA-7420) at ATCC March 7, 2006. In accordance with another embodiment, the antibody is produced by a hybridoma selected from the group consisting of a hybridoma deposited as Alpha5 / beta1 7H5.4.2.8 (ATCC No. PTA-7421 ), and hybridomas deposited as Alpha5 / beta1 7H12.5.1.4 (ATCC No. PTA-7420) at ATCC on March 7, 2006. In accordance with yet another embodiment m implementation, the antibody contains a sequence of variable heavy (VH) and variable light (VL) domains of antibodies produced by a hybridoma deposited as Alpha5 / beta1 7H5.4.2.8 (ATCC No. PTA-7421) in ATCC March 7, 2006. In another an embodiment, the antibody comprises a sequence of variable heavy (VH) and variable light (VL) domains from antibodies produced by a hybridoma deposited as Alpha5 / beta1 7H12.5.1.4 (ATCC No. PTA-7420) in ATCC on March 7, 2006. Human is also contemplated. and chimeric forms of antibodies deposited by hybridomas.

In accordance with one embodiment, the antibody binds to human alpha5beta1 with a Kd of between 500 nM and 1 pM. According to another embodiment, the antibody does not bind alphaVbeta3, or alphaVbeta5, or alphaVbeta1. According to another embodiment, the antibody comprises an Fc sequence of human IgG, for example, human IgG1 or human IgG4. In another embodiment, the Fc sequence has been modified or altered in another way so that it loses the effector function of antibody dependent cell cytotoxicity (ADCC), often associated with binding to Fc receptors (FcR). There are many examples of changes or mutations in Fc sequences that can alter effector functions. For example, WO00 / 42072 (Presta) and Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001) describe antibody variants with improved or weakened binding to FcR. The contents of these publications are hereby expressly incorporated by reference. The antibody may be in the form of Fab, Fab ', F (ab)' ₂ , single chain Fv (scFv), Fv fragment; diabodies and linear antibodies. Also, the antibody may be a multispecific antibody that binds alpha5beta1 and is an alpha5beta1 antagonist, but also binds one or more other targets and inhibits their function (e.g., VEGF). An antibody can be conjugated to a therapeutic agent (e.g., cytotoxic drug, radioisotope, and chemotherapeutic drug) or a label to detect alpha5beta1 in patient samples or in vivo by imaging (e.g., radioisotope, fluorescent dye and enzyme).

Also contemplated are nucleic acid molecules encoding anti-alpha5beta1 antibodies, expression vectors including nucleic acid molecules encoding one or both of the variable domains, and cells including nucleic acid molecules. These antibodies can be used in the therapy described here and for determining alpha5beta1 protein in patient samples (e.g., FACS, immunohistochemistry (IHC), ELISA) or in the patients themselves.

New combinations

New combinations to suppress angiogenesis and / or vascular permeability in a subject suffering from a disease include an alpha5beta1 antagonist and a VEGF antagonist. The VEGF antagonist and alpha5beta1 antagonist can be administered in simultaneous or sequential treatment cycles. Such combination treatment is applicable for the treatment of diseases, including those diseases that are characterized by abnormal angiogenesis and / or vascular permeability and suffering from which patients will benefit from antiangiogenesis therapy. Similar diseases include, but are not limited to cancer, eye diseases, and autoimmune diseases. Alternatively, the subject may be treated with a VEGF antagonist followed by administration of an alpha5beta antagonist, for example, treatment with a VEGF antagonist until the subject no longer responds to treatment with a VEGF antagonist, after which treatment of the subject with an alpha5beta antagonist. In one embodiment, the patient is treated with a VEGF antagonist when the cancer is non-invasive and an alpha5beta1 antagonist when the cancer is invasive. Some patients who have elevated alpha5beta1 levels initially or in response to VEGF antagonist therapy for non-disease patients or controls may be especially sensitive to this combinatorial treatment. Combinations are also contemplated that further include a therapeutic drug (e.g., an antineoplastic drug, a chemotherapeutic drug, a growth inhibitory drug, and a cytotoxic drug). For example, patients who should undergo chemotherapy (such as irinotecan) and are treated with alpha5beta1 antagonists or who have been treated with chemotherapy and are treated with alpha5beta1 antagonists may benefit from VEGF antagonist therapy. Alternatively, patients who have undergone chemotherapy and treatment with VEGF antagonists can benefit from therapy with an alpha5 beta1 antagonist. In one preferred embodiment, the anti-VEGF antibody is the Avastin ^®. In another preferred embodiment, anti-alpha5beta1 antibodies are anti-alpha5beta1 antibodies described herein. Also contemplated are kits comprising a VEGF antagonist, an alpha5beta1 antagonist and, optionally, a chemotherapeutic drug.

Pharmaceutical Compositions

The therapeutic antibody compositions used in accordance with the present invention are prepared for storage by mixing an antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) as lyophilized preparations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include buffers such as phosphate, citrate or other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride, benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other hydrocarbons, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming reverse ions such as sodium; metal complexes (e.g., Zn protein complexes); and / or non-ionic surfactants such as TWEEN ^™ , PLURONICS ^™ or polyethylene glycol (PEG). The exemplary antibody compositions described in WO98 / 56418 are directly incorporated herein by reference. Lyophilized preparations intended for subcutaneous administration are described in WO97 / 04801. Such lyophilized preparations can be reconstituted with a suitable solvent to high protein concentrations, reconstituted formulations can be administered subcutaneously to the mammal undergoing treatment here.

The composition described herein may also contain more than one active compound, which is necessary for the treatment of specific indications, where preferably the compounds have complementary activities that do not have undesirable effects on each other. For example, it may also be desirable to add a cytotoxic drug, chemotherapeutic drug, cytokine, or immunosuppressive drug (for example, acting on T cells, such as cyclosporin or an antibody that binds T cells, for example, binding LFA-1). An effective amount of these other factors depends on the amount of antibody present in the composition, the type of disease or disorder being treated, and other factors described above. They are usually used in the same dosages and using the same routes of administration as those described above or from about 1% to 99% of the dosage used previously.

The active ingredients can also be enclosed in microcapsules obtained, for example, using coacervation or interfacial polymerization techniques, for example, hydroxymethyl cellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, nanoparticles, nanoparticles, nanoparticles, or macroemulsions. Similar techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Slow release formulations may be prepared. Suitable examples of sustained release preparations include semi-permeable bases of solid hydrophobic polymers containing an antagonist, these bases are made in the form of shaped objects, for example, films or microcapsules. Examples of sustained release bases include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alkanoyl)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degrading ethylene- vinyl acetate, degrading copolymers of lactic and glycolic acids, such as LUPRON DEPOT ^™ (injectable microspheres consisting of a copolymer of lactic and glycolic acids and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid.

Compositions used for in vivo administration must be sterile. This can be easily achieved by filtering through sterile filtering membranes.

Products and kits

Another embodiment of the invention is an article containing materials useful for treating tumors, eye diseases, and autoimmune diseases and related conditions. The product may include a container and a label or liner inserted or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. Containers can be made of various materials, such as glass or plastic. Typically, the container contains a composition that is effective in treating the condition and may have a sterile access opening (for example, the container may be an IV bag or a vial having a plug that is permeable with a hypodermic needle). At least one active ingredient in the composition is a VEGF antagonist or alpha5beta1 antagonist, or a VEGFR agonist, or alpha5beta1 agonist in accordance with the invention. The label or package insert will also contain instructions for administering the antibody composition to the patient. The label or insert may further contain instructions for administering an antibody composition to a patient. Also contemplated are products and kits comprising the combination therapy described herein.

Packaging inserts refer to instructions usually included in commercial packages of therapeutic products that contain information about indications for use, use, dosage, administration, contraindications and / or warnings regarding the use of such a therapeutic product. In one embodiment, the package insert indicates that the composition is used to treat non-Hodgkin lymphoma.

Additionally, the product may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may further contain other materials desirable from a commercial or consumer point of view, including other buffers, solvents, filters, needles and syringes.

Kits are also available for various applications, for example, to isolate or detect alpha5beta1 and / or VEGF in patients, possibly in combination with other products. For isolation and purification of alpha5beta1, the kit may contain antibodies against alpha5beta1 linked to spheres (e.g., sepharose spheres). Kits may be provided that include antibodies for the detection and quantification of alpha5beta1 and / or VEGF in vitro, for example, in an ELISA or Western blot. As with the product, the kit contains a container and a label or package insert on the container or associated with it. For example, the container contains a composition comprising at least one anti-alpha5beta1 antibody in accordance with the invention. Additional containers may be included, containing, for example, solvents or buffers, control antibodies. The label or package insert may contain a description of the composition, as well as instructions for the intended in vitro or diagnostic use.

Monoclonal antibodies

Monoclonal antibodies can be obtained, for example, using methods based on hybridomas, for example, as described by Kohler and Milstein, Nature, 256: 495 (1975), or can be obtained using methods based on recombinant DNA (US patent No. 4816567 ), or can be obtained by the methods described here in the "Examples" section. In a hybridoma method, a mouse, hamster, or other suitable host animal is usually immunized with an immunizing agent to stimulate lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

An immunizing preparation will typically include a polypeptide or fusion protein from a protein of interest or a composition comprising a protein. Typically, either peripheral blood lymphocytes (PBLs) are used if cells of human origin are required, or spleen or lymph node cells are used if non-human mammals are desired as a source. The lymphocytes are then fused to an immortalized cell line using a suitable fusion preparation, such as polyethylene glycol, to form hybridoma cells. Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press, 1986), pp. 59-103. Immortalized cell lines are typically transformed mammalian cells, in particular myeloma cells derived from rodents, cows or humans. Rat and mouse myeloma cells are commonly used. Hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of non-transmissible immortalized cells. For example, if the original cells lack the hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzyme, the hybridoma culture medium will usually contain hypoxanthine, aminopterin and thymidine (HAT medium), these substances prevent the growth of HGPRT-deficient cells.

Preferred lines of immortalized cells are efficiently fused, maintaining stable expression of high levels of antibodies by selected antibody-producing cells and being sensitive to a medium, such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. The production of human monoclonal antibodies in the cell lines of human myeloma and mouse-human heteromyeloma has also been described. Kozbor. J. Immunol., 133: 3001 (1984): Brodeur et al. Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc .: New York, 1987) pp. 51-63.

The culture medium in which the hybridoma cells were cultured can be tested for the presence of monoclonal antibodies directed against the polypeptide. The specificity of binding of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding studies such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Similar techniques and analyzes are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Sketchard analysis by Munson and Pollard, Anal. Biochem. 107: 220 (1980).

After identifying the desired hybridoma cells, the clones can be subcloned using serial dilution procedures and grown using standard methods. See earlier Goding. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as mammalian ascites.

Monoclonal antibodies secreted by subclones can be isolated or purified from culture medium or ascites fluid using conventional immunoglobulin purification procedures, such as, for example, protein A Sepharose chromatography, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibodies can also be prepared using recombinant DNA techniques, such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be rapidly isolated and sequenced using conventional procedures (for example, using oligonucleotide probes, which are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention are a preferred source of such DNA. Once isolated, DNA can be inserted into expression vectors, which are then transfected with host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to achieve the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substitution by sequences encoding the human constant domains of the heavy and light chains of homologous murine sequences (US Pat. No. 4,816,567; Morrison et al., Supra) or by covalent binding to an immunoglobulin coding sequence of all or part of the coding sequences of a non-immunoglobulin peptide. Such a non-immunoglobulin polypeptide may be substituted for the constant domain of the invention or may be substituted with the variable domains of the antigen binding site of an antibody of the invention to create a chimeric bivalent antibody.

Antibodies may be monovalent antibodies. Methods for producing monovalent antibodies are known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is usually shortened at any point in the Fc region in order to prevent crosslinking of the heavy chains. Alternatively, important cysteine residues are replaced by other amino acid residues or undergo deletion to prevent crosslinking.

In vitro methods are also applicable to obtain monovalent antibodies. The cleavage of antibodies to obtain fragments thereof, in particular Fab fragments, can be performed using, but is not limited to, methods known in the art.

Human and Humanized Antibodies

Antibodies can be humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') ₂ , or other antigen-binding subsequences of antibodies) that typically contain a minimal sequence derived from inhuman immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which the CDR residues of the recipient are replaced by residues from the CDR of a non-human species (donor antibody), such as a mouse, rat or rabbit, having the desired specificity, affinity and capabilities. In some cases, the bases of the Fv backbone of a human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in either the recipient antibody or in the borrowed CDR or backbone sequences. Typically, humanized antibodies may comprise essentially all of at least one, and usually two, variable domains in which all or substantially all of the CDR regions correspond to analogues of non-human immunoglobulin, and all or substantially all of the FR regions correspond to analogues from consensus human immunoglobulin sequences. Humanitariannet antibodies will preferably also include at least part of the constant region of immunoglobulin (Fc), usually human immunoglobulin. Jones et al., Nature. 321: 522-525 (1986); Riechmann et al., Nature. 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

Some methods of humanizing non-human antibodies are described in the art and below in the examples. Typically, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as “import” residues, which are usually taken from the “import” variable domain. In accordance with one embodiment, humanization can be mainly performed according to the method of Winter and co-authors (Jones et al., Nature. 321: 522-525 (1986); Riechmann et al., Nature. 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing rodent CDRs or CDR sequences of corresponding sequences in a human antibody. Accordingly, such “humanized” antibodies are antibodies (US Pat. No. 4,816,567), in which a much smaller part than the entire human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.

As an alternative to humanization, human antibodies can be obtained. For example, it is currently possible to produce transgenic animals (e.g., mice) that are capable, after immunization, of producing a complete set of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that a homozygous deletion in a gene of a heavy chain (JH) site in mouse chimeric mice and germline mutants results in complete inhibition of endogenous antibody production. Transferring a set of human immunoglobulin germline genes into a similar germline germline mutant mouse results in the production of human antibodies upon antigenic stimulation. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); US patents No. 5545806, 5569825, 5591669 (all belong to GenPharm); 5,545,807; and WO 97/17852. Alternatively, human antibodies can be obtained by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes are partially or completely inactivated. During stimulation, production of human antibodies was observed, closely resembling that observed in humans in all respects, including gene rearrangement, assembly, and presentation of the antibody repertoire. This approach is described, for example, in US patent No. 5545807; 5,545,806; 5,569,825; 5,625,126; 5633425 and 5661016, and in the following scientific publications: Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol 13: 65-93 (1995).

Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 [1990]) can be used to obtain human antibodies and in vitro antibody fragments from the repertoire of immunoglobulin variable domain (V) gene immunoglobulins from non-immunized donors. In accordance with one embodiment of this technique, the V domain sequences of an antibody are cloned to a large or small coat protein of a filamentous bacteriophage, such as M13 or fd, into the gene and are presented as functional antibody fragments on the surface of the phage particle. Phage displays can be made in various formats, for example, as described below in the Examples section or as discussed, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Some sources of gene V segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a wide range of antioxazolone antibodies from a small random combinatorial library of V genes derived from spleens of immunized mice. A set of V genes from non-immunized human donors can be constructed, after which antibodies against a wide range of antigens (including native antigens) can be isolated using essentially the methods described by Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993). See also US Pat. Nos. 5,565,332 and 5,573,905.

As previously discussed, human antibodies can also be produced by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies can also be obtained using various methods known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Methods for making human monoclonal antibodies by Cole et al. Are also available. and Boerner et al. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol, 147 (1): 86-95 (1991).

Multispecific antibodies

Multispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificity for two or more different antigens (for example, bispecific antibodies have binding specificity for two or more different antigens). For example, one of the binding specificities can be like an anti-alpha5beta1 antibody, the other can be like any other antigen. In accordance with one preferred embodiment, the other antigen is a cell surface protein, receptor, or receptor subunit. For example, a cell surface protein may be a cellular receptor of a natural killer cell. Thus, in accordance with one embodiment, the bispecific antibody of the invention can bind alpha5beta1 and VEGF.

Examples of methods for making bispecific antibodies have been described. Traditionally, the recombinant production of bespecifically antibodies is based on the joint expression of two pairs of the heavy / light chain of immunoglobulin, where the two heavy chains have different specificities. Milstein and Cuello, Nature, 305: 537-539 (1983). Due to the random set of immunoglobulin heavy and light chains, these hybridomas (quadromas) potentially produce a mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out using the steps of affinity chromatography. A similar procedure is disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

Variable antibody domains with desired binding specificity (antigen-antibody connection sites) can be fused to immunoglobulin constant domain sequences. Preferably, the fusion is performed with the constant domain of the immunoglobulin heavy chain comprising at least a portion of the hinge region and CH2 and CH3 regions. Preferably, the first constant region of the heavy chain (CH1) containing the region necessary for binding the light chain is present in at least one fusion product. DNAs encoding immunoglobulin fusion heavy chains and, if desired, immunoglobulin light chains are inserted into separate expression vectors and co-transfected into a suitable host organism. For a more detailed description of the preparation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

Various methods have also been described for the manufacture and isolation of bispecific antibody fragments directly from a recombinant cell culture. For example, bispecific antibodies can be made using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Fastener leucine peptides from Fos and Jun proteins were linked to Fab 'fragments of two different antibodies by gene fusion. Antibody homodimers were reduced in the hinge region to form monomers, and then reoxidized to form antibody heterodimers. This method can also be used to produce homodimers of antibodies. Datel technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), proposed an alternative mechanism for the preparation of bispecific antibody fragments. Fragments contain VH connected to VL via a linker that is too short to allow pairing of two single chain domains. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen-binding sites. Another strategy has also been described for making bispecific antibody fragments by using single chain Fv dimers (sFv). See Gruber et al., J. Immunol., 152: 5368 (1994).

Antibodies having more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

Heteroconjugate antibodies

Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have, for example, been proposed for targeting immune system cells to unwanted cells (US Pat. No. 4,676,980) and for treating HIV infection. WO 91/00360; WO 92/200373; EP 03089. It is contemplated that antibodies can be prepared in vitro using methods known in the chemistry of synthetic proteins, including methods involving cross-linking drugs. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate, and are disclosed, for example, in US Pat. No. 4,676,980.

Creation of effector functions

It may be desirable to modify the antibody of the invention with respect to its effector function to improve, for example, the effectiveness of the antibody in the treatment of cancer. For example, one or more cysteine residues can be introduced into the Fc region, thereby making it possible to form a disulfide bond between the chains in this region. The homodimeric antibodies thus obtained may have an improved ability to internalize and / or increase complement-mediated cell lysis and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with increased antitumor activity can also be obtained using heterobifunctional cross-linking, as described in Wolff et al., Cancer Research. 53: 2560-2565 (1993). Alternatively, antibodies can be constructed that have double Fc regions and can thus have increased complement lysis and ADCC lysis capabilities. See, Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

To improve binding to FcR (e.g., FcgammaR, FcRn), mutations or sequence changes of the Fc region can be performed. In accordance with one embodiment, an antibody of the invention has at least one effector function altered from native IgG or parent antibody selected from the group consisting of ADCC, CDC, and improved binding to FcRn. Examples of some useful specific mutations are described, for example, in Shields, RL et al. (2001) JBC 276 (6) 6591-6604; Presta, L.G., (2002) Biochemical Society Transactions 30 (4): 487-490; and WO00 / 42072.

In accordance with one embodiment, the Fc receptor mutation is a replacement in at least one position selected from the group consisting of: 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 Fc sections according to the EU numbering system.

Immunoconjugates

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic drug, such as a chemotherapeutic drug, a toxin (e.g., having an enzymatic activity of a toxin of a bacterial, fungal, vegetable or animal origin, or a fragment thereof), or a radioactive isotope (e.g., a radio conjugate) .

Chemotherapeutic drugs used to create such immunoconjugates have been described above. Toxins having enzymatic activity and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, endotoxin chain A (from Pseudomonas aeruginosa), ricin chain A, abrin chain A, modestsin chain A, alpha-sacrin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), an inhibitor from Momordica charantia, kurtsin, crotin, an inhibitor from Sapaonaria officinalis, gelonin, mitogellin, restricttocin, phenomecin, enomycin and. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

Antibody and cytotoxic drug conjugates are made using various bifunctional protein-coupled substances, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolan (IT), bifunctional imidoester derivatives (such as active dimethyl adipid hydrochloride) esters (such as disuccinimyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanine you (such as (toliene 2,6-diisosuccinate), and bis-active fluoride compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. , Science. 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetyl acid (MX-DTPA) is an example of a chelating compound for conjugating a radionucleotide to an antibody. See WO94 / 11026.

In another embodiment, the antibody can be conjugated to a “receptor” (such as streptavidin) for use in pre-targeting a tumor when an antibody-receptor conjugate is administered to a patient, then the unbound conjugate is removed from the circulation using a cleanser, and then the “ligand” is administered "(Eg, avidin) conjugated to a cytotoxic drug (eg, a radionucleotide).

Immunoliposomes

Antibodies disclosed in this invention can also be made in the form of immunoliposomes. Antibody liposomes are made by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and US patent No. 4485045 and 4544545. Liposomes with increased circulation time are described in US patent No. 5013556.

Practical liposomes can be obtained by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and the PEG derivative of phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with a defined pore size to obtain liposomes with the desired diameter. Fab 'antibody fragments of the present invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982), by conducting a disulfide exchange reaction. A chemotherapeutic drug (such as doxorubicin) may be contained in the liposome. See Gabizon et al., J. National Cancer Inst., 81 (19): 1484 (1989).

Pharmaceutical compositions of antibodies and polypeptides

Antibodies specifically bind to the polypeptides described in this description, as well as other molecules identified by selection from the studies described in this invention earlier, can be assigned to treat various disorders, as described above and below in the form of pharmaceutical compositions.

Lipofectins or liposomes can be used to deliver the polypeptides or antibodies or compositions of this invention to cells. When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the sequences of the variable region of an antibody, peptide molecules that retain the ability to bind a target protein sequence can be constructed. Such peptides can be synthesized chemically or obtained using recombinant DNA technologies, see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993).

The composition described herein may also contain more than one active compound, if necessary for the treatment of specific symptoms, preferably compounds that have complementary activity that do not have undesirable effects on each other. Alternatively, or in addition, the composition may contain a drug that enhances its effect, such as, for example, a cytotoxic drug, a chemotherapeutic drug, or a growth inhibitory drug. Accordingly, such molecules are present in the composition in amounts that are effective for the intended purpose.

The active ingredients can also be enclosed in microcapsules, obtained, for example, using coacervation or interfacial polymerization techniques, for example, hydroxymethyl cellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, microbial microspheres, and nanocapsules) or macroemulsions. Similar techniques are disclosed in Remington's Pharmaceutical Sciences, see above.

Compositions used for in vivo administration must be sterile. This can be easily achieved by filtering through sterile filtering membranes.

Slow release formulations may be prepared. Suitable examples of sustained release preparations include semi-permeable bases of solid hydrophobic polymers containing an antibody, these bases are made in the form of shaped objects, for example, films or microcapsules. Examples of sustained release bases include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinylalkanoyl)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degrading ethylene vinyl acetate, degrading copolymers of lactic and glycolic acids, such as LUPRON DEPOT ^™ (injectable microspheres consisting of a copolymer of lactic and glycolic acids and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for more than 100 days, some hydrogels release proteins in shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 ° C, which leads to a loss of biological activity and possible changes in immunogenicity. Rational strategies for stabilization may be developed depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism consists in the formation of intermolecular SS bonds due to thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, controlling the moisture content, using suitable additives and developing specific polymer matrix formulations.

Diagnostic use and visualization

Labeled antibodies and their derivatives and analogs that specifically bind to a polypeptide can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or disorders associated with the expression, expression disorders and / or activity of the polypeptides of the invention. In accordance with one preferred embodiment, the antibodies of the invention can be used in diagnostic studies or imaging studies involving administration of antibodies to a subject. The invention provides a method for detecting abnormalities in expression of a VEGF polypeptide or alpha5beta1, comprising (a) studying the expression of a polypeptide in cells (e.g., tissues) or body fluids of an individual using one or more antibodies of the invention and (b) comparing the level of gene expression with the standard level of gene expression, where an increase or decrease in the level of expression of the test gene compared to the standard expression level is indicative of expression deviations.

Antibodies in accordance with this invention can be used to study protein levels in a biological sample using classical immunological methods known to specialists in this field of technology (for example, see Jalkanen, et al., J. Cell. Biol. 101: 976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105: 3087-3096 (1987)). Other antibody-based methods for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Labels suitable for analysis using antibodies are known in the art and include enzymatic labels such as glucose oxidase; radioisotopes such as iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^115m In, ^113m In, ¹¹² In, ¹¹¹ In ), and technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F) , ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru; luminol; and fluorescent labels, such as fluorescein, and rhodamine, and biotin.

Methods known in the art can be used to label antibodies of the invention. Such methods include, but are not limited to, the use of bifunctional conjugating agents (see, for example, US Pat. which are hereby incorporated by reference in full.

Diagnosing a disease or disorder associated with the expression or abnormalities of VEGF and / or alpha5beta1 expression in an animal, preferably a mammal, and most preferably a human, may include the step of detecting alpha5beta1 and / or VEGF molecules in a small-feeding one. In one embodiment, after administration of a VEGF antagonist, the diagnosis comprises (a) administering (eg, parenterally, subcutaneously or intraperitoneally) to the mammal an effective amount of a labeled anti-alpha5beta1 antibody; (b) waiting for a time interval after administration in order to allow labeled molecules to preferentially concentrate on the sites in the subject where the alpha5beta1 molecule is expressed (and to allow the concentration of unbound labeled molecule to drop to the background level); (c) determination of the background level; and (d) determining a labeled molecule in a subject, wherein detecting a labeled molecule in a subject above a background level indicates that the subject has a particular disease or disorder associated with the expression or aberrant expression of alpha5beta1. The background level can be determined using various methods, including comparing the amount of labeled molecule detected with a standard value previously determined for a particular system.

In accordance with one specific embodiment, the expression or overexpression of an alpha5beta1 polypeptide is determined in diagnostic or prognostic studies after administration of a VEGF antagonist therapeutic drug by assessing the levels of alpha5beta1 present on the cell surface (e.g., by immunochemical testing using antibodies against alpha5beta1). Alternatively or additionally, it is possible to measure the levels of an alpha5beta1 polypeptide encoding a nucleic acid or mRNA in a cell, for example by in situ fluorescence hybridization using a nucleic acid based probe corresponding to or complementary to an alpha5beta1 nucleic acid (FISH; see WO98 / 45479 published October 1998), Southern blot, Northern blot, or polymerase chain reaction (PCR) methods such as real-time quantitative PCR (RT-PCR). Excessive expression of alpha5beta1 can also be studied by measuring “peeling” antigen in biological fluids such as serum, for example, using antibody-based assays (see also, for example, US Patent No. 4933294, issued June 12, 1990; WO91 / 05264, published April 18, 1991; U.S. Patent 5,401,638, issued March 28, 1995; and Sias et al., J. Immunol. Methods 132: 73-80 (1990)). In addition to the above analyzes, a variety of in vivo studies are available to the practitioner. For example, you can expose cells in a mammal to an antibody that is possibly labeled with a detectable label, such as a radioactive isotope, after which the binding of antibodies to mammalian cells can be evaluated, for example, by an external scan of radioactivity or by analysis of a biopsy taken from a mammal previously treated antibodies.

All publications (including patents and patent applications) cited herein are hereby incorporated by reference in their entireties, including in particular US provisional application No. 60/784704, filed March 21, 2006, US provisional application No. 60/785330, filed 22 March 2006, and US Provisional Application No. 60/871743, filed December 22, 2006.

The following DNA sequences were deposited under the terms of the Budapest Agreement to the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA, as described below:

Material Deposit number Date of deposit

Alpha5 / beta1 7H5.4.2.8 PTA-7421 March 7, 2006 Alpha5 / beta1 7H12.5.1.4 PTA-7420 March 7, 2006

The deposit was made under the terms of the Budapest Agreement on the International Recognition of Microorganism Deposits for the Purposes of Patent Procedures and the Rules based on this agreement (Budapest Agreement). This ensures that a viable deposit culture is maintained for 30 years from the date of deposit. Deposits were made available to ATCC under the terms of the Budapest Agreement and are subject to agreement between Genentech, Inc. and ATCC, which guarantees the constant and unlimited accessibility of the descendants of the deposit culture to the public from the moment of granting the corresponding US patent or from the moment of publication of any US or foreign patent application, whichever comes first, and guarantees the availability of offspring for those with the right of access, as defined Commissioner for Patents and Trademarks in accordance with 35 USC 122 and its corresponding Commissioner rules (including 37 C.F.R. 1.14 with special reference to 886 OG 638).

The representative of the party submitting this application agreed that if the culture or material of the deposit dies, is lost, or is destroyed during cultivation under acceptable conditions, the materials will be quickly replaced by other ones upon notice. The availability of deposited materials should not be construed as a license to use the invention in violation of the rights granted by the authority of any government in accordance with its patent laws.

The commercially available reagents referred to in the Examples were used in accordance with the manufacturer's instructions, unless otherwise indicated. The source of cells indicated in the following examples and in the specification for ATCC access numbers is the American Type Culture Collection, Manassas, VA. Unless otherwise indicated, the present invention uses standard recombinant DNA technology procedures, such as those described hereinabove and in the following manuals: Sambrook et al., Above; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N. Y., 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc .: N.Y., 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

In this specification and claims, the word “comprise” or variations, such as “includes” or “including”, should be understood to mean the inclusion of the indicated whole or group of whole, but not the exclusion of any other whole or group of whole.

The foregoing text description is considered sufficient to enable a person skilled in the art to practice the invention. The following Examples are provided for illustrative purposes only and are not intended to limit the scope of the presented invention in any way. Moreover, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Examples

Example 1. The restoration of the population expressing alpha5beta1 stromal cells after therapy against VEGF

Xenograft sections of human colorectal HT-29 carcinoma that were monotherapy with anti-VEGF B20-4.1 antibody in athymic mice were stained for expression of anti-alpha5beta1 antibodies. Compared to the control group treated with the control antibody (anti-ragweed anti-ragweed antibody), in this study, B20-4.1 monotherapy yielded mean time to end point (TTE), which corresponded to absent or minimal activity. Tumors were measured twice a week for 58 days. Animals were euthanized when their tumors reached the endpoint volume of 1000 mm ³ or on day 58, depending on what happened before, after which (TTE) was calculated for each mouse. The treatment outcome was determined by the percentage of tumor growth inhibition (% TGD), defined as the percentage increase in the average TTE in the treated mice compared to the control, the differences were considered significant at 0.01 ≤ P ≤ 0.05, and highly significant at P ≤ 0 01 using Logrank analysis. The mean TTE of the control group was 20.6 days. Treatment with monotherapy B20-4.1 gave an average TTE of 20.1 days, which corresponds to a lack of activity.

1 shows tumor sections stained with anti-alpha5beta1 antibodies. An increased recovery of the stromal cell population after anti-VEGF treatment was observed. These stromal cells were positive for integrin alpha5beta1 (light green staining).

Example 2. Antibodies against alpha5beta1

Purified human alpha5beta1 (Chemicon CC1027) was administered to mice. Plasmocytoma cells expressing antibodies against alpha5beta1 were isolated and transformed into hybridoma cell lines. Two hybridoma cell lines, designated 7H5.4.2.8 and 7H12.5.1.4, were placed in ATCC, see above. Antibodies derived from 7H5.4.2.8 hybridoma were mIgG2a Kappa antibodies (also referred to herein as “7H5 antibodies"). Antibodies derived from 7H12.5.1.4 hybridoma were Kappa mIgG2b antibodies (also referred to herein as “7H12 antibodies").

Example 3. Direct binding studies with HUVEC cells

Tissue cultures containing growing human umbilical vein endothelial cells (HUVEC) were washed twice with PBS. Cells were separated from the culture vial using 3-4 ml of a solution of 5 mm EDTA / FSB. The cells were mixed with fresh culture medium. The number of cells in an aliquot of the mixture was determined. Cells were centrifuged and washed with wash buffer (50 mM Tris, 150 mM NaCl, pH 7.5) once. The cell concentration was chosen so that the cells could be plated at 25 μl per well on 96-well MSD plates with a high binding of 25,000 cells per well or 4000 cells per well in a 384-well plate (Cat # L11XB-1 or # L11XB-2, respectively, Meso Scale Diagnostics, LLC). Cells were incubated for 1 hour at room temperature so that capture occurred. For blocking the wells in the well was added 25 .mu.l of the basic buffer (30% fetal bovine serum (FBS) in TBS (50 mM Tris, 150 mM NaCl) +1 mM CaCl _2/1 mM MgCl _2, pH 7,5), then the plate was incubated at room temperature from 30 minutes to 1 hour.

It was prepared a series of antibodies against alfa5beta1 dilutions in assay buffer (TBS with 1mM CaCl _2/1 mM MgCl _2, pH 7,2 + 2-4% PBS) for obtaining various antibody concentrations. The wells were washed twice with washing buffer, after which the remaining liquid was removed using absorbent material. 25 μl of antibody dilutions were added to the well, after which the plate was incubated on ice for one hour. Then the wells were washed three times with TBS.

25 μl of a 0.5 μg / ml xmuFc-sulfo-tag solution was added to each well, after which the plate was incubated on ice for 45 minutes to one hour (xmuFc-sulfo-tag is a goat anti-mouse IgG antibody, catalog number R23-AC-5). MSD-SA-tag (catalog number R32-21-AD-5) was added and incubated on ice from 45 minutes to 1 hour. Wells were washed three times with TBS. 150 μl of 2x read buffer was added to each well (4x MSD read buffer diluted with dH ₂ O to 2x, R92TD-1 catalog number (no surfactants)). The subsequent electroluminescent (ECL) signal was measured using photodiodes and expressed in relative light units using an MSD scanner (default protocol 6000). Figure 2 shows the results of studies on direct binding to HUVEC cells. The EC50 of the 7H5 antibody was 0.22 nM. The EC50 of the 7H12 antibody was 0.38 nM.

Example 4. The study of FACS antibodies against alpha5beta1

7H12 or 7H5 antibodies were incubated with either RAJI cells (a cell line that does not express alpha5beta1 mRNA) or HUVEC cells (a cell line that expresses high levels of alpha5beta1 mRNA) in 100 μl. Bound cells were detected using conjugated secondary antibodies. Figure 3 shows by FACS analysis that 7H12 and 7H5 bind to HUVEC cells, but not to RAJI cells. Using the same methods with rabbit synoviocytes (HIG-82) or rhesus macaque cells (CL-160 Macaca mulatta fibroblasts or CRL-1780 retinal endothelial cells), we observed the binding of 7H12 and 7H5 to rabbit or monkey cells.

Example 5. Adhesion of cells to fibronectin in the presence of antibodies against alpha5beta1

Fibronectin (Sigma F1141 (bovine) or Roche 1080938 (human)) was diluted to 1 μg / ml in sodium carbonate buffer. 100 μl of fibronectin solution per well was added to a 96-well NUNC maxisorp plate, after which the plate was left to bind at 4 ° C overnight ((96-well NUNC MaxiSorp N / Ster 439454 immunological plates (VWR 62409-002)) The wells were then washed with phosphate-buffered saline (FSB) and blocked with 1% BSA (Sigma A9418) for at least 30 minutes, then the plates were washed three times with FSB, and 20,000 HUVEC cells were added to each well, followed by incubation with different concentrations of 7H5 or 7H12 in growth medium containing 1.4 mM MgCl ₂ and 1.4 mM CaCl _2. Then incubus Zion mixture was added to the fibronectin coated plate. Approximately 20,000 cells in the same growth medium were added to each control well without adding inhibitory antibodies.

The plates were centrifuged for 5 min at 140 g to synchronize cell contact with the substrate. Cells were incubated in a CO ₂ incubator for different lengths of time (from 0 to 120 minutes). The incubation time varied for each cell line. Then the tablets were washed with FSB three times. All liquid was removed from the cells, after which the plate was frozen at -80 ° C. The tablets were thawed at room temperature. CyQuant buffer (Molecular Probes CyQuant C7026) was added to the wells, after which the plate was incubated at room temperature for 10 minutes. OD measurement was performed. Figure 4 shows that the IC50 of the 7H5 antibody was 0.85 μg / ml (3.44 nM), and the IC50 of the 7H12 antibody was 0.7 μg / ml (4.38 nM).

Example 6. Reproduction studies using HUVEC cells

96-well plates were coated with fibronectin (1 μg / ml) overnight. Then the tablet was washed with FSB. 3000-5000 endothelial cells (EC) were added to each of the 96 wells, after which the plate was left to allow complete attachment of cells to the wells. Anti-alpha5 antibodies were added (including control isotypes). For each condition, 3 wells were used. Then the cells were incubated with antibodies for 1-24 hours. Antibodies against integrin alpha5beta1 were tested in several concentrations (for example, 0 μg / ml, 4 μg / ml, 16 μg / ml, 60 μg / ml 120 μg / ml).

The cells were then labeled with BrdU by incubating them with 2 μl of the original BrdU solution (25 mg / ml in PBS) in 1 ml of tissue culture medium (EGM2 + all additional nutrients from Clonetics (catalog number CC-4176). the cells were fixed with 4% PFA, treated with 1N HCl for 20 minutes, washed several times with FSB, and then blocked with 10% goat serum (FSB with 0.2% Triton) for 1-2 hours, then the cells were stained with monoclonal antibodies against BrdU (catalog number BD 347580, 1:40) FSB with 0.2% Triton and 5% goat serum), and then incubated in those Night at 44 ° C. The next day, the cells were washed with FSB 3 times and incubated with Alexa-594 conjugated secondary anti-rabbit antibodies (1: 800) at room temperature in the dark for 4 hours. Wells were washed again and incubated with DAPI (1: 10,000 in PBS) for 10 minutes. After the last FSB wash, the total number of cells in the well was counted by photographing DAPI staining at 5x. Cells that were BrdU positive in the same areas were photographed using a red filter. Reproduction was estimated as the percentage of cells positive for BrdU in the plot. Then the results were analyzed using Excel. Figure 5A shows the total number of HUVEC cells after 32 hours with an initial number of 5000 cells. Figure 5B shows the total number of HUVEC cells after 24 hours at an antibody concentration of 20 μg / ml.

Example 7. Migration study protocol

HUVEC cells were grown to the formation of a continuous layer in EGM2 with the addition of all additional nutrients from Clonetics (catalog number CC-4176) on 24-well plates coated with 5 μg / ml fibronectin. Cells in the center of each well were scraped off with 2 μl pipette tip, cells removed by scraping were washed off. Cell culture medium with a control antibody, 7H5 or 7H12, was added to different wells. All tested antibodies were used at a concentration of 20 μg / ml. Then the cells were allowed to grow for 1 to 2 days. The injured areas were monitored. Figure 6 shows a photograph of the migration of HUVEC cells at 5 μg / ml fibronectin with 20 μg / ml anti-alpha5 (7H5) antibodies in ECM-2 at 0 hours and 30 hours. Figure 7 is a graph of% migration after 30 hours for cells treated with 7H5 or 7H12 antibodies.

Example 8. Studies of apoptosis of activated HUVEC cells using caspase-3 immunostaining

96-well plates were coated with fibronectin (1 μg / ml) overnight. The tablets were washed by the FSB. Then, 3000-5000 HUVEC cells were seeded in each of the 96 wells and grown overnight in complete medium (EBM-2 medium (Cambrex CC-3156) with EGM-2 SingleQuots (Cambrex CC-4176)). If mouse endothelial cells 2H-11 are used for apoptosis studies, the medium will be 50/50 medium with 10% FBS.

The next day, in one set of wells, the medium was replaced with serum-free and the cells were incubated for 4-6 hours to deprive the cells of nutrition and bring them into a non-breeding state. Another set of wells was maintained in complete medium and represented actively proliferating cells. After 4-6 hours, antibodies were added (including control isotypes). Typically, 3 wells were used for each condition. Then the wells were incubated with antibodies for 1-48 hours. Antibodies against integrin alpha5beta1 were usually tested at the following concentrations: 0 μg / ml, 4 μg / ml, 16 μg / ml, 60 μg / ml and 120 μg / ml.

After incubation, the cells were fixed with 4% PFA, blocked with 10% goat serum (PBS with 0.2% Triton) for 1-2 hours, and then stained with a monoclonal antibody that specifically recognizes the activated form of Caspase-3 (for example, rabbit anti-rabbit antibody active caspase-3 from Bio Vision, diluted 1:50 in FSB with 0.2% Triton and 5% goat serum). Caspase-3 antibodies and fixed cells were incubated at 4 ° C overnight. The next day, the cells were washed with FSB 3 times and incubated with Alexa-594 conjugated secondary anti-rabbit antibody (1: 800) for 4 hours at room temperature in a dark place. Wells were washed again and incubated with DAPI (1: 10,000 in PBS) for 10 minutes. After the last FSB wash, the total number of cells in the well was counted by photographing DAPI staining at 5x. Cells that tested positive for active caspase-3 in the same areas were photographed using a red filter. Reproduction was estimated as the percentage of cells positive in the study for active caspase-3 on the site. Then the results were analyzed using Excel. Figure 8 shows that 7H5 and 7H12 do not actively induce apoptosis.

Example 9. Colometric analysis of caspase 3/7 HUVEC

Caspase 3/7 activity studies were performed using antibodies 7H5 and 7H12 (Caspase 3/7 Apo-One from Promega, see Technical Bulletin No. 295 for instructions on routine analysis in 96 wells).

Typically, 96-well plates were coated with 1 μg / ml fibronectin overnight. The tablets were washed by the FSB. Then, 3000-5000 HUVEC cells were seeded in each of 96 wells, and then grown overnight in complete medium (EBM-2 medium (Cambrex CC-3156) with EGM-2 SingleQuots (Cambrex CC-4176)). If mouse endothelial cells 2H-11 are used for apoptosis studies, the medium will be 50/50 medium with 10% FBS.

The next day, in one set of wells, the medium was replaced with serum-free and incubated cells to deprive the cells of nutrition and bring them into a non-breeding state. Another set of wells was maintained in complete medium and represented actively proliferating cells. After 4-6 hours, antibodies were added (including control isotypes). Typically, 3 wells were used for each condition. Then the cells were incubated with antibodies for 24-48 hours. Antibodies against integrin alpha5beta1 were usually tested at the following concentrations: 0 μg / ml, 4 μg / ml, 16 μg / ml, 60 μg / ml and 120 μg / ml.

After this incubation, 100 μl of reagent for caspase 3/7 Apo-One was added to each well, after which the contents of the plate were carefully mixed using a plate shaker at 300 rpm for 30 seconds. Then the tablet was incubated at room temperature for 1 to 8 hours, after which the data were recorded using a flatbed scanner. The fluorescence in each well was measured upon excitation by light with a wavelength of 485 nm and radiation with a wavelength of 530 nm.

The fluorescent signal resulting from cleavage of the substrate caspase 3/7, meant apoptosis. The figure 9 shows that 7H5 and 7H12 do not cause active apoptosis.

Example 10. The study of the formation of tubes

The ability of antibodies against alpha5beta1 to inhibit tube formation can be investigated. The following is an example of a tube formation study based on HUVEC cell growth and a tube formation study described by Nakatsu et al. (2003) Microvascular Research 66 (2003) 102-112.

Typically, HUVEC cells can be mixed with Cytdex 3 detoxified microcarriers (Amersham Pharmacia Biogech, Piscataway, NJ) at a concentration of 400 cells per bead in 1 ml of EGF-2 medium. Microbeads with cells can be gently shaken every 20 minutes for 4 hours at 37 ° C and 5% CO ₂ . After incubation, the beads can be transferred to a 25 cm ² culture bottle (BD Biosciences, Bedford, MA) and left for 12-16 hours in 5 ml of EGM-2 medium at 37 ° C and 5% CO ₂ . The next day, the beads with cells can be washed three times with 1 ml of EGM-2 and resuspended at a concentration of 200 coated cells / ml in 2.5 mg / ml fibrinogen (Sigma, St. Louis, MO). Five hundred microliters of fibrinogen solution with beads was added to 0.625 units of thrombin (Sigma) in one well of a 24-well culture plate. Fibrinogen with microspheres can form clots for 5 minutes at room temperature, and then at 37 ° C and 5% CO ₂ for 20 minutes. One milliliter of EGM-2 (containing 2% FBS) can be added to each well and then equilibrated with a fibrin clot for 30 minutes at 37 ° C and 5% CO ₂ . The medium was removed from the cell and replaced with 1 ml of fresh medium. About twelve thousand skin fibroblast cells (Detroit 551, ATCC, Rockville, MD) can be seeded onto the surface of the clot. Wednesday can be replaced every other day. Samples with beads can be tracked for 7 days.

HUVEC-coated particles can be cultured in fibrin gels with or without 500 μl of anti-alpha5beta1 antibodies (7H5 and 7H12) on the gel surface for 2-3 days, after which they are transferred to a Nicon Eclipse TE300 platform, equipped with axes in several dimensions and maintained at 37 ° C and 5% CO ₂ for 72 hours. The final concentration of antibodies used can be calculated taking into account the volume of the fibrin gel, i.e. final antibody concentration = total antibody weight / volume of medium + volume of fibrin gel. Using Metamorph software, you can capture multiple particles every 20 minutes. In vitro vascular quantification can be done using high resolution particle images (for example, the Olympus IX70 microscope with a 4x lens). The number of primordia on each particle can be determined compared to the untreated control, the primordium of a vessel can be defined as a vessel with a length equivalent to the diameter of the particle. The length of the embryo of the vessel can be measured in arbitrary units.

Example 11. Combination studies on tumor models based on xenografts and allografts .

Competitive and sequential administration of therapy with alpha5beta1 and VEGF antagonists can be evaluated in xenograft / allograft tumor models. Preferably, the models have a low or absent response to monotherapy with a VEGF antagonist. The following are examples of models that can be used: (a) Fo5 allograft in nude mice (breast tumor obtained from mmtv-Her2 transgenic mouse) (Finkle, D., et al., (2004) Clin. Cancer Res. 10: 2499-2511); (b) HT29 xenotransplant in nude mice (human colorectal line); and (c) RIP-TbAg (pancreatic tumors in the Tg model). Treatment may be given intraperitoneally, subcutaneously or intravenously. For example, anti-VEGF antibodies can be administered at a dose of 10 mg / kg once a week or 5 mg / kg twice a week. The amount of alpha5beta1 antagonist administered, such as an antibody, can be determined based on its affinity and activity. In one experiment, a VEGF antagonist and an alpha5beta1 antagonist could be administered on a joint schedule for 5-6 weeks. Alternatively or additionally, a VEGF antagonist and an alpha5beta1 antagonist could be administered sequentially (for example, anti-VEGF antibodies for three weeks, followed by dosing of anti-alpha5beta1 antibodies for three weeks).

The effectiveness of treatment can be evaluated based, inter alia, on tumor progression, tumor blood supply, tumor vascular density, morphology and / or survival. Tumor progression can be measured, for example, by tumor volume and / or tumor weight. To evaluate the vascular changes associated with neoplastic progression, perfusion of FITC-lecithin, as well as staining of vascular markers, can be used.

Example 12. The human breast cancer model - MDA-MB231

5 × 10 ⁶ human breast cancer cells were subcutaneously injected into female flies of HRLN nude mice (HRLN is the name of the line). Tumors were allowed to grow until they reached an average size of 80-120 mm ³ . Then the tumor bearing mice were divided into 4 groups, treatment was started when the average tumor volume in the group was approximately 100 mm ³ .

Tumor volumes were measured during the studies twice a week. Tumor volume was measured using a standard caliper method. Monoclonal hamster antibodies against mouse integrin alpha5, known as 10E7, were obtained from Genentech. The end point of the experiment was achieved when the tumor mass was 1.5 grams or after 60 days, depending on what happened before. In some cases, response to therapy has been monitored longer. Upon reaching the end point, the animals were euthanized.

Details of the treatment are described below.

(1) Control group: control monoclonal antibodies against ragweed ragweed (10 mg / kg, intraperitoneally (WB), once a week) were introduced.

(2) Single anti-VEGF drug group: monoclonal antibodies against VEGF B20.4.1 (10 mg / kg, wb, once a week) were administered.

(3) Combined group: B20.41. (10 mg / kg, WB, once a week) and monoclonal hamster antibodies against mouse integrin alpha5 10E7 (10 mg / kg, WB, twice a week).

(4) Single drug group against integrin alpha5: monoclonal hamster antibodies against mouse integrin alpha5 10E7 (10 mg / kg, wb, twice a week).

Control group data:

Study day one four 9 13 16 twenty 23 27 animal number Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) one 63 75 196 405 550 486 600 1080 2 63 75 126 196 320 320 446 527 3 75 126 288 666 666 936 1080 2048 four 75 126 196 320 320 446 527 936 5 75 126 288 446 486 787 908 1764 6 88 144 221 288 288 405 550 550 7 88 144 320 550 726 1008 1352 2025 8 88 144 144 446 600 1268 1268 1913 9 108 144 245 486 650 700 908 1437 10 144 162 320 527 527 847 1352 2138 average 86.5 126.6 234.4 432.8 513.2 720,2 898.9 1441.6 Sem 7.7 9.3 22 43.5 49.7 96.6 112.3 198.2 N 10 10 10 10 10 10 10 10

The data of the group of a single drug against VEGF:

Study day one four 9 13 16 twenty 23 27 animal number Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) one 63 108 144 320 405 550 256 500 2 63 63 one hundred 162 221 288 288 550 3 75 196 365 405 500 320 500 550 four 75 75 196 256 288 500 550 1099 5 75 126 196 320 500 500 550 787 6 88 144 221 320 352 446 446 600 7 88 196 365 405 405 666 726 864 8 88 196 288 320 352 384 288 365 9 108 172 256 500 405 320 288 320 10 144 245 416 567 750 968 1296 1296 average 86.5 152 254.6 357.5 417.8 494.1 518.8 693 Sem 7.7 18.7 32.6 36.9 45.8 64.5 99.1 one hundred N 10 10 10 10 10 10 10 10

The data of the group of the drug against VEGF and alpha5beta1:

Study day one four 9 13 16 twenty 23 27 animal number Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) one 63 63 63 63 63 108 126 108 2 63 108 172 256 288 288 288 288 3 75 126 126 221 245 245 320 320 four 75 75 75 126 196 288 245 446 5 75 108 172 405 352 650 650 908 6 88 196 221 320 320 288 196 196 7 88 75 196 256 196 288 288 446 8 88 88 144 320 320 288 320 405 9 108 126 144 196 256 320 320 486 10 144 221 270 446 600 650 600 787 average 86.5 118.5 158.1 260.8 283.6 341.3 335.3 438.8 Sem 7.7 16.5 19.9 37.3 44 54.7 52,2 78.1 N 10 10 10 10 10 10 10 10

The data of the group of a single drug against integrin alpha5:

Study day one four 9 13 16 twenty 23 27 animal number Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) Tv
(mm ³ ) one 63 75 196 320 486 787 1008 2025 2 63 108 126 365 365 726 1008 1352 3 75 75 144 288 288 550 600 1008 four 75 108 144 320 320 847 1152 1960 5 75 108 172 365 365 550 486 1152 6 88 196 352 650 787 908 1352 1666 7 88 one hundred 162 245 352 486 1764 End point 08/12/06, the tumor exceeded 1,500 mm ³ 8 88 126 162 320 446 486 650 650 9 108 288 446 600 1008 1352 936 3179 10 144 162 245 384 352 486 486 288 average 86.5 134.6 214.8 385.6 476.7 717.7 944.2 1475.6 Sem 7.7 20.7 33.1 42 74,2 86.7 129.7 286.4 N 10 10 10 10 10 10 10 9

These preliminary data show early signs of the combined activity of alpha5 and VEGF antagonists.

After the study, the average tumor volumes for each group were calculated (Figure 11A). Kaplan-Meyer charts were also constructed showing the percentage of animals remaining in the experiment as a function of time (Figure 11B). The data show that antibodies against integrin α5βl increase the potency of the VEGF antagonist in a breast cancer model.

Example 13.7H12 and bevacizumab in a rabbit ear wound healing model

New Zealand white rabbits were weighed and anesthetized with isofluorane. Each rabbit had a hair trimmed from the inner surface and along the edges of both auricles. The remaining coat was removed from the surgical field with a depilatory lotion. The surgical fields were cleaned with a betadine scrub, followed by wetting with alcohol. A round 8 mm biopsy piercing instrument was used to produce cartilage-deep wounds in each ear. The perichondria below were removed using a periosteal lift and thin scissors. Opsite ^® adhesive dressings were placed on each wound. After which the rabbit was left until it came out of anesthesia. Opsite ^® dressings were removed daily, wounds were examined, treatment was applied locally, and fresh dressings were applied. The size of the wound was determined by measuring the diameter of the wound at 0 (immediately after surgery), 7, 10, 14 and 18 days.

The treatment groups were as follows:

Bevacizumab (anti-VEGF antibody) 100 μl in 30 μl in each wound daily (n = 4)

7H12 (anti-alpha5beta1 antibody) 100 μg in 30 μl per wound daily (n = 4)

Bevacizumab 100 μg in 15 μl and 7H12 100 μg in 15 μl in each wound daily (n = 4)

Trastuzumab (anti-HER2 antibody) 100 μg in 30 μl to each wound daily (n = 3)

The data show that combination therapy with a VEGF antagonist and an alpha5beta1 antagonist had a striking effect on this model of angiogenesis compared to drugs alone (Fig. 10).

Example 14. Combination therapy with antibodies against alpha5beta1 and antibodies against VEGF in colon cancer

Female HRLN nu / nu mice were injected with 1 mm ^{3 of} HT29 tumor fragments (intestinal tumor) subcutaneously in the flank. Tumors were allowed to grow until they reached an average size of 80-120 mm ³ after treatment. Then, tumor-bearing mice were divided into 4 groups:

Group Number
mice Treatment regimen 1 Treatment regimen 2 A drug mg / kg Way Schedule A drug mg / kg Way Schedule one 10 The control 10 wb 1 x week x 7 FSB - wb 2 x weeks x 7 2 10 B20-4.1 10 wb 1 x week FSB - wb 2 x weeks 3 10 B20-4.1 10 wb 1 x week 10E7 10 wb 2 x weeks four 10 FSB - wb - 10E7 10 wb 2 x weeks

Tumor volume was measured twice a week using a standard caliper measurement method. Monoclonal anti-mouse alpha5 hamster antibodies, known as 10E7, were obtained from Genentech. Monoclonal antibodies against ragweed ragweed were used as control IgG. Body weight was measured 5 times during the first 2 days, then twice a week until the end of the study. The experiment ended with a tumor volume of 1 gram or 90 days, depending on what came before. Some treatment-responsive animals were observed longer. Upon reaching the end point, the animals were euthanized. The dosage was 10 ml / kg (or 200 μl per 20 gram mouse), the volume was selected in accordance with body weight. For animals showing complete regression (CR), at the end point of the tissue from the site of implantation, the tumors were collected and fixed with formalin, followed by storage in 70% EtOH for further study. All samples for freezing were placed in a cryomold, wrapped in foil and quickly frozen in liquid nitrogen.

After the end point of the study, mean tumor volumes for each group were calculated (Figure 12A). Kaplan-Meyer charts were also constructed showing the percentage of animals remaining in the experiment as a function of time (Figure 13B). The data show that the anti-integrin α5βl antibody increases the effectiveness of VEGF in a model of intestinal cancer.

Example 15. Antibodies against alpha5beta1 and chemotherapy for bowel cancer.

Female HRLN nu / nu mice were injected with 5 × 10 ⁶ HCT116 tumor cells (intestinal tumor) subcutaneously in the flank. Tumors were allowed to grow until they reached an average size of 80-120 mm ³ , after which therapy was started. Then, tumor-bearing mice were divided into 4 groups:

Group Number
mice Treatment regimen 1 Treatment regimen 2 A drug mg / kg Way Schedule A drug mg / kg Way Schedule one 10 FSB - wb 2 x week x7 - - wb - 2 10 10E7 10 wb 2 x week x7 - - wb - 3 10 FSB - wb 2 x week x7 irinotecan one hundred wb 1 x week x 3 four 10 10E7 - wb 2 x week x7 irinotecan one hundred wb 1 x week x 3

Tumor volume was measured twice a week using a standard caliper measurement method. Monoclonal anti-mouse alpha5 hamster antibodies, known as 10E7, were obtained from Genentech. Body weight was measured 5 times during the first 2 days, then twice a week until the end of the study. The experiment ended with a tumor volume of 1.5 grams or on day 60, whichever comes first. Some treatment-responsive animals were observed longer. Upon reaching the end point, the animals were euthanized. The dosage was 10 ml / kg (or 200 μl per 20 gram mouse), the volume was selected in accordance with body weight. 10E7 was administered 30 minutes before the administration of irinotecan. For animals showing complete regression (CR), at the end point of the tissue from the site of implantation, the tumors were collected and fixed with formalin, followed by storage in 70% EtOH for further study. All samples for freezing were placed in a cryomold, wrapped in foil and quickly frozen in liquid nitrogen.

After the end point of the study, mean tumor volumes for each group were calculated (Figure 13A). Kaplan-Meier diagrams were also constructed showing the percentage of animals remaining in the experiment as a function of time (Figure 13B). The data show that antibodies against integrin alpha5beta1 do not increase the effectiveness of the chemotherapeutic drug (irinotecan) in the model of intestinal cancer, but also does not interfere with the action of the chemotherapeutic drug. The observations were consistent with our assumptions that vascular damage that occurs before treatment with an alpha5beta antagonist can be very useful in antiangiogenesis, and in particular in antiangiogenesis in an oncological environment. Similar vascular injury may be caused by an antagonist of VEGF, such as AVASTIN ^® antibody. The chemotherapeutic drug itself in this model does not cause noticeable vascular damage. The use of all these drugs (VEGF antagonist / alpha5beta1 antagonist / chemotherapeutic drug) should be contemplated simultaneously or sequentially so that the VEGF antagonist is present and causes vascular damage.

Example 16. Sketchard Graphics for alpha5beta1

Antibodies against alpha5beta1 were iodinated using the lodogen method, radioactively labeled antibodies were purified from free ¹²⁵ I-Na by gel filtration using a PD-10 column. R9ab cells, rabbit fibroblast cell line (purchased from ATCC, No.CCL-193) were seeded at approximately 50,000 per well in 24-well plates and incubated for 48 hours in 5% CO ₂ at 37 ° C. Cells were washed three times with binding buffer (50:50 DMEM / F12 medium containing 2% FBS and 50 mM HEPES, pH 7.2), and then incubated on ice for 15 minutes. The washed cells were incubated for 4 hours on ice with approximately 50 pM ¹²⁵ I-monoclonal antibodies against alpha5beta1 containing decreasing concentrations of unlabeled monoclonal antibodies against alpha5beta1 diluted serially with 0.5 μM in binding buffer in 13 concentrations, taken in triplicate. Cells were washed three times with binding buffer, after which 200 μl of lysing buffer with SDS was solubilized (1% SDS, 8 M urea, 100 mM glycine, pH 3.0). The radioactivity of cell lysates was determined on a Wallac Wizard 1470 gamma counter. Binding data were evaluated using the Genentech NewLigand program, which uses the Manson and Robard curve approximation algorithm (Munson, P. and Robard, D. (1980) Anal. Biochem 107: 220-239) for determining the binding affinity of an antibody and the concentration of binding sites. Figures 14 and 15 show that in these binding studies, 7H5 antibodies have a Kd of 0.10 nM and 7H12 antibodies have a Kd of 0.30 nM, respectively.

Example 17. Studies mapping epitopes / competitive binding of IgG against integrin alpha5beta1

First, a series of three-fold dilutions of IgG against integrin α5β1 was incubated in a 96-well Nunc Maxisorp plate coated with antigen - human integrin α5β1 (1 μg / ml, R&D) in PBST buffer (FSB and 0.5% (w / v) BSA, and 0.05% (volume) Tween 20) for 1-2 hours at room temperature, followed by the addition of 0.3 nM biotinylated hIgG1 h7H5.vl (a variant of the 7H5 antibody obtained by Genentech, Inc.), which was first determined by submaximal binding signal (50-70%), within 15 minutes. The plate was then washed 5 times with PBT buffer (FSB and 0.05% (volume) Tween 20). Bound biotinylated h7H5.vl hIgG1 was detected using streptavidin conjugated horseradish peroxidase (Pierce) diluted 1: 2500 in PBST buffer developed using 3.3 ', 5.5'-tetramethylbenzidine substrate (TMB, Kirkegaard & Perry Labs, Gaithersburg, MD) for approximately 5 minutes, the reaction was terminated with 1.0 M H ₃ PO ₄ , after which measurements were carried out on a spectrophotometer at 450 nm. Curves were selected using a nonlinear regression curve fitting program with four parameters (Kaleidagraph, Synergy Software).

Figure 16 shows that bound h7H5.v1 compete with increasing amounts of cold m7H5. In fact, the m7H5 competition curve was almost identical to the h7H5.v1 competition curve (data not shown). Cold m7H12 also competed with biotin-h7H5.vl for binding to alpha5beta1, demonstrating that the alpha5beta1 binding epitopes of h7H5.v1 and m7H12 overlap. On the other hand, the control antibody did not compete with the bound h7H5.vl.

Claims (37)

1. The anti-alpha5beta1 antibody produced by the hybridoma deposited with ATCC accession number PTA-7421, where the antibody is IgG2 and has a Kd value of 0.10 nM, measured using R9ab cells with ATCC accession number CCL-193 according to the Sketchard method. 2. The anti-alpha5beta1 antibody produced by the hybridoma deposited with ATCC accession number PTA-7420, where the antibody is IgG2 and has a Kd value of 0.30 nM, measured using R9ab cells with ATCC accession number CCL-193 according to the Sketchard method. 3. An antibody conjugated to a therapeutic agent for inhibiting angiogenesis and / or vascular permeability, wherein the antibody is produced by a hybridoma deposited with an ATCC access number No. PTA-7421, or a hybridoma deposited with an ATCC accession number No. PTA-7420, where the antibody is IgG2, where the antibody produced by ATCC hybridoma No. PTA-7421 has a Kd value of 0.10 nM, and the antibody produced by ATCC hybridoma No. PTA-7420 has a Kd value of 0.30 nm, as measured using R9ab cells with ATCC access number No. CCL-193 according to m Tod Scatchard. 4. The antibody according to claim 3, where the therapeutic agent is selected from the group consisting of a cytotoxic agent, a radioisotope, and a chemotherapeutic agent. 5. An antibody conjugated to a label for use in the diagnosis of a disease associated with the expression of alpha5beta1, where the antibody is produced by a hybridoma deposited with an access number of ATCC No. PTA-7421, or a hybridoma deposited under an access number of ATCC No. PTA-7420, where the antibody is IgG2, where the antibody produced by ATCC hybridoma No. PTA-7421 has a Kd value of 0.10 nM, and the antibody produced by ATCC hybridoma No. PTA-7420 has a Kd value of 0.30 nm, as measured using R9ab cells with access number ATCC No. CCL-193 according to method from Scatchard. 6. The antibody according to claim 5, where the label is selected from the group consisting of a radioisotope, a fluorescent dye and an enzyme. 7. An isolated nucleic acid molecule encoding an antibody according to claim 1 or 2. 8. The expression vector encoding the nucleic acid molecule according to claim 7. 9. Recombinant cell containing a nucleic acid molecule according to claim 7. 10. The cell according to claim 9, where the cell is a hybridoma deposited under the access number of ATCC No. PTA-7421, or a hybridoma deposited under the access number of ATCC No. PTA-7420. 11. A method for producing an antibody, comprising culturing cells according to claim 9 or 10 and isolating antibodies from the cell culture. 12. A pharmaceutical composition for inhibiting angiogenesis and / or vascular permeability, comprising an effective amount of an antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier. 13. A method for detecting alpha5beta1 protein in an in vitro sample from a patient by contacting a sample with an antibody according to claim 1 or 2 and detecting an anti-alpha5beta1 antibody bound to alpha5beta1 protein. 14. The method according to item 13, where the antibody is used in immunohistochemical studies (IHC) or in studies using ELISA. 15. The use of an antibody according to claim 1 or 2 in the manufacture of a medicament for inhibiting angiogenesis and / or vascular permeability in a subject. 16. The use of antibodies according to claim 1 or 2 in the manufacture of a medicament for the treatment of a disease in a subject, where the disease is accompanied by pathological angiogenesis or pathological vascular permeability. 17. The use of antibodies against alpha5beta1 according to claim 1 or 2 in the manufacture of a medicinal product for inhibiting angiogenesis and / or vascular permeability in a subject suffering from a disease accompanied by pathological angiogenesis or pathological vascular permeability, where the anti-alpha5beta1 antibody according to claim 1 or 2 is administered the specified subject in combination with a VEGF antagonist. 18. The use of claim 17, wherein the disease is selected from cancer, eye disease, and autoimmune disease. 19. The use of claim 17, wherein the subject is administered a VEGF antagonist, and then an anti-alpha5beta1 antibody. 20. The use of claim 17, wherein the VEGF antagonist and anti-alpha5beta1 antibody are administered to the subject simultaneously. 21. The use according to claim 17, wherein the subject is treated with a VEGF antagonist until the subject ceases to respond to treatment with a VEGF antagonist, after which the subject is treated with an anti-alpha5 beta1 antibody. 22. The use of claim 17, wherein the disease is cancer and the subject is treated with a VEGF antagonist when the cancer is not invasive and treated with an anti-alpha5 beta1 antibody when the cancer is invasive. 23. The use of claim 17, wherein the subject has elevated levels of alpha5beta1 in the affected tissue compared to the tissue of a subject not suffering from a disease. 24. The use of claim 17, wherein the subject is further administered a therapeutic agent selected from the group consisting of an anti-neoplastic agent, a chemotherapeutic agent that inhibits the growth of the agent, and a cytotoxic agent. 25. The application of claim 17, wherein the VEGF antagonist is bevacizumab. 26. The use of claim 17, wherein the anti-alpha5beta1 antibody is conjugated to a cytotoxic agent. 27. The use of claim 26, wherein the cytotoxic agent is a radioactive isotope, chemotherapeutic agent, or toxin. 28. A pharmaceutical composition for inhibiting angiogenesis and / or vascular permeability, comprising an effective amount of a VEGF antagonist, an effective amount of an anti-alpha5 beta1 antibody according to claim 1 or 2, and a pharmaceutically acceptable carrier. 29. A kit for detecting alpha5beta1 in a subject that has been treated with a VEGF antagonist, containing
an antibody against alpha5beta1 according to claim 1 or 2, and
instructions for detecting alpha5beta1 in a subject who has been treated with a VEGF antagonist. 30. The use of the composition according to p. 28 in the manufacture of a medicament for inhibiting angiogenesis and / or vascular permeability in a subject suffering from a disease. 31. The application of claim 30, wherein the disease is selected from the group consisting of cancer, eye disease, and autoimmune disease. 32. The application of clause 30, where the disease is selected from the group consisting of a solid tumor, metastatic tumor, soft tissue tumor, a disease associated with neovascularization of the eye, an inflammatory disease accompanied by pathological angiogenesis, a disease that occurs after transplantation to a subject, and a disease, accompanied by pathological development of vascular fibrous tissue. 33. The use of claim 31, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, intestinal cancer, lung cancer, non-Hodgkin lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, head and neck cancer, melanoma , ovarian cancer, mesothelioma, soft tissue cancer and multiple myeloma. 34. The application of claim 30, wherein the disease is selected from the group consisting of retinopathy caused by age-related macular degeneration, rubeosis, psoriasis, psoriatic arthritis and inflammatory kidney diseases, hemolytic uremic syndrome, diabetic nephropathy, arthritis, inflammatory bowel disease, chronic inflammation chronic retinal detachment, chronic uveitis, chronic vitritis, corneal transplant rejection, corneal neovascularization, corneal transplant neovascularization, Crohn’s disease , myopia, ocular neovascular disease, osteoarthritis, Paget's disease, pemphigoid, polyarthritis, post-laser radial keratotomy, neovascularization of the retina, Sjogren's syndrome, ulcerative colitis, transplant rejection, pneumonia, nephrotic syndrome, edema, ascites, tumors associated with malignant tumors and neovascular glaucoma. 35. The use of the antibody according to claim 1 or 2 in the manufacture of a medicament for the treatment of a subject suffering from a disease, where the subject responded to the treatment of the disease with a VEGF antagonist, but partially or completely stopped responding to the VEGF antagonist. 36. The application of clause 35, where the subject has elevated levels of alpha5beta1 in diseased tissue compared with a subject not suffering from a disease. 37. The application of clause 36, where the subject is additionally administered a therapeutic agent selected from the group consisting of an antineoplastic agent, a chemotherapeutic agent that inhibits the growth of the agent and a cytotoxic agent.

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