Combined therapy with using alpha5beta1 antagonists

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

 

The scope of the invention

The presented invention relates to the use of VEGF antagonists and antagonists alfaretta for cancer treatment and suppression of angiogenesis and/or suppression of vascular permeability, including abnormal angiogenesis in certain diseases. The presented invention also relates to the use of VEGFR agonists and agonists alfaretta to enhance angiogenesis and vascular permeability. The presented invention also relates to antibodies against alfaretta, including their compositions and kits and methods for their manufacture and application.

The prior art inventions

Recognized the important role of VEGF-A in pathological and non-pathological angiogenesis. The introduction of VEGF in models in vivo causes 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 allele of a gene VEGF-A 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 vascular permeability factor due to its ability to cause bleeding (Senger, D.R. et al., (1995) Science 219:983-985; Dvorak, H.F., et al., (1995) Am. J. Pathol. 146: 1029-1039). Thus VEGF-A is involved in angiogenesis during development, reproduction and bone angiogenesis, in addition to other non-pathological forms of angiogenesis.

VEGF-A binds to the two receptor tyrosine kinases (RTK), VEGFR-1 (Flt-1) and VEGFR-2 (KDR, Flk-1). It is usually assumed that VEGFR-2 is the major mediator of the mitogenic, angiogenic and increasing the permeability of the actions of VEGF-A. In February 2004, the US Food and Drug Administration (FDA) approved bevacizumab, a humanized monoclonal antibody against VEGF (growth factor vascular endothelial)-A, for the treatment of metastatic colorectal cancer in combination with based on 5-fluorouracil (FU) chemotherapy regimens. Subsequently, the FDA approved pegaptanib, aptamer that blocks the isoforms of VEGF-A from 165 amino acids, for the treatment of wet (neovascular) form of age-related macular degeneration (AMD).

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

Brief description of the invention

The presented invention relates to medicaments and methods of treatment of patients who may benefit from a reduction of angiogenesis is, suffering from abnormal angiogenesis and/or suffering from neoplasia. In accordance with one embodiment of the presented invention provides a method of inhibiting angiogenesis and/or vascular permeability in a subject, comprising the stage of introducing to the subject a therapeutically effective amount of a VEGF antagonist and antagonist alfaretta, simultaneously or sequentially. In accordance with another embodiment of the invention, the presented invention provides a method of treatment of a subject suffering from a disease, and the subject or previously responded to treatment with the VEGF antagonist, but only partially, or no longer responds to the VEGF antagonist, comprising the stage of introducing to the subject a therapeutically effective amount of the antagonist alfaretta. In accordance with another embodiment, the presented invention provides a method of treatment of a subject suffering from a disease resistant or intractable by treatment with antagonist alfaretta, alone or in combination with chemotherapy, including the stage of introducing to the subject a therapeutically effective amount of a VEGF antagonist.

The presented invention also relates to new antibodies against alfaretta, kits and compositions, comprising, and methods for their manufacture or use. In the accordance with one variant of implementation of new antibodies against alfaretta are described here antibody 7H5 or 7H12, or humanized or chimeric form. In accordance with another separate embodiment the antibody 7H5 or 7H12 or chimeric or humanized forms can be in the form of Fab fragments, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment, diately, polyspecific antibodies and a linear antibody. In accordance with another variant of implementation of new antibodies against alfaretta can be conjugated with another object, but not limited to, such as a therapeutic drug, or a fluorescent dye or other marker for detection alfaretta patients or in samples from patients. These new antibodies against alfaretta can be used in various therapeutic and diagnostic methods. For example, such antibodies against alfaretta can be used in the treatment of abnormal angiogenesis, neoplasia, eye diseases and autoimmune diseases. Such antibodies can be used to detect protein alfaretta patients or in samples from patients through the implementation of contact of such antibodies with protein alfaretta patients or patient samples, and quantitative or qualitative determination of related protein alfaretta antibodies against alfaretta.

In accordance with another embodiment, provided is Lenna invention provides a method of treating cancer in a subject, including the state administration of VEGF antagonist and antagonist alfaretta simultaneously or sequentially. In accordance with one preferred embodiment the cancer responds to treatment with VEGF antagonist. Another variant implementation of the invention is a method of treating age-related macular degeneration in a subject suffering from AMD, including the stage of introducing a therapeutically effective amount of a VEGF antagonist and antagonist alfaretta simultaneously or sequentially. According to another variant implementation of the method of treating autoimmune disease in a subject, comprising the stage of introducing a therapeutically effective amount of a VEGF antagonist and antagonist alfaretta simultaneously or sequentially.

In one embodiment, the implementation of the treated subject may be first introduced the VEGF antagonist, and then treated with antagonist alfaretta. In another embodiment, the subject is subjected to treatment with the VEGF antagonist and the antagonist alfaretta at the same time. In accordance with another embodiment of the subject being treated with the VEGF antagonist as long as the subject is no longer responsive to treatment with a VEGF antagonist, after which the subject is subjected to treatment with the antagonist alfaretta. In one individual variations is the implementation of the subject treated with the VEGF antagonist, if the cancer was non-invasive or at an early stage and treated alfaretta antagonist when the cancer was invasive. In another variant implementation of the subject, which were treated with the antagonist alfaretta had an increased level alfaretta in diseased tissue compared to tissue from a subject not suffering from the disease. In this case, the method may further include phase detection alfaretta the subject, for example in the diseased tissue after treatment with VEGF antagonist. In accordance with one embodiment of invasive cancer is metastatic cancer. In accordance with another embodiment, the cancer is at an early stage is cancer curable auxiliary therapy (such as chemotherapy or surgical removal).

In one preferred embodiment, the subject suffering from a disease is a pathological angiogenesis. In accordance with another embodiment, the disease is selected from the group consisting of cancer, immune diseases or eye diseases. In accordance with one preferred embodiment, the disease is selected from the group consisting of solid tumors, metastatic tumors, soft tissue tumors, diseases associated with neovascularization, inflammatory eye disease associated with the abnormal angiogene the Ohm, diseases that occur after transplantation to a subject, and diseases associated with abnormal development of vascular fibrous tissue. In accordance with another preferred option, the disease is selected from the group consisting of breast cancer (including metastatic breast cancer), cervical cancer, colon cancer (including metastatic colon cancer), lung cancer (including small cell lung cancer), non-Jackinsky lymphoma (NHL), chronic lymphocytic leukemia, pochernkletocny cancer, prostate cancer, including resistant to hormones, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, cancer of the soft tissues, gastrointestinal tumor stroma, glioblastoma multiforme, and multiple myeloma. In accordance with another preferred embodiment, the disease is selected from the group consisting of retinopathy, caused by age related macular degeneration (e.g., wet AMD), diabetic macular edema, reddening of the iris, psoriasis, inflammatory kidney disease, haemolytic uraemic syndrome, diabetic retinopathy (e.g., proliferative diabetic retinopathy), arthritis (e.g., psoriatic arthritis, osteoarthritis, rheumatoid arthritis), inflammatory bowel disease, chronic inflammation, chronic retinal detachment retinal XP is in clinical uveitis, chronic vitrite, corneal rejection of transplanted tissue, corneal neovascularization, corneal neovascularization transplanted tissue, Crohn's disease, myopia, ocular neovascular disease, Paget's disease, pemphigoid, arthritis, post-laser radial keratotomy, neovascularization of the retina, Sjogren syndrome, ulcerative colitis, rejection of the implant, lung inflammation, nephrotic syndrome, edema, ascites associated with malignancy, stroke, angiofibromas and neovascular glaucoma. In one of the embodiments, the subject is added therapeutic drug is selected from the group consisting of anti-neoplastic drug, a chemotherapeutic drug and a cytotoxic drug.

In accordance with a preferred embodiment of the present invention, subjected to treatment with the antagonist alfaretta the subject is suffering from a relapse after treatment with VEGF antagonist or has become unresponsive to treatment with an antagonist of VEGF. In accordance with another embodiment, subjected to treatment with the antagonist alfaretta and a VEGF antagonist to the subject suffering from a metastatic cancer or had previously undergone adjuvant therapy. In one embodiment, a disease in which the candidate patient is recurrence, vospreimchev or resistant to chemotherapeutic drugs, such as irinotecan. Examples of such diseases include, but are not limited to, metastatic colon cancer, the recurrence of metastatic colon cancer, metastatic breast cancer, the recurrence of metastatic breast cancer, metastatic breast cancer HER2+, adjuvant breast cancer, adjuvant breast cancer HER2+, metastatic pancreatic cancer, 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, metastatic renal cell carcinoma and adjuvant renal cell carcinoma.

In accordance with one embodiment of the subject suffering from these diseases after treatment with the VEGF antagonist is supportive, where supportive therapy involves the use of antagonist alfaretta by itself or after, or simultaneously with a VEGF antagonist.

In accordance with one preferred embodiment, the VEGF antagonist may be selected from the group consisting of antibodies, immunoadhesins, Patiala, small molecules and nucleic acids, which hybridizes with a nucleic acid molecule that encodes a VEGF under severe conditions (e.g., a ribozyme, siRNAs or aptamers). In accordance with one site is titanium of the embodiment, the VEGF antagonist is an antibody. In accordance with another embodiment the antibody is a monoclonal antibody. In accordance with one preferred embodiment, the binding of the antibody against VEGF to human VEGF can be completely ingibirovalo antibody Avastin®. In accordance with one preferred embodiment antibodies against VEGF are human, humanitarianism or chimeric. In accordance with one specific embodiment the antibody against VEGF antibody is Avastin®. In accordance with another embodiment of antibodies against the VEGF is selected from the group consisting of Fab, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment; dyatel and a linear antibody. In accordance with another embodiment, the VEGF antagonist is bespecifically antibody that binds to VEGF and Alfama and is an antagonist alfaretta.

In accordance with one preferred embodiment, the antagonist alfaretta can be selected from the group consisting of antibodies, immunoadhesins, Patiala, small molecules and nucleic acids, which hybridizes with a nucleic acid molecule that encodes alfaretta under hard conditions.

In accordance with one preferred embodiment, the antagonist Alfama is an antibody. In the accordance with another embodiment the antibody is a monoclonal antibody. In accordance with the following embodiment, the monoclonal antibody is a chimeric antibody, such as antibody against human alfaretta, known as the M200 or F200. In accordance with one embodiment of antibodies against alfaretta include a sequence of VH SEQ ID NO:1 and a VL sequence SEQ ID NO:2. In accordance with another embodiment of antibodies against alfaretta include the sequence of SEQ ID NO:3 or the sequence SEQ ID NO:4. In accordance with another embodiment of antibodies against alfaretta include the sequence of SEQ ID NO:4, and the sequence SEQ ID NO:5. In accordance with a preferred embodiment of the invention, binding of the antibody against alfaretta with human alfaretta can be completely ingibirovalo antibody 7H5 or antibodies 7H12. In accordance with one preferred embodiment of antibodies against alfaretta are human, humanitarianism or chimeric. In accordance with one specific embodiment the antibodies against human alfaretta are antibody 7H5, antibody 7H12 or chimeric, or humanitariannet antibody based on them. In accordance with another embodiment of antibodies against alfaretta selected from the group consisting of Fab, Fab', F(ab)'2, single-stranded F (scFv), the Fv fragment; dyatel and a linear antibody. In accordance with another embodiment, the antagonist Alfama is bespecifically an antibody that binds VEGF and Alfama and is an antagonist of VEGF. In accordance with another embodiment, the antagonist anti-Alfama has an altered effector function. In accordance with one embodiment of antibodies against alfaretta changed in order to reduce or suppress antibody-dependent cellular cytotoxic activity (ADCC) or complementation cytotoxic effect (CDC) (for example, by modifying a nucleic acid sequence that encodes a protein Fc antibody). In accordance with another embodiment, antibodies against alfaretta have been modified to increase the half-life in the human body (for example by modifying the nucleic acid sequence that encodes a plot Fc antibody).

In accordance with one embodiment, the VEGF antagonist or antagonist alfaretta anywhereman with a cytotoxic drug or chemotherapeutic drug. In accordance with another embodiment of the cytotoxic drug is a radioisotope or a toxin.

The presented invention provides compositions comprising a VEGF antagonist, the antagonist alphabet and a pharmaceutically acceptable carrier. The presented invention also provides a product comprising instructions for detecting alfaretta the subject, which were treated with a VEGF antagonist.

Presents the invention also concerns the application of VEGFR agonists and agonists alfaretta for stimulation of angiogenesis and vascular permeability and compositions, including VEGFR agonists, and agonists alfaretta, and a pharmaceutically acceptable carrier. Combination therapy VEGFR agonists and agonists alfaretta can be used in the treatment of various diseases which can occur improved by increasing angiogenesis and vascular permeability, including, for example, the healing of wounds, such as chronic wounds, acute wounds and normal wounds.

Brief description of figures

Figure 1 shows the increase in the population expressing alfaretta stroma cells after treatment xenotransplantion HT29 tumors with antibodies against VEGF, B20-4.1.

Figure 2 is a graph showing the binding of the antibody 7H5 and 7Hl2 with HUVEC cells in the study of direct binding.

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

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

Figure 5 depict is to place a (A) a column chart demonstrating the action 7H5 and 7H12 on the development of HUVEC cells by the total number of cells and (B) bar chart showing the effect 7H5 and 7H12 on the development of HUVEC cells by staining Alamar blue in another study.

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

Figure 7 is a vertical bar chart, quantitatively demonstrating the migration of HUVEC cells after treatment 7H5 and 7H12.

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

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

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

Figure 11 shows the results of treatment with antibodies against VEGF with the addition of antibodies against alfaretta or without them on the model of breast cancer in the form of (A) graph showing the mean tumor volume in the group which were treated mice, or (B) graph of Kaplan-Meier, showing the percentage of animals remaining in the study as a function of time. Animals were removed from the study when their Messiah. the tumor has reached or exceeded 1500 mm 3.

Figure 12 shows the results of treatment with antibodies against VEGF with the addition of antibodies against alfaretta or without them on the model of colon cancer in the form of (A) graph showing the mean tumor volume in the group which were treated mice, or (B) graph of Kaplan-Meier, showing the percentage of animals remaining in the study as a function of time. Animals were removed from the study when their tumors reached or exceeded 1500 mm3.

Figure 13 shows the results of treatment with antibodies against alfaretta or chemotherapeutic drug on the model of colon cancer in the form of (A) graph showing the mean tumor volume in the group which were treated mice, or (B) graph of Kaplan-Meier, showing the percentage of animals remaining in the study as a function of time. Animals were removed from the study when their tumors reached or exceeded 1500 mm3.

Figure 14 shows a graph of Scatchard binding125I-7H5 with alfaretta on cell line of fibroblasts rabbit R9ab.

Figure 15 shows a graph of Scatchard binding125I-7H12 with alfaretta on cell line of fibroblasts rabbit R9ab.

Figure 16 shows the results of epitope mapping of IgG against integrin alfaretta and research of competitive binding different and the antibodies against alfaretta.

Detailed description of the invention

Without being bound by theory, the authors suggested that the increase in the recovery of the population of stromal cells can be delivered to patients in the areas other growth factors vessels that can compensate for the loss of activity of VEGF in patients subjected to treatment with VEGF antagonists. Impact on expressing alfaretta stromal cells by antibodies against alfaretta can lead to a reduction in the number of stromal cells, thus reducing the production of potentially offsetting factors vascular growth. Alternative or additionally, the authors suggested that the inhibition of interactions between the endothelium and the extracellular matrix and partial inhibition of binding interactions alfaretta will increase the effectiveness of treatment with VEGF antagonists by inhibiting recurrence of angiogenesis along tracks in the extracellular matrix, left regressing following treatment with a VEGF antagonist vessels. Thus, treatment with antagonists alfaretta simultaneously with any treatment with VEGF antagonists or after may inhibit the restoration of the vessels after treatment with VEGF antagonist and, therefore, the resumption of neovascular growth.

"Alfama", or "α5β1," or "AB" is an integrin comprising two different proteins (i.e. when jedinica Alfa and beta). For alfaretta shown that it binds to fibronectin, L1-CAM and fibrinogen. Integrin alfaretta also called protein very late activation-5, VLA-5, alfaretta, CD49e/CD29, fibronectin receptor, FNR and GPIc-IIa. In accordance with the preferred embodiment Alfama is human alfaretta.

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

"Beta" also known as CD29, beta, platelet GPIIa; beta-chain of VLA; beta-1 chain integrin, CD29; FNRB; MDF2; at vlab; GPIIA; MSK12 and VLA5B. Amino acid sequence of human beta can be found, for example, under Genbank access number X06256.

The term "VEGF" or "VEGF-A"as used here, refers to the 165-amino acid human growth factor, endothelial cells of vessels and related 121-, 189-, and 206-amino acid human growth factors, endothelial cells of blood vessels, as described in Leung et al. Science 246:1306 (1989), and Houck et al. Mol. Endocrin., 5: 1806 (1991), along with naturally occurring allele is diversified and changed their forms. The term "VEGF" also refers to VEGF non-human species such as mouse, rat or Primate. Sometimes VEGF from a specific type is indicated by terms such as hVEGF for human VEGF, mVEGF for murine VEGF, and the like, the Term "VEGF" is also used to denote the truncated forms of the polypeptide comprising amino acids 8 to 109, or 1 to 109 of the 165-amino acid human growth factor, endothelial cells of blood vessels. Reference to any such forms may be identified in the present application, for example, as "VEGF (8-109)", "VEGF (1-109)or VEGF165". The position of amino acids in a "shortened" natural VEGF numbered as they appear in the natural sequence of VEGF. For example, the amino acid at position 17 (methionine) in a shortened natural VEGF corresponds to the 17 amino acid (methionine) in the natural VEGF. Shortened natural VEGF has affinity binding to receptors KDR and FIt-1, comparable with the natural VEGF. In accordance with a preferred embodiment of VEGF is human VEGF.

The term "VEGF antagonist" refers to a molecule capable of neutralizing, blocking, inhibiting, deactivate, reduce or prevent the action of VEGF, the method including its binding to VEGF or one or more VEGF receptors or their coding nucleic acids. Preferably, the VEGF antagonist binds to VEGF or R is zeptogram VEGF. The VEGF antagonists include antibodies against VEGF and their antigen-binding sites, polypeptides that bind to VEGF or VEGF receptors and block the interaction of the ligand with the receptor (for example, immunoadhesin, Patiala), antibodies against the receptor for VEGF and the VEGF receptor antagonists such as small molecule inhibitors tyrosinekinase VEGFR, aptamers that bind VEGF and nucleic acids that hybridize under defined conditions to the sequences of nucleic acids 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. In accordance with one preferred embodiment, the VEGF antagonist binds to VEGF or a VEGF receptor with greater affinity than not with VEGF or not with VEGF receptor. In accordance with one preferred embodiment, the VEGF antagonist binds to VEGF or a VEGF receptor with a Kd of between 1 μm and 1 PM. In accordance with another preferred embodiment, the VEGF antagonist binds to VEGF or a VEGF receptor with a Kd between 500 nm and 1 PM.

In accordance with the preferred embodiment, the VEGF antagonist is selected from the group consisting of a polypeptide, such as an antibody, Patiala, it is unadvised, low-molecular compound or an aptamer. In a preferred embodiment, the antibody is an anti-VEGF antibody, such as antibody AVASTIN®or an antibody against VEGF receptor, such as antibodies against VEGFR2 or against VEGFR3. Other examples of antagonists include VEGF 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 able to bind 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 prevent diseases and conditions that involve the action of VEGF. Antibodies against VEGF usually not associated with any other VEGF homologues, such as VEGF-B or VEGF-C or with other growth factors, such as P1GF, PDGF or bFGF. Preferred antibodies against VEGF are monoclonal antibodies that bind to the same epitope as the monoclonal antibody against VEGF A.4.6.1 produced by hybridoma ATCC HB 10709. More preferably, antibodies against VEGF are recombinant humanitarianism monoclonal antibody against VEGF, generated according to Presta et al. (1997) Cancer Res. 57:4593-4599, including, but not limited to antibody known who Tim as bevacizumab (BV; Avastin®). In accordance with another embodiment, antibodies against VEGF, which can be used include, but are not limited to, antibodies that are disclosed in the application WO 2005/012359. In accordance with one embodiment, antibodies against VEGF include variable heavy and variable light areas of any of the antibodies disclosed in figures 24, 25, 26, 27 and 29 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 diseases of the eye, such as diabetic neuropathy and AMD.

The anti-VEGF antibody "bevacizumab(BV), also known as "rhuMAb VEGF" or "Avastin®"that is a recombinant humanized monoclonal antibody against VEGF, obtained according to Presta et al. (1997) Cancer Res. 57:4593-4599. It includes mutant structural parts of the human IgG1 and antigennegative complementarity determining areas of mouse monoclonal antibodies against hVEGF A.4.6.1 that blocks binding of human VEGF to its receptors. Approximately 93% amino acid sequence bevacizumab, including most structural sections is derived from human IgG1, and approximately 7% of the sequence is derived mouse anti the La A4.6.1. Bevacizumab has a molecular mass of approximately 149000 Dalton and glycosylated. Other antibodies against VEGF include the antibodies described in the patent of the United States No. 6884879 and WO 2005/044853.

Antibodies against the VEGF ranibizumab or antibodies LUCENTIS®or rhuFab V2 are humanitarianism antibodies with established affinity against human VEGF fragment Fab. Ranibizumab produced with conventional methods of recombinant technology using the expression vector for Escherichia coli and bacterial fermentation. Ranibizumab not glycosylated and has a molecular mass of approximately 48000 daltons. Cm. WO98/45331 and US20030190317.

The term "antagonist alfaretta" refers to any molecule that inhibits the biological activity of Alfama. In accordance with one preferred embodiment, the molecule antagonist specifically binds alfaretta. In accordance with one preferred embodiment, the molecule antagonist binds to alpha. In accordance with one preferred embodiment, the antagonist alfaretta preferably associated with alfaretta with greater affinity than with integrins, which are not alfaretta. In accordance with one preferred embodiment, the antagonist is selected from the group consisting of a polypeptide, tcog is how the antibody, Patiala, or immunoadhesin, low-molecular compounds or aptamer that inhibits the binding Alfama with its ligands (in particular, fibronectin), or nucleic acid, which hybridizes under stringent conditions with a nucleic acid molecule that encodes alfaretta (e.g., mRNA, interfering expression Alfa). Biological effects alfaretta can be any of the effects, their combination or all of the effects selected from the group consisting of (1) binding to fibronectin, (2) increase cell migration on fibronectin, (3) increase the survival rate of cells containing alfaretta in the presence of fibronectin, (4) increasing the proliferation of cells containing alfaretta in the presence of fibronectin, and (5) increasing the formation of tubes from cells containing alfaretta in the presence of fibronectin.

Examples of antagonists, antibodies against alfaretta include M200 and F200 (WO 2004/089988A2)described herein antibodies N and antibodies N, and recombinant, fully human and humanized antibodies based on them. For example, antibodies M200 and F200 can be derived from the variable heavy and variable light chains of murine antibodies against human alfaretta, IIA1 (Pharmingen, San Diego, Ca). Examples of low molecular weight inhibitors alfaretta include Ac-PHSCN-NH2 (WO-9822617A1) and (S)-2-[(2,4,6-trimetilfenil)sulfonyl]-amino-3-7-benzyloxycarbonyl-8-(2-pyridinylmethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-EN-3-yl]carbylamine]propionic acid. In accordance with one of preferred embodiments of the invention, the antagonist alfaretta associated with alfaretta, but is not associated with V3, V5 or V1. In accordance with one of preferred embodiments of the invention, the antagonist alfaretta associated with alfaretta with a Kd of between 1 μm and 1 PM. In accordance with another preferred embodiment of the invention the antagonist alfaretta associated with alfaretta with a Kd between 500 nm and 1 PM. In accordance with one preferred embodiment the antibody against Alfama is an antibody that can compete with the antibody N or antibody N when linking with alfaretta in the study of competitive binding. In accordance with another preferred embodiment, the antibody is an antibody, the binding of which alfaretta can be completely ingibirovalo antibodies produced by hybridomas deposited as Alfa/beta 7H5.4.2.8 (ATCC No. PTA-7421), or hybridoma deposited as Alfa/beta 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. The VEGFR agonists include, but are not limited to, agonists-ligands of VEGFR, VEGF variants, antibodies and active fragments.

The term "agonist and hareta" refers to a molecule, which can activate alfaretta or increase its expression. Agonists alfaretta include, but are not limited to, for example, ligand agonists alfaretta.

Molecules, such as antibodies, characterized by binding to overlapping or similar target areas can be identified through research competitive inhibition/binding.

In one embodiment, HUVEC cells or other cells expressing alfaretta used in studies of competitive inhibition, to estimate the location of the binding of two antibodies against alfaretta relative to each other was used FACS. For example, HUVEC cells can be washed in a conical tube and precipitated by centrifugation at 100 rpm for 5 minutes the Precipitate was usually washed twice. Then the cells can be resuspendable calculated and stored on ice until use. A hole may be added to 100 μl of the first antibody against alfaretta (for example, starting with a concentration of 1 μg/ml or lower concentration). Then in each well can be added to 100 μl of cells (for example, 20×105cells), followed by incubation on ice for 30 minutes Then in each well can be added to 100 ál of biotinylated antibodies against alfaretta (original solution of 5 μg/ml)media and conducted by incubation on ice for 30 minutes Then cells were washed and precipitated by centrifugation for 5 min at 1000 rpm, the Supernatant was removed. In the hole was added (100 μl, 1:1000) conjugated with R-phycoerythrin streptavidin (Jackson 016-1 10-084). Then the tablet was wrapped in foil and incubated on ice for 30 min After incubation the precipitate can be washed and precipitated by centrifugation for 5 min at 1000 Rev/min the Precipitate was resuspendable and transferred into a microtube 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, the growth of endothelial cells, stability of blood vessels, and/or vasculogenesis etc. Angiogenic factors include, but are not limited to, e.g., VEGF and members of the family of VEGF, P1GF, PDGF family, the family of fibroblast growth factor (FGF), TIE ligands (angiopoietins), afrina, Del-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF), follistatin, factor stimulation of colonies of granulocytes (G-CSF), a growth factor, hepatocyte (HGF)/scattering factor (SF), interleukin 8 (IL-8), leptin, midkine, placental growth factor, platelet-derived growth factor endothelial cells (PD-ECGF), platelet-derived growth factor, in particular PDGF-BB or-DERIVED beta, pleiotrophin (PTN), progranulin, proliferin, transform what they growth factor alpha (TGF-alpha), proliferin, transforming growth factor beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), a growth factor, vascular endothelial (VEGF)/factor vascular permeability (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 members of his family, 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) (for example, table 1 lists the known risk factors); and Sato Int. J. Clin. Oncol., 8:200-206 (2003).

The term "Kd" or "Kd value" for antibodies against VEGF in accordance with this invention in one preferred embodiment, is measured by examining the binding of radioactively labeled VEGF (RIA)carried out between the Fab version of the antibody molecule and VEGF, as described in the next dimension in which the affinity of binding in solution of Fab against VEGF is determined by balancing Fab minimum concentration of labeled (125I) VEGF(109) in the presence of a series of dilutions of unlabeled VEGF, with the subsequent capture of bound VEGF coated antibodies against Fab tablet (Chen, et al., (1999) J. Mol Biol 293:865-881). To set conditions for analysis microtiter plates (Dynex) were coated with exciting the mi antibodies against Fab (Cappel Labs) by incubation overnight with 5 μg/ml of antibody in 50 mm sodium carbonate (pH 9,6), then blocked with 2% (by weight) of bovine serum albumin in the FSB within two to five hours at room temperature (approximately 23°C). In non-absorbing tablet (Nunc No. 269620) 100 PM or 26 PM [125I] VEGF(109) was mixed with serial dilutions of interest Fab, for example, Fab-12 (Presta et al., (1997) Cancer Res. 57:4593-4599). Then interest Fab incubated over night; however, incubation can last up to 65 hours in order to ensure that equilibrium is achieved. Then the mixture was transferred to an exciting tablet and incubated at room temperature for one hour. Then the solution was removed, after which the plate was washed eight times with 0.1% Tween-20 in FSB. When the tablets were dried was added 150 μl of scintillation fluid (MicroScint-20; Packard) per well, and then the tablets were counted on a gamma counter Topcount(Packard) for ten minutes. The concentration of each of the Fab, which gave the link to, less than or equal to 20% of the maximum were selected for use in research on competitive binding. In accordance with another embodiment the Kd or Kd value were measured using assays using surface plazminovogo resonance using BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized hVEGF (8-109), CM5 chips and ~10 edit is Izumi response (RU). Briefly, biosensor chips on carboxymethylamino dextran (CM5, BIAcore Inc.) were activated by the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Human VEGF was diluted 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) before the introduction of a flow rate of 5 μl / minute to obtain approximately 10 response units (RU) of the associated protein. After the introduction of human VEGF was introduced 1M ethanolamine to block unreacted groups. For kinetic measurements was introduced a series of twofold dilutions of Fab (0.78 nm to 500 nm) in the FSB with the addition of 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl/min Rate of Association (konand the rate of dissociation (koff) were calculated using a simple model linking one-to-one of Langmuir (BIAcore Evaluation Software version 3.2) by simultaneous substitution sensogram Association and dissociation. The equilibrium dissociation constant (Kd) was calculated as the ratio of koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the velocity of the Association exceed 106M-1S-1according to research by surface plasmon resonance, as described earlier, the rate of Association can be determined using the technique of fluorescence quenching, in which smiryaetsya increase or decrease the intensity of fluorescence emission (excitation 295 nm, emission 340 nm, 16 nm bandwidth) at 25°C 20 nm antibodies against VEGF (in the form of Fab) in the FSB, pH 7.2 in the presence of increasing concentrations of short forms of human VEGF (8-109) or murine VEGF measurements using a spectrometer, such as a spectrometer, equipped with a stop-flow (Aviv Instruments) or SLM-Aminco spectrophotometer 8000 series (ThermoSpectronic) with a stirred up cuvette. Similar studies linking can be performed to determine the Kd Fab or antibody against alfaretta using as a target alfaretta.

In used here is treated the subject is a mammal (e.g., human, Primate, non-human, rat, mouse, cow, horse, pig, sheep, goat, dog, cat etc). The subject may be a clinical patient, volunteer clinical trials, experimental animals, etc. the subject may be suspected with the presence or present the risk of cancer, immune diseases or any other diseases characterized by pathological angiogenesis. Many ways to diagnose cancer, immune diseases, or other diseases, demonstrating abnormal angiogenesis and clinical presentation of these diseases that are well known in this field. In accordance with one preferred embodiment subjected to treatment with bject is the man.

The term "abnormal angiogenesis" is used when new blood vessels grow or excessive or inappropriate (e.g., plot, time, or the beginning of angiogenesis are undesirable from the point of view of medicine) of disease or in such a way that it causes the state of the disease. Excessive, inappropriate or uncontrolled angiogenesis occurs when the growth of new blood vessel contributes to the deterioration of the disease or is the cause of the condition of the disease, such as cancer, especially vacuolation solid tumors and metastatic tumors (including colon cancer, lung (especially small cell lung cancer, or prostate cancer), diseases caused by neovascularization of the eye, in particular, diabetic blindness, retinopathy, primarily diabetic retinopathy or age-related macular degeneration, choroidal neovascularization (CNV), diabetic macular edema, pathological myopia, disease van Hippel-Lindau, histoplasmosis of the eye, occlusion of retinal vein (CRVO), corneal neovascularization, neovascularization of the retina and robes; psoriasis, psoriatic arthritis, haemangioblastoma, such as hemangioma; inflammatory renal diseases, such as glomerulonephritis, especially mesangiocapillary Glo is arulappa, haemolytic uraemic syndrome, diabetic nephropathy or hypertensive nephrosclerosis; various inflammatory diseases such as arthritis, especially rheumatoid arthritis, inflammatory bowel disease, psoriasis, sarcoidosis, arterial arteriosclerosis and diseases occurring after transplants, endometrios or chronic asthma and more than 70 other conditions. New blood vessels can nourish the affected tissue, destroy normal tissue, and, in the case of cancer, new vessels may allow tumor cells to enter the circulation and deposited in other organs (metastatic tumors). The presented invention considers the treatment of patients with the risk of the aforementioned diseases.

Other patients that are candidates for obtaining antibodies or polypeptides according to this invention, have or are at risk of developing, abnormal proliferation of vascular fibrous tissue, red eel, acquired immunodeficiency syndrome, clogged arteries, atopic keratitis, bacterial ulcers, disease behceta, blood-borne tumors, obstructive carotid artery disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitrite, excessive wearing of contact lenses is tworzenia transplant of a cornea, the corneal neovascularization, neovascularization of corneal transplant, Crohn's disease, diseases of Ailes, epidemic keratoconjuctivitis, fungal ulcers, infection, simple herpes, infection with herpes zoster, syndromes with high viscosity, Kaposi's sarcoma, leukemia, degeneration of lipids, Lyme disease, marginal keratolysis, ulcers Moray, mycobacterial infections except leprosy, myopia, ocular neovascular disease, congenital pits of the optic nerve syndrome Osler-Weber (Osler-Weber-Rendu), osteoarthritis, Paget's disease, inflammation of the ciliary circle, pemphigoid, electrolyze, polyarteritis, complications after laser radiation, infections protozoa, elastic pseudoxanthoma, pterygia, dry ceratite, pterygium keratitis sicca, radial keratotomy, neovascularization of the retina, retrolental fibroplasia, sarcoid, sclerite, sickle cell anemia, Sjogren syndrome, solid tumors, diseases of Stargate, disease Steven Johnson, the higher limbic keratitis, syphilis, systemic lupus erythematosus, regional degeneration of the cornea, toxoplasmosis, trauma, tumors Ewing sarcoma, neuroblastoma tumors, tumors of osteosarcoma, tumors of retinoblastoma, tumors of rhabdomyosarcoma, ulcerative colitis, vein occlusion, vitamin a deficiency and sarcoidosis of Wegner, unwanted angiogenesis SV is related to the diabetes, parasitic diseases, abnormal wound healing, post-surgical hypertrophy, injury or trauma, inhibition of hair growth, inhibition of ovulation and the formation of a yellow body, suppression of implantation and inhibition of embryo development in the uterus.

Therapy anti-angiogenesis is useful for a General treatment of transplant rejection, lung inflammation, nephrotic syndrome, preeclampsia, effusion in the region of the pericardium, such as that associated with pericarditis, and pleural effusion, diseases and disorders characterized by undesirable vascular permeability, e.g., edema associated with brain tumors, ascites associated with malignant tumors, Meigs syndrome, lung inflammation, nephrotic syndrome, exudative pericarditis, pleural effusion, permeability associated with cardiovascular diseases such as post-infarction state, shock and the like.

Other dependent angiogenesis diseases in accordance with this invention include angiofibroma (abnormal bleeding from prone to bleeding vessels), neovascular glaucoma (the growth of blood vessels in the eye), arteriovenous malformations (abnormal communication between arteries and veins), nerastas fractures (fractures that have not healed), atheroscler the political plaques (hardening of the arteries), pyogenic of granulomas (frequent pathological changes of the skin, composed of blood vessels), scleroderma (disease of the connective tissue), hemangioma (tumor composed of blood vessels), trachoma (the main cause of blindness in the third world), hemophilic arthropathy, coagulation of blood vessels and hypertrophic scars (improper formation of the scar).

The term "treatment" refers to therapeutic treatment and prophylactic or preventative measures. In need of treatment for patients include those who already have the disorder, and those with the disorder should be prevented.

The terms "repeat", "relapse" or "recurrent" refers to the return of the cancer or disease after clinical definition of extinction of the disease. Diagnosis of distant metastasis or local recurrence can be considered a relapse.

The terms "immune" or "sustainable" refers to cancer or disease that does not respond to treatment.

The term "adjuvant therapy" refers to treatment given after the primary treatment, usually surgical. Adjuvant therapy in cancer or disease may include immune therapy, chemotherapy, radiotherapy or hormone therapy.

The term "supportive therapy" refers to the planned re-l is teaching, which is to aid in maintaining the effects of previous therapy. Supportive therapy is often performed to keep cancer in remission or to extend the response to a specific 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 began, in the 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 cancer that has not spread beyond the tissue that occurs.

The term "survival without progression in Oncology refers to the period of time during and after treatment, during which the cancer is not growing. Survival without progression includes the amount of time during which the patient experienced a complete or partial response, as well as the period of time during which the patient experienced stable disease.

The term "progressive disease" in Oncology may relate to tumor growth by more than 20 percent since the start of treatment or because of the increased mass of the tumor, or the tumor spread.

The term "disorder" is any condition in which treatment with antibodies will benefit. For example, mammals, strad is either from abnormal angiogenesis (redundant, inappropriate or uncontrolled angiogenesis or vascular permeability or need in their prevention. This includes chronic and acute disorders or diseases including pathological conditions which predispose the mammal to discuss the disorder. Non-limiting examples being treated disorders include malignant and benign tumors, placemates and lymphoid malignancies; neuronal, glial, astatically, hypothalamic and other glandular, mikrofalowe, epithelial, stromal and bestialiska disorders; and inflammatory, angiogenic and immunologic disorders.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Some examples of such cancers include squamous cancer cells, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, bowel cancer, colon cancer, endometrial sarcoma, sarcoma salivary glands, kidney cancer, cancer of the renal tubules, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, head and neck cancer, colorectal cancer, p is to the intestines, lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, squamous cancer cells (for example, cancer of the squamous epithelial cells), prostate cancer, cancer of the peritoneum, liver cancer, stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, retinoblastoma, astrocytoma, Tacoma, arrhenoblastoma, hepatoma, hematologic malignancy, including nahodkinskuju lymphoma (NHL), multiple myeloma and acute hematologic malignant conditions, endometrial or uterine carcinoma, endometriosis, fibrosarcomas, horiokartsinoma, carcinoma salivary gland cancer of the vulva, cancer of the thyroid gland, cancer of the esophagus, liver carcinoma, carcinoma of the rectum, carcinoma of the penis, nasopharyngeal carcinoma, carcinoma of the larynx, Kaposi's sarcoma, melanoma, skin carcinomas, sandamu, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, carcinoma urinary bladder carcinoma thyroid, Wilm tumor, as well as B-cell lymphoma (including immature/follicular nahodkinskuju lymphoma (NHL); small lymphocytic (SL) NHL; sredneperesechennoy/follicular NHL; diffuse NHL moderate; well-differentiated immunoblastic NHL; viskovitz is stimulated lymphoblastic NHL; well-differentiated small-cell NHL with unsplit cores; NHL with massive lesions, lymphoma mantle cell; AIDS-associated lymphoma, the disease Mandelstam (macroglobulinemia); chronic impositions leukemia (CLL); acute lymphoblastic leukemia (ALL)leukemia, hairy cell; chronic leukemia of myeloblasts; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal growth of blood vessels associated with phakomatoses and Meigs syndrome.

The term "tumor"as used here, refers to neoplastic growth and cell proliferation, malignant and benign, and all pre-cancerous and cancerous cells and tissues.

The term "anti-neoplastic composition" or "anti-neoplastic drug" refers to compositions that are useful in the treatment of cancer, comprising at least one active therapeutic factor, such as "anti-cancer drug". Examples of therapeutic agents (anti-cancer drugs include, but are not limited to, for example, chemotherapeutic drugs, any abscopal growth drugs, cytotoxic drugs, drugs used in radiotherapy, drugs against angiogenesis, apoptotic drugs, protivostoyanie drugs, and other drugs for the treatment of RA is a, such as antibodies against HER-2, antibodies against CD20, an antagonist of the growth factor receptor epidermal (EGFR) (e.g., a tyrosine kinase inhibitor), an inhibitor of HER1/EGFR (for example, erlotinib (TarcevaTM), inhibitors of platelet-derived growth factor (e.g., GleevecTM(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 purposes - with the receptor(s) of ErbB2, ErbB3, ErbB4, DERIVED-beta, BAFF, BR3, APRIL, BCMA or VEGF, TRAIL/ Apo2, and other bioactive and organic chemical factors, and other combinations are also included in the invention.

Used herein, the term "factor suppressing (inhibiting) growth" refers to a compound or composition which inhibits growth of cells in vitro and/or in vivo. Thus, the suppression factor of growth may be a factor that greatly reduces the percentage of cells in S-phase. Examples of the factors inhibiting the growth include the factors that block the progress of the cell cycle (point other than S phase), such as factors that can stop G1 and stop in M-phase. Classic blockers M-phases include the Vinca alkaloids (vincristine and vinblastine), TAXOL®and inhibitors topo, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Factors (agents), nl is kirousis G1, also apply to block S - phase, for example, DNA alkylating agents 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" authors Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13.

The term "cytotoxic drug (agent)," as it is used here, refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. It is assumed that the term shall include radioactive isotopes (e.g., I131I125, Y90and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

The term "chemotherapeutic drug" refers to a chemical compound used in the treatment of cancer. Examples of chemotherapeutic agents include chemical compounds that can be used for cancer treatment. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN®cyclophosphamide; alkyl sulphonates such as busulfan, improsulfan, and pilosula; aziridine, such as benzodepa, carb is Kwon, matureup, and uredepa; ethylenimines and methyleneimine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenin (mainly bullatacin, bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including synthetic analogs of adozelesin, carzelesin and bizelesin); cryptophycin (namely cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin a; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine chlorpropamide, estramustine, ifosfamide, mechlorethamine, oxide hydroxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, uramustine; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; spiramycin; and the chromophore neocarzinostatin and related chromoprotein andinavia antibiotic chromophores), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, erinomisen casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino doxorubicin, cyanomethane doxorubicin 2 pyrroline doxorubicin and deoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol,mepitiostane, testolactone; inhibitors of the adrenals such as aminoglutethimide, mitotane, trilostane; DOPOLNITEL folic acid, such as prolinnova acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; acetate elliptica; epothilone; etoposide; gallium nitrate; GI is roximation; lentinan; londini; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; polysaccharide complex PSK®(JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tinoisamoa acid; triaziquone; 2,2',2"-trihlortrietilamin; trichothecenes (in particular toxin T-2, verrucarin And, roridin and unguided); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoid, for example, paclitaxel TAXOL®(Bristol-Myers Squibb Oncology, Princeton, NJ.), ABRAXANETM without cremophor, constructed on the basis of albumin form of paclitaxel nanoparticles (American Pharmaceutical Partners, Schaumberg, Illinois), and docetaxel TAXOTERE®(Rhone - Poulenc Rorer, Antony, France); chlorambucil; gemcitabine GEMZAR®; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine NAVELBINE®; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment with irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinue the traveler acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the treatment regimen with oxaliplatin (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras and EGFR (for example, erlotinib (TarcevaTM)that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of all the above drugs.

Also included in this description protivokomarinye drugs that regulate or inhibit hormone action on tumors such as antiestrogens and selective regulators of estrogen receptors (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates the production of estrogen in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol-acetate MEGASE®ackzemestan AROMASIN®, formestane, fadrozole, vorozole RIVISOR®, letrozole FEMARA®and anastrozole ARIMIDEX®; antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane, a nucleoside analogue of cytosine); antisense oligonucleotides, particularly inhibiting the expression of genes in signaling pathways implicated in the reproduction of aberrant cells, such as, for example, PKC-alpha, Raf and H-Ras; ri is winter, such as the inhibitor of the expression of VEGF (e.g., ribozyme ANGIOZYME®) and the inhibitor of HER2 expression; vaccines such as vaccines, genetic therapy, such as vaccine ALLOVECTIN®the vaccine , LEUVECTIN®and the vaccine VAXID®; PROLEUKIN®rIL-2; topoisomerase inhibitor 1 LURTOTECAN®; ABARELIX®rmRH; vinorelbine and espiramicina (see U.S. patent No. 4675187), and pharmaceutically acceptable salts, acidic forms or derivatives of the above drugs.

The term "prodrug"used in this application refers to a precursor or derivative form of pharmaceutically active substances (for example, low-molecular compounds), which is less cytotoxic compared to the diseased cells compared to the original drug and can be enzymatically activated or converted into the more active its original 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, modified D-amino acid prodrugs, glycosylated prodrugs, β-lacto what-containing prodrugs, contains possibly substituted phenoxyacetamide prodrugs or may contain substituted phenylacetamide prodrugs, 5-fertilizing and other prodrugs of 5-ptoluidine that can be converted into a more cytotoxic free drug. Examples of cytotoxic drugs that can be transferred in the form of prodrugs for use in this invention include, but are not limited to the above chemotherapeutic drugs.

The term "isolated"when used to describe the various polypeptides disclosed here, means polypeptide that has been identified and separated and/or isolated from cells or cell culture, in which he expressively. Contaminant components of its natural environment are materials that normally inhibit the diagnostic or therapeutic application of the polypeptide, and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, the implementation of the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a sequencing machine with rotating cups or (2) to homogeneity according to SDS-PAGE under non and reducing conditions with what ispolzovaniem blue Kumasi or preferably, the color silver. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the natural environment of the polypeptide is not present. Typically, however, an isolated polypeptide is produced using at least one purification stage.

The term "isolated" encodes the polypeptide, nucleic acid or other encoding a polypeptide, nucleic acid refers to a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule, which is usually associated in the natural source encodes a polypeptide nucleic acid. Isolated encoding a polypeptide molecule of nucleic acid is in a different form, or environment than those in which it occurs in nature. Isolated encoding a polypeptide molecule of the nucleic acid thus differs from that encodes a polypeptide molecule of nucleic acid in natural cells. However, encoding the isolated polypeptide molecule of nucleic acid include encoding the polypeptide molecule is a nucleic acid that normally Express the polypeptide in the case of, for example, when the nucleic acid molecule is in position on a chromosome other than the provisions in natures is the R cells.

The term "control sequences" refers to DNA sequences necessary for the expression of the operatively linked coding sequence in a particular organism, the host. The control sequences that are suitable for prokaryotes, for example, include a promoter, you can sequence operator, and the binding site of the ribosome. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is operatively linked"when it is in a functional relationship with another nucleic acid sequence. For example, predpolozhytelno DNA or secretory leader sequence operatively linked to DNA that encodes a polypeptide if it expresses preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operatively associated with the sequence if it affects the transcription of the sequence; or the binding site of the ribosome operatively linked to the coding sequence if it is located in such a way that contributes to broadcast. In General, "operatively linked" means that the DNA sequence of connected and continuous, and, in the case of a secretory leader sequence, continuous and are in the same phase of reading. One is to enhancers do not have to be adjacent. The binding occurs by ligating the appropriate restriction sites. If such sites do not exist, in accordance with normal practice used synthetic linkers and adapters.

"Stringent conditions" or "terms " high rigidity", as described here, can be defined as (1) including low ionic strength and high temperature for washing, for example of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulphate at 50°C; (2) including denaturing factor, such as formamide, in the process of hybridization, for example, 50% (volume) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mm sodium phosphate buffer pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42°C.; or (3) hybridization overnight in a solution containing 50% formamide, 5x SSC (0,75 M NaCl, Of 0.075 M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5x denhardt's solution, processed by the ultrasound salmon sperm (50 µg/ml), 0.1% of SDS and 10% dextran sulfate at 42°C. with washing for 10 minutes at 42°C in 0,2x SSC (sodium chloride/sodium citrate) followed 10 min washing under conditions of high stringency, consisting of 0.1 x SSC containing EDTA at 55°C.

"Percent (%) amino acid sequence identity" with respect to listed here polypeptide sequences is determined by the ka is the percentage in the discussed sequence of amino acid residues, identical amino acid residues compared to the polypeptide after alignment and, if necessary, to achieve the maximum percent sequence identity, the introduction of gaps, and conservative substitutions are not considered part of the identity of the sequences. Alignment for purposes 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 technical field, for example, using publicly available software, such as software BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in the art can determine appropriate parameters for measuring homology, including the algorithms required to achieve maximum homology within the full length of the compared sequences. However, to solve there problems % identity of amino acid sequences were obtained using the computer program of sequence comparison program ALIGN-2. A computer program for comparing sequences ALIGN-2 was developed by Genentech, Inc, source code (table 1) was entered in the register together with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it was registered under the registration number U.S. TXU51087. The program ALIGN-2 is publicly available through Genentech, Inc., South San Francisco, California. The program ALIGN-2 must be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All the parameters of comparison sequences were set by the program ALIGN-2 was not changed.

Amino acid sequences described herein are continuous amino acid sequences, unless otherwise noted.

Used here, the term "immunoadhesin" means such antibody molecules which combine the binding specificity of a heterologous protein (adhesin") with the effector functions of the constant domain of immunoglobulin. Structurally, immunoadhesin include merged sequence consisting of amino acid sequence with the desired binding specificity, other than the plot of the recognition and binding of antigen antibodies (i.e. which is "heterologous"), and the sequences of the constant domains of immunoglobulin. Adhesiva part of the molecule immunoadhesin is usually a continuous amino acid sequence comprising at least the binding site of the receptor or ligand, such as VEGFR or fibronectin ligand. Constant sequence region of immunoglobulin in immunoadhesin can be obtained from any of them is noglobulin, such as the subtypes of IgG-1, IgG-2, IgG-3, or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. Patiala, often containing a sequence obtained by sampling from a sequence of phage display, which are specifically associated with the target, and their fusion with the Fc part of the immunoglobulin can also be considered here immunoadhesins.

The term "antibody" is used in the most General sense and, in particular, implies, for example, single monoclonal antibodies (including agonist, antagonist and neutralizing antibodies), antibody composition with mnogoetapnoe specificity, polyclonal antibodies, single-chain antibodies and fragments of antibodies (see below), as long as they specifically associated with native polypeptide and/or demonstrate the biological or immunological activity according to this invention. In accordance with one embodiment, the antibody binds to the oligomeric form of the protein target, for example with the trimeric form. In accordance with another embodiment, the antibody specifically binds with the protein, this binding can be ingibirovalo monoclonal antibodies in accordance with this invention (e.g., deposited antibody according to this invention and the like). The phrase "functional fragment or analog of the" antibody refers to a compound that to the national biological activity, in common with the antibody with which it is compared. For example, a functional fragment or analog of an antibody according to the invention can specifically bind to VEGF or alfaretta. In one embodiment, the antibody can prevent or significantly reduce the ability of VEGF to induce proliferation of cells.

An "isolated antibody" is an antibody that has been identified and separated and/or purified from components of its natural environment. Contaminant components of its natural environment are materials that can interfere with diagnostic or therapeutic application of antibodies, including enzymes, hormones and other protein or non-protein soluble substances. In preferred embodiments, the implementation of the antibody will be purified (1) to more than 95% by weight of antibody, when determining by the method of Lowry, and, most preferably, up to more than 99% by weight, (2) to a degree sufficient to obtain at least 15 amino acid residues N-terminal or internal amino acid sequence by using a sequencing machine with rotating cups, or (3) to homogeneity according to SDS-PAGE under reducing or non conditions using blue staining of Kumasi or, preferably, silver. Isolated antibody includes the antibody in situ in R is combinant cells, since at least one component of the natural environment antibodies will not be present. Typically, however, isolated antibody is obtained using at least one purification stage.

The basic 4-chain unit antibodies is heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (IgM antibody consists of 5 regular heterotetrameric units, along with an additional polypeptide called chain J, and, therefore, contains 10 binding sites of the antigen, while secreted IgA antibodies can cure, forming a multivalent structure, including 2-5 basic 4-chain units along with J chain). In the case of IgG, 4-chain unit usually has a weight of approximately 150,000 daltons. Each circuit L is connected with a chain of H one covalent disulfide bond, while two chains H are connected to each other by one or more disulfide bonds, depending on the isotype of the H-chain. Each of the chains H and L also has regularly spaced intrachain disulfide bridges. Each H chain has at the N end of the variable domain (VH), followed by three constant domains (CHfor each of the α and γ chains and four constant domains (CHfor isotypes μ and ε. Each L chain has at the N end of the variable domain (VL), after the existing constant domain (C L) on the other end. VLlocated opposite the VHand CLis opposite the first constant domain of the heavy chain CH1. It is believed that certain amino acid residues form the interaction between the variable parts of the heavy and light chains. Paired together, VHand VLform the binding site of the antigen. Structure and properties of different 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 differentiated 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 (CN), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated, respectively, α, δ, γ, and μ. Classes γ and α are further divided into subclasses on the basis of relatively minor differences in the sequence CHand functions, for example in humans is expressed by the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term "variable" refers to the fact that some segments of the variable domain is to have significant differences in sequence among antibodies. Domain V provides the binding to the antigen and determines the specificity of a particular antibody to a specific antigen. However, the variability is not evenly distributed within the 110-th amino acid of the variable domain. Instead, the plots of V consist of relatively regular intervals, called frame sections (FR) of 15-30 amino acids separated by shorter sections of the exceptional variability called "hypervariable areas", each of which has a length of 9-12 amino acids. Each of the variable domains of the natural heavy and light chain consists of four FR, basically taking a configuration of a beta-sheet associated with three hypervariable sites, which form loops connecting, and in some cases forming part of the structure of beta-sheet. In each chain hypervariable region are held together in close proximity by using FR and, with the hypervariable sites with the other chain, contribute to the formation of the antigen-binding site 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 involved directly in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody-specific antibody cellular cytotoxicity (ADCC).

Ter is in the hypervariable area", used here, refers to the amino acid residues in the antibody responsible for binding to the antigen. Hypervariable area typically comprises amino acid residues from a "plot that defines complementarity" or "CDR" (e.g. residues about 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VLand about 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the VH(in one embodiment, H1 is about approximately 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 residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VLand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

Used here, the term "monoclonal antibody" refers to an antibody obtained from a population of mostly homogeneous antibodies, the antibodies contained in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies of vysokospetsifichnymi and directed against one section of the antigen. Moreover, in contrast to the preparations of polyclonal antibodies, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinants. The stage is the implementation to their specificity, monoclonal antibodies have the advantage that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies that are applicable in the present invention, can be produced using hybridoma technology, first described by Kohler et al., Nature, 256:495 (1975), or may be obtained using methods of recombinant DNA in bacterial, eukaryotic animal or plant cells (see, for example, U.S. patent No. 4816567). "Monoclonal antibodies" may also be isolated from phage libraries of antibodies using techniques described, for example, 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 portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass, while the remainder of the chain (chain) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of these antibodies is, as long as they demonstrate biological activity in accordance with this invention (see U.S. patent 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA. 81:6851-6855 (1984)). Chimeric antibodies of interest in this invention include "primaryservername" antibodies, including antigen-binding sequence of the variable domain derived from a Primate, non-human (e.g., Old world monkeys, apes, etc), and human constant sequence of sections.

"Intact" antibody is an antibody containing the antigen-binding site, and CLand at least the constant domains of the heavy chain CH1, CH2 and CH3. The constant domains may be constant domains of the natural sequence (e.g., human constant domains of the original sequence) or variant of the amino acid sequence. Preferably, the intact antibody has one or more effector functions.

"Antibody fragments" comprise a portion of whole antibodies, preferably antigennegative or variable plot of a whole antibody. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')2and Fv; dyatel, linear antibodies (see U.S. patent No. 5641870, example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-stranded molecule of the antibody; multispecific antibodies collected from fragments of antibodies.

The expression "linear antibodies" generally refers to the antibodies described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies have a pair of tandem Fd segments (VH-CH1-VH-CH1)which, together with complementary light chain polypeptides, form a pair antigenspecific areas. Linear antibodies can be bispecific or monospecific.

Cleavage of antibodies with papain produces two identical antigen-binding fragment, called " fragments "Fab", and the residual fragment "Fc", the designation reflects the ability to easily crystallize. The Fab fragment consists of an entire L chain along with the variable domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is a monovalent with respect to binding to the antigen, i.e. has a single binding site of the antigen. Processing of antibodies with pepsin gives a large fragment F(ab')2, which roughly corresponds to the two linked by a disulfide bridges Fab fragments with divalent antigen-binding activity and is still capable of cross-knit antigen. Fragments, Fab' differ from Fab fragments by the fact that they have a few additional amino acid residues at the C-end of the domain CH1, including one or more cysteine residues of saryrn is the second portion of the antibody. Fab'-SH is used here the notation for Fab'in which one or more cysteine residues of the constant domain are free thiol group. Antibody fragments F(ab')2were originally obtained as a pair of fragments, Fab'having a hinge cysteine residues between them. Also known other chemical compounds fragments of antibodies.

The Fc fragment comprises the C-terminal parts of both circuits H, connected together by disulfides. Effector functions of an antibody is determined by sequences in the plot of Fc, which is also part recognized by Fc receptors (FcR), found in some types of cells.

"Fv" is the minimum antibody fragment that contains a complete plot of the recognition and binding of the antigen. This fragment is a dimer consisting of one variable plot heavy and one light chain, which is in tight, non-covalent linkages. In linking these two domains are formed six hypervariable loops (3 loops from H and L chains), which contain the amino acid residues involved in the binding to the antigen, and attach the binding specificity of the antigen with 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, although at a lower crodt the om compared with intact binding site.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", are fragments of antibodies, which include the domains of the antibody VHand VLattached to a single polypeptide chain. Preferably, the sFv polypeptide contains a polypeptide linker between domains VHand VLthat enables the sFv to form the structure required for binding antigen. 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 "diately" refers to small fragments of antibodies obtained by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between domains VHand VLthus is achieved the mating domain V between circuits, and not in a chain that leads to the formation of a bivalent fragment, e.g., fragment having two binding site of the antigen. Bespecifically of diately are heterodimeric two "crossed" sFv fragments, in which the domains of the two antibodies VHand VLpresent in different polypeptide chains. Diately 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 nonhuman antibodies (e.g., rodent antibodies are chimeric antibodies that contain minimal sequence originating from nonhuman antibody. Mainly, humanized antibodies are human immunoglobulins (antibodies-recipients), in which the remains of the hypervariable area of the recipient replaced the remnants of the hypervariable area antibodies (donor antibody) from species that are not human, such as mouse, rat, rabbit or non-human Primate having the desired specificity, affinity, and capability. In some cases, remnants of the structural region (FR) of a human immunoglobulin are replaced by corresponding inhuman remnants. Furthermore, humanized antibodies may contain residues that are not found neither in the recipient or in the donor antibody. These modifications are made to further improve the effectiveness of antibodies. Usually, humanitariannet antibody includes basically all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to available in non-human immunoglobulin and all or substantially all of the FR is FR from a human immunoglobulin sequence. Humanized antibodies can also include at least part of the constant part of the immunoglobulin (Fc), typically from a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

"Videosanime antibody" is an antibody that has a higher binding affinity to the antigen from one species of mammals than with its homologs from other species of mammals. Usually, wideawake antibodies "specifically bind" to a human antigen (i.e., have the value of the binding affinity (Kd) no greater than 1×10-7M, preferably not more than 1×10-8and most preferably not more than 1×10-9M)but has a binding affinity with a homologue of the antigen from another, non-human mammal, which at least about 50-fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity to the human antigen. Wideawake antibody may belong to any of various types of antibodies, as defined above, but preferably are humanitarianism or human antibodies.

In such scenarios, the implementation, the degree of binding of the polypeptide, antibody, antagonist or composition with "non-target" protein will be less than approximately 10% of the binding of the polypeptide, antibody, antagonist or composition with protein, which is its direct purpose, according to analysis by the method of fluorescence-activated sorting of cells (FACS) and the and radioimmune precipitation (RIA). With regard to the binding of the polypeptide, antibody, antagonist or composition with molecule-target, the term "specific binding"or "specifically binds to"or is "specific for" a particular polypeptide or epitope on a particular polypeptide target means binding that is non-measurable non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding reference molecule, which is usually a molecule of similar structure that does not have binding activity. For example, specific binding may be determined by the method of competition c control molecule that is similar to the target, for example, with excess unlabeled target. In this case, specific binding will be shown, if the binding of the labeled target probe will be competitive suppressed by excess unlabeled target. Used here, the term "specific binding"or "specifically binds to"or is "specific for" a particular polypeptide or epitope on a particular polypeptide target can be illustrated, for example, a molecule having a Kd with respect to the target is not less than approximately 10-4M, alternatively not less than about 10-5M, the alternative is not less than the roughly 10 -6M, alternatively not less than about 10-7M, alternatively not less than about 10-8M, alternatively not less than about 10-9M, alternatively not less than about 10-10M, alternatively not less than about 10-11M, alternatively not less than about 10-12M or better. In one embodiment, the term "specific binding" refers to binding, where a molecule binds to a particular polypeptide or epitope-specific polypeptide in the absence of significant binding to any other polypeptide or epitope of the polypeptide.

The term "effector function" antibody refers to the biological activities attributed to plot Fc (site Fc natural sequence or the variant amino acid sequence plot Fc) antibodies and changes with isotype antibodies. Examples of effector functions of antibodies include: linking 1q and complementability cytotoxicity; the binding of the Fc receptor; mediated antibody-dependent cell cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors; and activation of b-cells. The term "natural sequence plot of Fc" means amino acid sequence identical to the amino acid after which outermost plot Fc, 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 "variant Fc parcel" means amino acid sequence that differs from the natural sequence plot Fc the benefit of having at least one amino acid modification described in this invention. Preferably, the variant section Fc has at least one amino acid substitution compared to the natural sequence of plot Fc or area Fc of the original polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the natural sequence plot Fc or area Fc of the original polypeptide. In one embodiment variant of the plot Fc, described here, 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 with a plot of Fc natural sequence. In accordance with another embodiment, a variant of the plot Fc, described here, will be not less than approximately 80% g is mologie, not less than about 85% homology, at least about 90% homology, at least about 95% homology, or at least about 99% homology with section Fc of the original polypeptide.

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

The term "polypeptide comprising a site of Fc" refers to the polypeptide, such as an antibody or immunoadhesin (see definitions above), including the site of the Fc. Lysine located at the C-end (balance 477, in accordance with the EU numbering system) section Fc may be removed, for example, during purification of the polypeptide or by recombinant construct nucleic acid that encodes a polypeptide. Accordingly, a composition comprising the polypeptides, including antibodies, with the plot of Fc in accordance with this invention, may contain a population of polypeptides, in which all residues K is dalene, polypeptide of a population in which not removed any residue C or polypeptide of the population with a mixture of polypeptides with and without residue C.

Within this specification and claims in relation to residues in the variable domain (approximately residues 1-107 light chain and residues 1-113 of the heavy chain) is commonly used numbering system according to Kabat (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). "EU numbering system" or "index EU" is usually used when referring to a residue in the constant section of the heavy chain of immunoglobulin (for example, the EU index described 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 stated, references to the numbers of residues in the variable domain of antibodies means the residue numbering according to the Kabat numbering system. Unless otherwise stated, references to the numbers of residues in the constant domain of antibodies means the residue numbering according to the EU numbering system.

The terms "Fc receptor" or "FcR" is used to describe a receptor that binds with a plot of Fc antibodies. In one embodiment, FcR according to this invention binds IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII, and FcγRIII, including allelic variants of the s and forms of alternative splicing of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains a cytoplasmic domain based on the tyrosine site activation (ITAM). Inhibiting receptor FcγRIIB contains a cytoplasmic domain based on the tyrosine inhibitory site (ITIM) (see review publication M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). The term includes allotype, such as allotype FcγRIIIA: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and/or FcγRIIA-H131. FcR 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 FcR, including those that will be discovered in the future, are also covered here by the term "FcR". The term also includes the neonatal receptor, FcRn, 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 major histocompatibility complex (MHC) and consists of ∀-chain, ecovalence related ∃2-microglobulin. Numerous functions of the neonatal Fc receptor FcRn considered Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays a passive role in the delivery of immunoglobulins IgG from mother to calf, regulate the levels of serum IgG. FcRn can act as a receptor recycling, linking and transporting minorityowned IgG in intact form into the cells and through them, allowing them to avoid the usual metabolic pathways leading to their degradation.

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

"CH1 domain of human land IgG Fc (also called domain "C1" or "H1") typically has a length of from about 118 amino acids to about 215 amino acids (according to the EU numbering system).

The term "hinge area" is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22: 161-206 (1985)). The hinge parts of other IgG isotypes may be aligned with the IgG1 sequence by setting the first and last bases of cysteine, forming connection S-S between the heavy chains in one position.

"The bottom hinge plot" plot Fc is typically defined as the length of the grounds directly on the C-end of the hinged section, i.e. residues 233 to 239 section Fc. In the previous studies linking FcR is usually attributed to amino acid residues in the lower hinge section section Fc of IgG.

"CH2 domain" of a human plot IgG Fc (also referred to as domain "C2" or"H2") is usually in the range from 231 to 340 amino acids of amino acids. Domain CH2 is unique in that it is not closely coupled with another domain. Instead, between the two CH2 domains of intact molecules of natural IgG contains two N-linked branched carbohydrate chains. An assumption was made that carbohydrates can replace the mating domain with domain and to promote stabilization of the CH2 domain. Burton, Molec. Immunol.22:161-206 (1985).

"CH3 domain" (also referred to as domain "C2" or "H3") includes the length of the residues on the C-end of the CH2 domain in the plot Fc (i.e. from about amino acid residue 341 to-end sequence antibodies, usually at amino acid residue 446 or 447 IgG).

"The functional site of Fc" has "effector functions" natural sequence plot of Fc. Examples of "effector functions" include linking 1q, complementability cytotoxicity; binding to Fc receptor; dependent antibody-mediated cell cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (i.e. receptors In cells, BCR), etc. Such effector functions generally require a combination of land with Fc binding domain (for example, variable domain antibodies) and can be assessed using various assays, for example as disclosed in this specification.

"1q" is a polypeptide that includes a website link to the ivania plot with immunoglobulin Fc. 1q together with two serine-proteases, C1r and C1s, form a complex C1, the first component of the path complementability cytotoxicity (CDC). Human 1q can be purchased commercially, for example from Quidel, San Diego, CA.

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

The antibody or Patiala variant IgG Fc with "modified" by the affinity of binding of the FcR or ADCC activity has increased or decreased ability to bind FcR (e.g., FcγR or FcRn) and/or ADCC activity compared to the starting polypeptide or polypeptide, including the original sequence plot Fc. Variant Fc, which shows improved binding with FcR binds at least one FcR with higher affinity (i.e. with a lower value of Kd or IC50)than the original polypeptide or IgG Fc with the natural sequence. In accordance with some of the options for implementation, the increase in binding compared to the starting polypeptide may be approximately 3-fold, preferably approximately 5, 10, 25, 50, 60, 100, 150, 200, or cat, or from about 25% to 1000% better binding. Variant of the polypeptide, which shows reduced binding with FcR binds at least one FcR with lower affinity (i.e. with a higher Kd or IC50)than the original polypeptide. In accordance with some of the options for implementation, the decrease in binding compared to the starting polypeptide may be approximately 40% or more.

The term "mediated antibody-dependent cell cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to receptors FcR present on certain cytotoxic cells (e.g., cells - natural killer cells (NK), neutrophils and macrophages), allowing these cytotoxic cells effectors specifically bind with bearing antigen on target cells and subsequently kill the target cell with cytotoxins. Antibodies vzvoda" cytotoxic cells and are absolutely necessary for such destruction. The basic cells are mediators of ADCC, NK cells Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression in hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of specific molecules can be carried out the study ADCC in vitro, as described in U.S. patent No. 5500362 or 5821337 or in the Examples below. Used for such studies effector cells include mononuclear cells of peripheral blood (PBMC) cells and natural killer (NK). Alternative or additionally, ADCC activity of specific molecules can be studied in vivo, for example in animal models, such as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

A polypeptide comprising a variant of the plot Fc, which shows increased ADCC" or is a mediator mediated antibody-dependent cell cytotoxicity (ADCC) in the presence of human cells-effector is more efficient than the polypeptide with Fc IgG wild-type or parent polypeptide is significantly more effective in vitro or in vivo to mediate ADCC, if the amount of the polypeptide variant of the section Fc and polypeptide with a plot of Fc wild-type (or parent polypeptide) in the study are almost identical. Typically, these options are defined using research ADCC in vitro, such as described here, however, refers to and other tests or methods for determining ADCC activity, for example in animal models, etc. In one embodiment, the preferred option is more effective as a mediator of ADCC compared to wild-type Fc (or the original polypeptide) in p is blithedale from 5 to about 100 times, for example, from about 25 to about 50 times.

The term "complementability cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical pathway of complement begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass)that are associated with them recognizable antigen. To assess activation of complement can be analyzed by the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202: 163 (1996).

Variants of the polypeptide with altered amino acid sequences of the section Fc and reduced or increased ability to bind C1q described in U.S. patent No. 6194551B1 and WO99/51642. The content of these publications directly here by reference. Cm. also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term "human cells-effector" refers leukocytes expressing one or more FcR and perform effector functions. According to one implementation, the cells Express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes, which are the mediators of ADCC include mononuclear cells from peripheral blood (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; preferred PBMC and NK cells. Effector cells can be provided which are from their natural source, for example, from blood or PBMC, as described here.

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997, Hinton 2004), as also described in the examples below. Binding to human FcRn in vivo and the period of life bind to human FcRn with high affinity polypeptides can be studied, for example, in transgenic mice or transfected lines of human cells expressing human FcRn, or primates, which introduced a variant Fc polypeptide. In one embodiment, antibodies against alfaretta with the modified IgG Fc in accordance with the invention, showed increased affinity binding to human FcRn compared to a polypeptide having an Fc IgG wild-type, not less than 2-fold, at least 5 times, at least 10 times, at least 50 times, at least 60, at least 70 times, not less than 80-fold, at least 100 times, at least 125 times, not less than than 150 times. In a specific embodiment, the affinity of binding to human FcRn increased approximately 170 times.

In one embodiment, EC50 or apparent Kd (pH 6,0) polypeptide affinity for binding to FcRn 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 increased affinity of binding to FcγRIII (F158; i.e. sotip with low affinity) EC50 or apparent Kd was less than or equal to 10 nm, and FcγRIII (V158; isotype with high affinity) EC50 or apparent Kd was less than or equal to 3 nm. In accordance with another embodiment, a decrease in the binding of an antibody to an Fc receptor relative to a reference antibody (e.g., antibodies Herceptin®) can be considered significant in relation to the control antibody, if the ratio of the absorption values at the midpoint of the curves of binding of the test antibody and control antibodies (for example, a450 nm(antibody)/A450 nm(control antibody)) less than or equal to 40%. In accordance with another embodiment, an increase in the binding of an antibody to an Fc receptor relative to a control antibody (such as antibody Herceptin®) can be considered significant in relation to the control antibody, if the ratio of the absorption values at the midpoint of the curves of binding of the test antibody and control antibodies (for example, a450 nm(antibody)/A450 nm(control antibody)) greater than or equal to 125%. See, for example, Example 16.

"The original polypeptide" or "original antibody is a polypeptide or an antibody comprising the amino acid sequence from which arose the modified polypeptide or antibody with which comparison is made of the modified polypeptide or antibody. Usually the original polypeptide or source of antibodies does not have one or more modifications plot Fc, described here, and differs from the modified polypeptide described here effector functions. The original polypeptide can include natural sequence plot Fc or a portion of a Fc with existing modifications of the amino acid sequence (such as joining, deletions and/or substitutions).

Antibodies in accordance with this invention can be obtained using phage display. Used here, the term "library" refers to the set of all sequences of the antibodies, or fragments of antibodies, or nucleic acids that encode these sequences, the sequences are differences in the combination of modified amino acids, which were introduced in this sequence in accordance with the methods of the invention.

"Phage display" is the method by which the variants of the polypeptides are displayed as fusion proteins as at least part of the envelope protein of phage particles, e.g., 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 antigen target with high affinity. The display of peptide and protein libraries on phage is used to test millions of polypeptides on the subject has the appropriate specific binding. Methods polyvalent phage display was used to demonstrate small random peptides and small proteins by fusing them or with the gene III or gene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992) and references cited therein. In a monovalent phage display protein or peptide library fused with the gene III or part thereof and is expressed at low levels in the presence of the gene III wild type, so that phage particles show a single copy or do not demonstrate fusion proteins. Compared with polyvalent phage avidity reduced, so is used to sort based on significant affinity for ligands, and famiglie vectors that facilitate the manipulation of DNA. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

"Fahmida" is a plasmid vector having a bacterial point of initiation of replication, such as ColE1, and a copy of the intergenic site of the bacteriophage. Fahmida can be used with any known bacteriophage, including filamentous bacteriophages and lambdoid bacteriophages. The plasmid also usually contains a token selection for antibiotic resistance. Segments of DNA, cloned in these vectors may be referred to as plasmids. When cells containing these vectors are supplied all genes necessary for the production of phage particles, method of replication of plasmids changes to replicas is the operation principle of the rolling rings for copies of one strand of plasmid DNA and packaging of phage particles. Family can form infectious and non-infectious phage particles. This term implies family containing the gene of phage envelope protein or fragment associated with the gene heterologous polypeptide so that the resulting gene fusion heterologous polypeptide is demonstrated on the surface ragovoy particles.

The term "phage vector" refers to double-stranded replicative form of bacteriophage containing a heterologous gene and is able to replicate. Phage the phage vector has a point of initiation of replication which allows replication of phage and education ragovoy particles. The phage is preferably filamentous bacteriophage, such as M13, fl, fd phage Pf3 or their derivatives, or lambdoid phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc, or their derivatives.

Covalent modification of polypeptides, such as Patiala, immunoadhesin, antibodies and short peptides that are included in the scope of this invention. One way of covalent modification includes carrying out the reaction of the target amino acid residues of the polypeptide with an organic modifying reagent capable of reacting with selected side chains or the N - or C-terminal residues of the polypeptide. Modification of bifunctional agents is useful, for example, to create cross-linking of the polypeptide is water-insoluble support matrix or surface for use in the method of purification of antibodies and Vice-versa. Commonly used cross-linking reagents include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, esters of N-hydroxysuccinimide, for example, esters with 4-azidoaniline acid, homobifunctional imidiately, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylester), bifunctional maleimide, such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propionamide.

Other modifications include deliciousa glutaminergic and asparaginase residues to the corresponding glutaminase and aspartame residues, respectively, hydroxylase of Proline and lysine, phosphorylation of hydroxyl groups serinovyh or travelover residues, methylation of the α-amino group lysine, arginine and his-tag side chains [T. E. Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, the amidation of any C-terminal carboxyl group.

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

Another type of covalent modification of polypeptides comprises linking the polypeptide to one or with a variety of nepateikiami polymers, such as polyethylene glycol (PEG), polypropyleneglycol or poly is calcimine, the method described in the U.S. patents№№ 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337.

The polypeptide in accordance with the invention may also be modified, if it will benefit, therefore, to form a chimeric molecule comprising a polypeptide fused to another, the heterologous polypeptide or amino acid sequence (for example, immunoadhesin or Patiala).

In one embodiment, such a chimeric molecule include the polypeptide is fused with a domain transduction protein, which directs the polypeptide to the delivery to various tissues, and more specifically through the blood-brain barrier, using, for example, the transduction domain TAT protein of human immunodeficiency virus (Schwarze et al., 1999, Science 285: 1569-72).

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

In an alternative embodiment, the chimeric molecule may include a polypeptide, fused with an immunoglobulin or a particular segment of the immunoglobulin. For a bivalent form of the chimeric molecule (for example, "immunoadhesin") such a merger can be carried out with a plot of Fc of IgG molecules. Fused Ig according to the invention include polypeptides that comprise approximately or only residues 94-243, residues 33-53, or residues 33-52 from a person instead of at least one of the variable segment in the Ig molecule. In a particularly preferred embodiment, a fusion immunoglobulin includes articulated in the Astok and plots CH2 and CH3, or the hinge area and the areas CH1, CH2 and CH3 of IgG1 molecules. Receive a sentence of immunoglobulins are also described in U.S. patent No. 5428130, from June 27, 1995

The invention provides methods and compositions for inhibiting or preventing relapse tumor growth or relapse cancer cells from spreading. In a different implementation, the cancer is a relapse tumor growth or relapse growth of cancer cells if the number of cancer cells is not significantly decreased, or increased, or the size of the tumor was not significantly decreased, or increased, or are unsuccessful attempt to further reduce the size or number of cancer cells. Determining whether the cancer cells relapse tumor growth or relapse growth of cancer cells, is carried out in vivo or in vitro by any method for evaluating the effectiveness of impact on cancer cells, are known in the art. The tumor is resistant to treatment against VEGF, is an example of a recurrence of tumor growth.

"Effective amount" of a polypeptide, antibody, antagonist or composition, described herein, corresponds to a quantity sufficient for a specific stated purpose. "Effective amount" may be determined empirically and by means of known methods, related to the stated purpose.

The term "therapeutically effective amount" refers to the number of antibodies, polypeptide or antagonist according to this invention, effective to "treat" a disease or disorder in a mammal (i.e. the patient). In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the size or weight of the tumor; to suppress (i.e. slow down to some extent or preferably stop) cancer infiltration into peripheral organs; inhibit (i.e., to slow down to some extent or preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more symptoms associated with cancer. To ensure that the drug could prevent growth and/or kill existing cancer cells, it must be cytostatic and/or cytotoxic. In one embodiment, therapeutically effective amount is the amount by overwhelming growth. In another embodiment, therapeutically effective amount is an amount that improves the survival of patients without progression. In another embodiment, therapeutically effective amount is the amount by which prolongs the life of the patient.

In the case of treatment of wounds, the term "effective amount" or "therapeutically effective amount" relates the I to the quantity of the drug, effective for speeding up or improving wound healing in the subject. therapeutic dose is a dose that demonstrates a therapeutic effect on the patient, and subtherapeutic dose is a dose, do not demonstrate a therapeutic effect on the patient undergoing treatment.

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, bedsores, trophic ulcers, etc. Acute wound can develop into a chronic wound. Acute wounds include, but are not limited to, wounds, caused, for example, thermal damage, injury, surgery, extensive destruction of skin cancer, deep fungal and bacterial infections, vasculitis, scleroderma, pemphigoid, toxic necrosis of the epithelium, etc. See for example, Buford, Wound Healing and Pressure Sores, HealingWell.com published: 24 October 2001. The term "conventional wound" refers to a wound that is exposed to normal 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, e.g., cancer of the o cells, both in vitro and in vivo. Inhibiting the growth of a number of polypeptide, antibody, antagonist or composition according to this invention for inhibiting the growth of neoplastic cells can be determined empirically or by known methods, or as in the examples.

The term "cytotoxic amount" of a polypeptide, antibody, antagonist or composition according to this invention refers to the amount that can cause destruction of cells, especially tumor, such as cancer cells, both in vitro and in vivo. 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 known in the field of engineering methods.

The term "autoimmune disease" means a disease or disorder, originating from its own tissues of the individual and directed against them or their joint manifestation or symptoms of, and resulting from this 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, and the infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, arthritis of the spine, and juvenile rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, arthritis deformans; primary chronic arthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as spotted psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiformis dermatitis, and atopic dermatitis, associated with the X chromosome syndrome increased IgM, urticaria such as chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic necrosis of the epidermis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS)such as Spino-optical MS, primary progressive MS, and MS relapsing-remitting course, progressive systemic sclerosis, atherosclerosis, arteriosclerosis, a disease Charcot-Vulpiani, and ataxic sclerosis, inflammatory bowel disease (IBD) (for example, Crohn's disease, colitis such as ulcerative colitis, microscopic colitis, collagenosis to whom it polypous colitis, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, syndrome of respiratory disorders in adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of any part of the choroid eyeball, Ericom, choroiditis, and autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE mediated diseases such as allergic, allergic and atopic rhinitis, encephalitis such as encephalitis of Ramussen and limbic encephalitis and/or encephalitis of the brain stem, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, agranulocytosis uveitis, valanchery uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with nephrotic syndrome or without it, such as chronic or acute glomerulonephritis such as primary GN, immunopositivity GN, membranous GN (membranous nephropathy), idiopathic membranous GN, membranous proliferative GN (MPGN), including type I and type II, and rapid progressive GN, allergic conditions, allergic reactions, eczema, R is allergic or atopic eczema, asthma, such as bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, chronic pulmonary inflammatory disease, autoimmune myocarditis, insufficient adhesion of lymphocytes of systemic lupus erythematosus (SLE) or systemic red volcanoe, such as cutaneous SLE, subacute systemic lupus erythematosus, lupus erythematosus neonatal (NLE)distributed lupus erythematosus, lupus (including neprinol, entsefalitnye, pediatric, non-renal, discoid, alopecias), juvenile diabetes (type I)diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM)diabetes mellitus in adults (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus; and immune responses associated with immediate hypersensitivity and delayed type, mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, including lymphomatoid granulomatous, granulomatous of Wegner, agranulocytosis, vasculitides, including vasculitis (including rheumatic primarliy and giant cell alterity (alterity Takayasu), vasculitis of medium vessels (including Kawasaki disease and Nowotny polyarthritis), microscopic arthritis, vasculitis of the Central nervous system, necroticism, skin and or allergic vasculitis, system necroticism vasculitis, and ANCA-associated vasculitis, such as vasculitis or syndrome Jurga-Strauss (CSS), temporal arthritis, aplastic anemia, autoimmune aplastic anemia, positive anemia Kumba autoimmune gemolitichesky anemia bulk thermal agglutinate, diamond-blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, Addison disease, true red cell anemia or aplasia (PRCA), a deficiency of factor VIII, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving diabetes leukocytes, inflammatory disorders CNS syndromes damage to multiple organs, such as secondary after sepsis, trauma or bleeding, diseases, mediated by a complex of antigen-antibody, a disease of the basal membrane of the glomeruli, the syndrome protivovospolitelnyh antibodies, allergic neuritis, Bechet disease and behceta, syndrome of Castellana, syndrome of Goodpaster, Raynaud's syndrome, Sjogren syndrome, and syndrome of Stevens-Johnson, pemphigoid, such as bullous pemphigoid and pemphigoid skin, hand, foot (including common bladderwort, the leaf bladderwort, pemphigoid mucous membranes and erythematous HSS is Ratko), autoimmune polyendocrinopathy, sickness or Reiter syndrome, immune complex nephritis, antibody-mediated jade, chronic neuropathy such as IgM neuropathy or IgM-mediated neuropathy, thrombocytopenia (developing, for example, in patients with myocardial infarction), including thrombocytopenic thromboembolitic purple (TTP) and autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP)including chronic or acute ITP, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polygranular syndromes such as autoimmune polygranular syndromes (or polygranular endocrinopathy syndromes), paraneoplastic syndromes, including neurological neuroplasticity syndromes, such as myasthenic syndrome Lambert-Eaton or the syndrome of Eaton-Lambert syndrome "hard man" or "hard person", encephalomyelitis, so the mi as allergic encephalomyelitis or encephalomyelitis allergica and eksperimentalnye allergic encephalomyelitis (EAE), malignant myasthenia gravis, spinal cerebellar degeneration, neuromyotonia, or needs to be opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, liposlim hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid intermediate pneumonitis, obliterating bronchitol (no transplant) or NVP (nonspecific intermediate pneumonia), Guillain-Barre syndrome, a disease Berger (IgA nephropathy), idiopathic IgA neuropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumoconiosis, autoimmune enteropathic syndrome, coeliac disease, celiac disease, abdominal sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophies lateral sclerosis (ALS, the disease Lou Gehrig's disease (als), coronary heart disease, autoimmune disease of the inner ear (AIED), or autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis, such as refractory or recidivistic polyhedric, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancer limfotsitoz, primary limfotsitoz, including monoclonal b-cell lymphocytosis (e.g., dobrokachestvenno is a monoclonal gammopathy and the monoclonal gammopathy unclear significance MGUS), peripheral neuropathy, paraneoplastic syndrome, canadatime, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis and canadatime CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune Hepatology disorder, fibromyalgia, multiple endocrine failure, syndrome Schmidt, adrenalitis, gastric atrophy, age-related dementia, demyelinizing diseases such as autoimmune demieliniziruyushchee disease, diabetic nephropathy, Dressler syndrome, alopecia areata, CREST syndrome (calcinosis, the phenomenon of Reynald, dysmotility of the esophagus, sclerodactyly and telangiectasia), male and female autoimmune infertility, mixed collagenosis, Chagas disease, rheumatic fever, recurrent abortion, "light farmer, erythema multiforme, post-cardiotoniceski syndrome, Cushing's syndrome, pulmonary allergic to poultry farmers, allergic granulomatosis anghit, benign lymph of anyit syndrome Alport, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, typanosomiasis shistosomiasis, ascaridiasis, aspergillosis syndrome Sumpter, syndrome Kaplan, dengue, endocarditis, endomyocardial fibrosis, pulmonary fibrosing-alveolitis, interstitially lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, congenital anemia of newborn, eosinophilic fasciitis, the syndrome, Shulman syndrome, still's, filariasis, cyclitol, such as chronic cycle, heterologous cycle, iridocyclitis or cycle Fuchs, purpura's disease-Shanina, infection with human immunodeficiency virus (HIV)infection echoviruses, cardiomyopathy, Alzheimer's disease, infection with parvovirus, a virus infection rubella, post-vaccination syndromes, congenital rubella infection, infection with Epstein-Barr, mumps, syndrome Evan, autoimmune deficiency sex glands, Korea Sydenham, post-streptococcal nephritis, obliterating thromboangiitis, thyrotoxicosis, the " dryness " of the spinal cord, chorioretinitis, the giant cell polymyalgia, ophthalmopathy, chronic allergic pneumonitis, dry keratoconjunctivitis, epidemic keratoconjuctivitis, idiopathic nephritic syndrome, nephropathy with minimal changes, benign family and ischemic reperfusion damage is observed autoimmune parsename retina, inflammation of the joints, bronchitis, chronic airway obstruction, silicosis, thrush, aphthous stomatitis, arteriosclerotic disorders, aspermatogenesis, autoimmune hemolysis, Beck disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylaxis endophthalmitis, allergic enteritis, type II lepromatosis reactions idiopathic facial palsy, chronic fatigue syndrome, rheumatic fever, a disease of Hamman-rich, sensoneural hearing loss, paroksizmalnoj hemoglobinuria, hypogonadism, regional eleita, radiation, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculoneuritis, pyoderma gangrenosum, subacute thyroiditis, acquired spencely atrophy, infertility due to the presence of antibodies against sperm, non-malignant thymoma, vitiligo, diseases associated with viruses, SCID and Epstein-Barr, acquired immunodeficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions involving infiltration of T cells, adhesion failure is aconito, immune responses associated with immediate hypersensitivity and delayed type, mediated by cytokines and T-lymphocytes, diseases involving diabetes leukocyte syndrome injuries to multiple organs, diseases, mediated by a complex of antigen-antibody diseases main antiglomerular membranes, allergic neuritis, autoimmune polyendocrinopathy, aforetime, primary myxedema, autoimmune atroficheskimi gastritis, sympathetic ophthalmia, rheumatic diseases, mixed collagenosis, nephrotic syndrome, insulite, polyendocrine failure, peripheral neuropathy, autoimmune polygranular syndrome type I, ideoptions hypoparathyroidism adults (AOIH), generalized alopecia, dilated cardiomyopathy, acquired bullous bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or negaunee sinusitis, acute or chronic sinusitis, epimedii, front, maxillary and openedmy sinusitis associated with eosinophils disorder such as eosinophilia, pulmonary infiltration eosinophilia syndrome eosinophilia-myalgia syndrome Leffler, chronic eosinophilic pneumonia, tropical legac the traveler eosinophilia, bronchopulmonary aspergillosis, aspergilloma or syndrome Kimura, anaphylaxia, seronegative spondylarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic skin and mucous candidiasis, Bruton syndrome, temporary infant hypogammaglobulinemia syndrome Wiskott-Aldrich, ataxia-telangiectasia, autoimmune disorders associated with collagen disorders, rheumatism, neurological disease, ischemic reperfusion disorder, response to low blood pressure, vascular dysfunction, angiectasia, wound tissue, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity, glomerulonephritis, reperfusion injury, reperfusion injury myocardial 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 induced by cytokines toxicity, acute inflammation, chronic intractable inflammation, peelite, pneumoconiosis, diabetic retinopathy, diabetic disorder of large arteries, intra-arterial hyperpl the Ziya, gastric ulcer and duodenal ulcer, valvulitis and endometriosis.

Methods of cancer treatment can be assessed by indicators, such as regression of a tumor, reduce the weight or size of the tumor, time to progression, survival time, survival without progression, the overall rate of response, duration of response, quality of life, expression and/or activity of the protein, but not limited to these values. Since anti-angiogenic drugs described here are aimed at the tumor vessels, and not necessarily on the neoplastic cells themselves, they represent a unique class of anti-cancer drugs and, thus, require unique criteria and definitions of clinical responses to drugs. For example, tumor shrinkage of more than 50% in 2-dimensional analysis is a standard boundary value to confirm the answer. However, antagonists alfaretta and antagonists of VEGF according to the invention can cause suppression of the spread of metastasis without reducing primary tumor or can simply call apocalittici effect. Accordingly, can be applied approaches to determining the effectiveness of therapy, including, for example, measurement of markers of angiogenesis in plasma or urine and measurement of the response method for radiological imaging.

Depending on the indications for treatment of faktorov, relevant to dosing with which the physician is a specialist in this area should be familiar, antibodies according to the invention will be introduced at a dosage which is effective for the treatment of symptoms while minimizing toxicity and side effects. For the treatment of cancer, autoimmune disease or immunodeficiency diseases a therapeutically effective dose can be, for example, in the range of 50 mg per dose to 2.5 g/m2. In one embodiment, introduced a dosage from about 250 mg/m2up to approximately 400 mg/m2or 500 mg/m2. In another embodiment, the dosage is approximately 250-375 mg/m2. In yet another embodiment, the range of dosages 275-375 mg/m2.

Methods of treating age-related macular degeneration (AMD) can be estimated, but are not limited to, a speed reduction or prevention of further loss of vision. For AMD treatment efficacy in vivo may be, for example, measured in one of the following ways: by estimating the average changes in best corrected visual activity (BCVA) from the main line within the specified time, the evaluation of the relative number of subjects who lost less than 15 characters visual activity in comparison with the main line for a certain period of time, evaluation of the Rel the relative number of subjects, acquired number of characters visual activity, more than or equal to 15 compared with the main line for a certain period of time, the evaluation of the relative number of subjects with equivalent visual activity Snellen 20/2000 over time, assessment using the questionnaire visual functioning NEI, evaluation of CNV size and the number of leaking CNV over time according to fluorescein angiography, etc.

The term "discovery" refers comprising determining the presence or absence of a substance or quantitative determination of the amount of substance. Thus, the term refers to the use of the materials, compositions and methods in accordance with the invention for qualitative and quantitative determinations. In General, the specific methodology used to determine, is not critical for the practical implementation of the invention.

For example, the "discovery" in accordance with the invention may include: observation of the presence or absence of the gene product alpha, mRNA molecules or polypeptide alpha; changes in the levels of the polypeptide alpha or number associated with the target; changes in biological function/activity of the polypeptide alpha. In some embodiments, the implementation of the "discovery" may include the detection levels of al the A5 wild type (for example, levels of mRNA or polypeptide). Detection may include quantitative determination of the change (increase or decrease) any value between 10% and 90%, or any value between 30% and 60%, or more than 100% compared to control. The determination may include a quantitative assessment of the change in any value from 2-fold to 10-fold, inclusive, or more, for example 100 times.

The term "label", when it is used here, refers to a detectable compound or composition that is conjugated directly or indirectly with the antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze which is detectable chemical change of the substrate, compound or composition.

New antibodies against alfaretta

Here the proposed new antibodies that can bind human alfaretta and competitively inhibit the binding of antibodies against alfaretta with human alfaretta. In accordance with one embodiment of antibodies against alfaretta produced by hybridomas selected from the group consisting of hybridoma deposited as Alpha5/beta1 7H5.4.2.8 (ATCC No. PTA-7421), and hybridoma deposited as Alpha5/beta1 7H12.5.1.4 (ATCC No. PTA-7420) in the ATCC on March 7, 2006, In accordance with another variant assests the tion, the antibody is produced by hybridoma selected from the group consisting of hybridoma deposited as Alpha5/beta1 7H5.4.2.8 (ATCC No. PTA-7421), and hybridoma deposited as Alpha5/beta1 7H12.5.1.4 (ATCC No. PTA-7420) in the ATCC on March 7, 2006, In accordance with another embodiment, the antibody contains a sequence of variable heavy (VH) and variable light (VL) domains of antibodies produced by hybridoma deposited as Alpha5/beta1 7H5.4.2.8 (ATCC No. PTA-7421) in the ATCC on March 7, 2006, In another embodiment, the antibody contains a sequence of variable heavy (VH) and variable light (VL) domains of antibodies produced by hybridoma deposited as Alpha5/beta1 7H12.5.1.4 (ATCC No. PTA-7420) in the ATCC on March 7, 2006, Also assumed the human and chimeric forms of the antibodies deposited hybridomas.

In accordance with one embodiment, the antibody binds to human alfaretta with a Kd between 500 nm and 1 PM. In accordance with another embodiment, the antibody does not bind V3, or V5, or V1. In accordance with another embodiment, the antibody comprises the sequence of the Fc of human IgG, e.g., human IgG1 or human IgG4. In another embodiment, the Fc sequence has been modified or changed in another way so that she lost effector f is NCLI antibody-dependent cellular cytotoxicity (ADCC), often associated with the binding to Fc receptors (FcR). There are many examples of changes or mutations in the Fc sequences, which can alter effector function. For example, WO00/42072 (Presta) and Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001) describe variants of antibodies with improved or weakened by binding to FcR. The content of these publications in a special way here by reference. The antibody may be in the form of Fab, Fab', F(ab)'2, single-chain Fv (scFv), Fv fragment; dyatel and a linear antibody. Also, the antibody can be multispecific antibody which binds Alfama and is an antagonist alfaretta, but also associates one or more other targets and inhibits their function (e.g., VEGF). The antibody may be conjugated to a therapeutic agent (e.g., a cytotoxic drug, a radioisotope and a chemotherapeutic drug) or a label for detection alfaretta in samples from the patient, or in vivo by imaging (e.g., radioisotope, fluorescent dye and enzyme).

Also provides for nucleic acid molecules encoding antibodies against alfaretta, expression vectors that include nucleic acid molecules encoding one or both of the variable domain, and cells comprising the nucleic acid molecule. These antibodies can be used in therapy, a description of the authorized here and for protein determination alfaretta in samples from patients (e.g., FACS, immunohistochemistry, tissues, analysis ELISA) or in the patients.

New combinations

New combinations to inhibit angiogenesis and/or vascular permeability in a subject suffering from diseases include antagonist Alfama and the VEGF antagonist. The VEGF antagonist and the antagonist alfaretta can be entered in simultaneous or consecutive cycles of treatment. Such a combined treatment is applicable for the treatment of diseases, including those diseases that are characterized by abnormal angiogenesis and/or vascular permeability and suffering which patients will benefit from antiangiogenesis therapy. Such diseases include, but are not limited to cancer, eye diseases and autoimmune diseases. Alternatively, the subject may be subjected to treatment with a VEGF antagonist, followed by the introduction of the antagonist alfaretta, for example treatment with VEGF antagonist to until the subject is no longer responsive to treatment with a VEGF antagonist, after which treatment the subject an antagonist alfaretta. In accordance with one embodiment, the patient is treated with the VEGF antagonist when the cancer was non-invasive and antagonist alfaretta when the cancer was invasive. Some patients who have elevated levels alfaretta iznachalno in response to treatment with VEGF antagonist relative to non-disease patients or control, may be particularly sensitive to this combinatorial treatment. It is also planned combination, additionally comprising a therapeutic drug (e.g., antineoplastics drug, a chemotherapy drug, which inhibits the growth of the preparation and cytotoxic drug). For example, patients who must undergo chemotherapy (e.g., irinotecan) and treatment with antagonists alfaretta or who have undergone chemotherapy and treatment with antagonists alfaretta can benefit from therapy with a VEGF antagonist. Alternatively, patients who have undergone chemotherapy and treatment with VEGF antagonists can benefit from therapy antagonist alfaretta. In one preferred embodiment, the anti-VEGF antibody is an antibody Avastin®. In another preferred embodiment, antibodies against alfaretta are described here by antibodies against alfaretta. Also supposed to be the sets containing the VEGF antagonist, the antagonist alfaretta and, optionally, a chemotherapeutic drug.

The pharmaceutical composition

Therapeutic compositions of the antibodies used in accordance with the present invention are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carrier and, fillers or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilised preparations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate or other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chloride hexadecane, benzalkonium chloride; chloride benzene; phenol, butyl or benzyl alcohol; alkalemia parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides with low molecular weight (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 carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya reverse ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants which, such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG). Illustrative compositions of the antibodies described in WO98/56418 directly here by reference. Liofilizovannye drugs intended for subcutaneous administration are described in WO97/04801. Such liofilizovannye drugs can be recovered using an appropriate solvent to high concentrations of protein, restored the compositions can be injected subcutaneously exposed here to the treatment of the mammal.

Described here, the composition may also contain more than one active connection that is necessary for the treatment of specific indications, where preferably the compounds have complementary activities that do not have undesirable actions on each other. For example, it may be desirable to also add a cytotoxic drug, a chemotherapeutic drug, a cytokine, or immunosuppressive drug (e.g., acting on T cells, such as cyclosporine or an antibody that binds to T-cells, for example, binding LFA-1). The effective amount of such other factors depends on the amount of antibody present in the composition, the type of disease or disorder treated, and other factors described above. They are usually used in the same dosages and with the use of the e pathways of introduction, as described above, or from about 1% to 99% used previously dosage.

The active ingredients may also be enclosed in microcapsules obtained, for example, using techniques koatservatsii or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymetylmetacrylate microcapsules, respectively, in colloidal systems drug delivery (for example, liposomes, albumen the microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsion. Such techniques are disclosed in Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Can be prepared medication slow release. Suitable examples of drugs slow release include a semi-permeable foundations of solid hydrophobic polymers containing the antagonist, these bases are made in form of objects, e.g. films, or microcapsules. Examples of the foundations for sustained release include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactide (U.S. patent No. 3773919) copolymers of L-glutamic acid and ethyl-L-glutamate, nadogradili ethylene-vinyl acetate-degrading copolymers of lactic and glycolic acids, such as LUPRON DEPOTTM(entered by the injection of microspheres consisting of a copolymer of lactic and Glick is left acids and acetate leuprolide) and poly-D-(-)-3-hydroxybutyrate acid.

Composition used for administration in vivo, must be sterile. This may be readily accomplished by filtration through sterile filtration membranes.

Products and kits

Another variant of the invention, this product contains materials that are applicable for the treatment of tumors, eye diseases and autoimmune diseases and related conditions. The product may include a container and a label or insert, inserted or associated with the container. Applicable containers include, for example, bottles, vials, syringes, and other Containers can be made of various materials, such as glass or plastic. Typically, the container holds a composition which is effective in treating the condition and may have a sterile access hole (for example, the container may be a bag for intravenous infusion or vial having a stopper penetrable by a needle hypodermic injector). At least one active ingredient in the composition is an antagonist of VEGF or antagonist alfaretta, or a VEGFR agonist, or agonist alfaretta in accordance with the invention. The label or the liner in the package will also contain instructions on the introduction of the composition of the antibody to the patient. The label or the liner may optionally contain instructions for the introduction of the compositions of antibodies to patients who NTU. Also refers to the products and kits, including combination therapy described herein.

Liners for packaging refer to the instructions that are usually included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, introduction, contraindications and/or warnings concerning the use of such therapeutic products. In one embodiment, the to the package insert indicates that the composition is used to treat non-jackinsky lymphoma.

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

Also offers kits for various applications, such as separation or detection alfaretta and/or VEGF in patients, possibly in combination with other products. For isolation and purification alfaretta the kit may contain antibodies against alfaretta related fields (for example, sivaratnam areas). Can be offered as kits comprising the antibodies for detection and the number is the result of evaluation alfaretta and/or VEGF in vitro, for example, in an ELISA or a Western blot. As in the case of product, the kit includes a container and a label or an insert in the packaging for the container or associated with it. For example, the container contains a composition comprising at least one antibody against alfaretta in accordance with the invention. May include additional containers containing, for example, solvents or buffers, control antibodies. The label or the liner in the package may contain a description of the composition, and instructions for intended use in vitro diagnostic use.

Monoclonal antibodies

Monoclonal antibodies can be obtained, for example, using methods based on the hybridomas, for example, as described by Kohler and Milstein, Nature, 256:495 (1975), or may be obtained using methods based on recombinant DNA (U.S. patent No. 4816567), or can be obtained by the methods described herein in the Examples section. In the method using hybridoma mouse, hamster, or other appropriate animal-owner usually immunities immunizing drug for stimulation of lymphocytes that produce or are capable of producing antibodies that specifically associated with immunizing drug. Alternatively, the lymphocytes may be immunized in vitro.

Immunizing preparation will typically be on the part of the polypeptide or fusion protein of the protein of interest or a composition including protein. Usually, used or peripheral blood lymphocytes (PBL), if required cells of human origin, or used cell spleen or lymph node, if the source is desirable mammals, non-human. Then lymphocytes merge with immortalizing cell line using a suitable preparation for the merger, such as polyethylene glycol, to form cells hybridoma. Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press, 1986), pp. 59-103. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells, originating from rodents, cows or humans. Commonly used rat and mouse myeloma cells. Cell hybridoma can be cultured in a suitable medium cultivation, which preferably contains one or more substances that inhibit the growth or survival unmerged immortalized cells. For example, if the original cells lacking the enzyme gipoksantin guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain gipoksantin, aminopterin and thymidine (Wednesday HAT), these substances prevent the growth of cells with deficiency of HGPRT.

The preferred lines immortalized cells are capable of effective in order to merge, support stable high level expression of antibody selected producing antibodies cells and is sensitive to the environment, such as environment HAT. More preferred termed the cell lines are murine lines myeloma, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California, and American Type Culture Collection, Manassas, Virginia. Was also described the production of human monoclonal antibodies in cell lines of human myeloma and mouse-human heteromyinae. Kozbor. J. Immunol., 133:3001 (1984): Brodeur et al. Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.

Cultural environment in which were cultivated cells hybridoma, can be tested for the presence of monoclonal antibodies directed against the polypeptide. The binding specificity of monoclonal antibodies produced by the hybrid cells may be determined by the method thus or with the assistance of research associate in vitro, such as radioimmune assay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such methods and assays known in the art. Affinity binding of monoclonal antibodies can be determined, for example, analysis of Scatchard by Munson and Pollard, Anal. Biochem. 107:220 (1980).

After identifying the desired cells hybridoma clones can be subcloned from the SIP is utilized procedures serial dilution and grown using standard methods. Cm. previously, Goding. Suitable environment cultivation for this purpose includes, for example, the modified Dulbecco Wednesday Needle and medium RPMI-1640. Alternatively, cells hybridoma can be grown in vivo as ascites mammals.

Monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascitic fluid using normal procedures purification of immunoglobulins, such as, for example, chromatography on sepharose with protein And, hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be obtained by using methods of recombinant DNA, such as described in U.S. patent No. 4816567. DNA encoding the monoclonal antibodies according to the invention can be quickly isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). Cell hybridoma according to the invention are the preferred source of such DNA. Being isolated, the DNA may be placed into expression vectors, which are then transfairusa cell host, such as cells COS monkey cells Chinese hamster ovary (Cho or myeloma cells that do not otherwise PR is doziruut immunoglobulin protein, to achieve the synthesis of monoclonal antibodies in the recombinant cell host. DNA can be modified, for example, by substitution of sequences encoding the human constant domains of the heavy and light chain of the homologous murine sequences (U.S. patent No. 4816567; Morrison et al., above) or by covalent binding to immunoglobulin coding sequence all or part of the coding sequence of the peptide, non-immunoglobulin. Such non-immunoglobulin polypeptide can be substituted for the constant domain according to the invention or can be substituted for the variable domains antigennegative site antibodies in accordance with the invention to create a chimeric bivalent antibody.

Antibodies may be monovalent antibodies. Methods of producing a monovalent antibody known in the art. For example, one method involves recombinant expression of light chain immunoglobulin and a modified heavy chain. A heavy chain is usually truncated at any point in the plot of Fc in order to prevent cross-linking the heavy chains. Alternatively, important cysteine residues are substituted with other amino acid residues, or are deleted to prevent cross-linkage.

To obtain monovalently antibodies applicable methods in vitro. Cleavage of the antibodies for their fragments, particularly fragments Fab, can be performed using, but not limited to methods known in the art.

Human and humanized antibodies

Antibodies can be humanitarianism antibodies or human antibodies. Humanized forms of inhuman (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments (such as Fv, Fab, Fab', F(ab')2or other antigennegative subsequences of antibodies)which usually contain minimal sequence derived from nonhuman immunoglobulin. Humanized antibodies include human immunoglobulins (antibodies-recipients), in which residues of a CDR of the recipient are replaced by residues from a CDR of nonhuman species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity, and capability. In some cases, the base of the skeleton of the Fv of the human immunoglobulin are replaced by corresponding inhuman remnants. Humanized antibodies may also include residues that do not occur in the antibody-recipient nor borrowed the sequences of the CDR or framework. Typically, humanized antibodies can include basically the sun is out, at least one, and typically two, variable domains, in which all or substantially all areas CDR match analogues nonhuman immunoglobulin and all or substantially all of the FR plots correspond to the counterparts of the consensus sequence of human immunoglobulin. Humanized antibodies preferably will also include at least part of the constant part of the immunoglobulin (Fc), typically a 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 ways to humanitarian nonhuman antibodies described in the art and below in the examples. Usually, humanitariannet antibody has one or more of the included amino acid residues from a source, not a person. These inhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. In accordance with one embodiment humanization can be mainly performed by the method of winter and co-workers (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 substituting rodent CDR or CDR sequences corresponding to sequences in the human antibody. Accordingly, such "humanized" anti-Christ. ate are antibodies (U.S. patent No. 4816567), which is significantly smaller than a human variable domain, are replaced by the corresponding sequence from nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues substituted by residues from analogous sites in rodent antibodies.

Alternatively, humanization can be obtained from human antibodies. For example, currently it is possible to produce transgenic animals (e.g. mice)that are capable, after immunization, of producing a full set of human antibodies in the absence of endogenous production of immunoglobulin. For example, it has been described that the homozygous deletion in the gene for the wiring from the heavy chain (JH) in chimeric mice and mutant germline of mice results in complete inhibition of endogenous production of antibodies. The transmission of a set of human genes germline immunoglobulin in this mouse mutant germ line leads to the production of human antibodies upon antigen 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); U.S. patent No. 5545806, 5569825, 5591669 (all owned by GenPharm); 5545807; and WO 97/17852. Alternatively, human antibodies can be obtained by introducing lococo the human immunoglobulin in transgenic animals for example, mice in which the endogenous immunoglobulin genes partially or completely inactivated. When stimulation was observed production of human antibodies, much like seen in humans in all respects, including gene rearrangement, Assembly, and presentation of the repertoire of antibodies. This approach is described, for example, in U.S. patents№№ 5545807; 5545806; 5569825; 5625126; 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, to obtain human antibodies and fragments of antibodies in vitro from the repertoire of genes variable domains (V) of immunoglobulins from unimmunized human donors can be used the technology of phage display (McCafferty et al., Nature 348:552-553 [1990]). In accordance with one embodiment of this method, a sequence of domain V antibodies are cloned maintaining the frame in a gene of large or small envelope protein of filamentous bacteriophage, such as M13 or fd, and presented in the form of functional fragments of the antibodies on the surface ragovoy particles. Phage display can be performed in a variety of formats, for example, as described below in the Examples section, or as considered, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Some sources of gene segments V can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) identified a broad set anticatholic antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Can be constructed set of V genes from unimmunized human donors, after which antibodies against a wide range of antigens (including its own 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 U.S. patent No. 5565332 and 5573905.

As discussed earlier, human antibodies can also be produced activated in vitro B-cells (see U.S. patent 5567610 and 5229275).

Human antibodies can also be produced using various methods known in the field of engineering, including libraries of phage display. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also available methods of producing human monoclonal antibodies Cole et al. 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

Multispecificity antibodies are monoclonal, preferably human or humanitarianism, antibodies that have specificity for the binding is of two or more different antigens (e.g., bespecifically antibodies have binding specificity to two or more different antigens). For example, one of specificdate binding can be as antibodies against Alfama, the other can be any other antigen. In accordance with one preferred embodiment the other antigen is a protein on the cell surface, the recipient or a subunit of the receptor. For example, a protein found on the cell surface may be cell receptor natural cell killer. Thus, in accordance with one embodiment, bespecifically antibody according to the invention can bind alfaretta and VEGF.

Have been described examples of methods of manufacturing bespecifically antibodies. Traditionally, the recombinant getting bespecifically antibodies based on simultaneous expression of two pairs of heavy/light chain immunoglobulin, where the two heavy chains have different specificity. Milstein and Cuello, Nature, 305: 537-539 (1983). Because of the random set of heavy and light chains of immunoglobulin, these hybridoma (quadroma) potentially produce a mixture of ten different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule is usually performed with the use of stages affinity chromatography. A similar procedure is disclosed in WO 93/0882, published 13 may 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

The variable domains of the antibodies with the desired binding specificity (lots of connections antigen-antibody) can be merged with the sequence of the constant domain of immunoglobulin. Preferably, the merge is performed with a constant domain of a heavy chain immunoglobulin comprising at least a portion of the hinged section and sections CH2 and CH3. Preferably, the first constant section of the heavy chain (CH1)containing the site necessary for binding to the light chain was present in at least one product of the merger. DNA encoding fused heavy chain immunoglobulin and, if required, light chain immunoglobulin, are inserted into separate expression vectors and co transfairusa in a suitable organism, the host. For a more detailed description of obtaining bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

Also described various methods of making and isolating bespecifically fragments of antibodies directly from a culture of recombinant cells. For example, bespecifically antibodies can be obtained using latinovich zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). Lacinova peptides-clasps of proteins Fos and Jun were associated with Fab fragments' of two different antibodies by t the s gene. Homodimeric antibodies were recovered at the site of the hinge to form monomers and then re-oxidized to education heterodimeric antibodies. This method can also be used for the production of homodimeric antibodies. Technology "datel"described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)has proposed an alternative mechanism for making bespecifically fragments of antibodies. The fragments contain VH connected to VL through a linker that is too short to allow pairing of the two domains of a single chain. Accordingly, the domains VH and VL of one fragment are forced to pair with complementary domains VL and VH of another fragment, thereby forming two antigenspecific plot. Was also described another strategy making bespecifically fragments of antibodies through the use of single-chain Fv dimers (sFv). Cm. Gruber et al., J. Immunol., 152:5368 (1994).

Also discusses antibodies with more than two valencies. For example, can be obtained thespecification antibodies. Tutt et al. J. Immunol. 147: 60 (1991).

Heteroconjugate antibodies

Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have, for example, proposed to target immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection. WO91/00360; WO 92/200373; EP 03089. It is assumed that antibodies can be obtained in vitro using methods known in the chemistry of synthetic proteins, including methods involving cross linking agents. For example, immunotoxins can be constructed using the exchange reaction of the disulfide or by forming a thioester linkages. Examples suitable for this purpose reagents include minotola and methyl-4-mercaptopyrimidine disclosed, for example, in U.S. patent No. 4676980.

Creating effector functions

It may be desirable to modify the antibody according to the invention in respect of its effector functions to improve, for example, the effectiveness of the antibodies in the treatment of cancer. For example, in the plot of Fc can be entered one or more cysteine residues, thus making it possible education in this area disulfide bonds between the chains. Thus obtained homodimeric antibodies may have improved the ability to internalize and/or increased complement-mediated cell lysis and antibody-dependent cellular cytotoxicity (ADCC). Cm. Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be obtained by applying heterobifunctional cross-links, as described in Wolff et al., Cancer Research. 53: 2560-2565(1993). Alternatively, it may be constructed of antibodies that have dual Fc plots and can thus have an increased ability to lysis with the participation of complement and ADCC. See, Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

To improve binding to FcR (for example, FcgammaR, FcRn) can be carried out mutation or sequence plot Fc. In accordance with one embodiment, the antibody according to this invention has at least one changed compared to native IgG or the original antibody effector function selected from the group consisting of ADCC, CDC and improved binding to FcRn. Examples of some useful specific mutations described, for example, Shields, RL et al. (2001) JBC 276(6)6591-6604; Presta, L.G., (2002) Biochemical Society Transactions 30(4):487-490; and publication WO00/42072.

In accordance with one embodiment, the mutation of the Fc receptor 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 section Fc in the EU numbering system.

Immunoconjugate

The invention also relates to immunoconjugates, including antibody conjugated with a cytotoxic drug, so is AK a chemotherapy drug toxin (for example, having the enzymatic activity of the toxin of bacterial, fungal, plant or animal origin, or their fragment), or a radioactive isotope (for example, radioconjugates).

Chemotherapeutic drugs used in the creation of such immunoconjugates were described above. Having the enzymatic activity of the toxins and fragments thereof that can be used include diphtheria A-chain, neisvaziuosiu active fragments of diphtheria toxin a chain of endotoxin (from Pseudomonas aeruginosa), a chain of ricin, a chain abrina, chain And modeccin, alpha-Scrin, proteins Aleurites fordii, diantimony proteins, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), the inhibitor from Momordica charantia, Curtin, krotin, inhibitor of Sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. For the production of radioconjugates antibodies are available a variety of radionuclides. Examples include212Bi131I131In90Y and186Re.

Conjugates of the antibody and cytotoxic drug are manufactured using a variety of bifunctional connecting with protein substances, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as hydrochloride of dimethylpiperidino), active esters (such as disuccinimidyl suberate), and degidi (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)-Ethylenediamine), diisocyanates (such as (talien 2,6-diisooctyl), and bis-active fluorine compounds (such as 1,5-Diptera-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., Science. 238: 1098 (1987). Labeled with carbon-14 1-isothiocyanatobenzene-3-methyldiethanolamine acid (MX-DTPA) is an example of chelating compounds for conjugation of radionucleotide with the antibody. See, WO94/11026.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in pre-targeting the tumor when the patient is injected conjugate antibody-receptor, then the unbound conjugate is removed from the circulation using a cleansing product, then introduces the "ligand" (e.g. avidin)that is conjugated with a cytotoxic drug (e.g., radionucleotide).

Immunoliposome

Antibodies disclosed in this invention can also be manufactured in the form of immunoliposome. Liposomes containing the antibody produced by methods known in the art, such as 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 patentable No. 4485045 and 4544545. Liposomes with enhanced circulation time is described in U.S. patent No. 5013556.

Practice liposomes can be obtained by the method of reverse phase evaporation with the use of a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivative of phosphatidylethanolamine (PEG-PE). Liposomes forced through filters with defined pore size to obtain liposomes with the desired diameter. Fragments Fab' antibody according to the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982), by the reaction of the disulfide exchange. The liposome may contain chemotherapeutic drugs (such as doxorubicin). Cm. Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).

The pharmaceutical compositions of the antibodies and polypeptides

Antibodies specifically bind to the polypeptides identified herein, as well as other molecules identified by screening the results of the research described in this invention can be prescribed to treat various disorders, as described above and below in the form of pharmaceutical compositions.

Lipofectin or liposomes can be used for delivery of polypeptides or antibodies or compositions in accordance with this invention into cells. When using fragments of antibodies, predpochtite the flax smallest inhibitory fragment, which specifically binds to the binding domain of the protein target. For example, on the basis of sequences of the variable segment antibodies can be designed peptide molecules that retain the ability to bind the protein sequence of the target. Such peptides can be chemically synthesized or obtained by the use of recombinant DNA technologies, see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The composition described herein may also contain more than one active connection, if it is necessary to treat specific symptoms, preferable are compounds having a complementary activity which does not exert undesirable influence on each other. Alternatively, or in addition, the composition may contain a drug that increases the action, such as, for example, a cytotoxic drug, a chemotherapy drug, or growth-inhibitory drug. Accordingly, these molecules are present in the composition in amounts that are effective for the intended purpose.

The active ingredients may also be enclosed in microcapsules obtained, for example, using techniques koatservatsii or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymetylmetacrylate microcapsules, sootvetstvenno is, in colloidal systems drug delivery (for example, liposomes, albumen the microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences, see above.

Composition used for administration in vivo, must be sterile. This may be readily accomplished by filtration through sterile filtration membranes.

Can be prepared medication slow release. Suitable examples of drugs slow release include a semi-permeable foundations of solid hydrophobic polymers containing the antibody, these bases are made in form of objects, e.g. films, or microcapsules. Examples of the foundations for sustained release include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactide (U.S. patent No. 3773919) copolymers of L-glutamic acid and γ ethyl-L-glutamate, nadogradili ethylene-vinyl acetate-degrading copolymers of lactic and glycolic acids, such as LUPRON DEPOTTM(entered by the injection of microspheres comprising a copolymer of lactic and glycolic acids and acetate leuprolide) and poly-D-(-)-3-hydroxipropionic acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid, is capable of releasing molecules is for more than 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denaturing or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved may be a rational strategy for stabilization. For example, if it is found that the mechanism of aggregation is the formation of intermolecular S-S linkages due thio-disulfide mutual interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, use of appropriate additives, and developing specific polymer compositions of the matrix.

Diagnostic use and visualization

Labeled antibodies, and derivatives and analogs that specifically bind to the polypeptide, can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with expression of bias, the expression and/or activity of the polypeptides in accordance with the invention. In accordance with one preferred embodiment, antibodies in accordance with Yes the NYM invention can be used in diagnostic studies or visualizeus research involving antibodies to a subject. The invention provides a method of detecting abnormalities in the expression of VEGF polypeptide or alfaretta, including (a) the study of the expression of the polypeptide in the cells (e.g., tissues) or bodily fluids of an individual using one or more antibodies in accordance with this invention and (b) comparing the level of gene expression with a standard expression level of the gene, where the increase or decrease in the level of expression of the test gene compared to the standard expression level is indicative in respect of the variance expression.

Antibodies in accordance with this invention can be used for studies of protein levels in a biological sample using classical immunological methods known to experts in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). The other is based on antibody detection methods gene expression of protein include immunological tests, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis (RIA). Suitable for analysis using the antibody labels known in the field of engineering and include enzyme labels, such as glucose oxidase; radioisotopes, such as iodine (131I125I123I121I), carbon (14C), sulfur (35S), tritium 3H), indium (115mIn113mIn112In111In), and technetium (99Tc99mTc), thallium (201Ti), gallium (68Ga67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F),153Sm177Lu,159Gd149Pm,140La,175Yb,166Ho,90Y47Sc,186Re,188Re,142Pr105Rh,97EN; luminal; and fluorescent labels, such as fluorescein, and rhodamine, and Biotin.

For labelling of antibodies according to the invention can be applied is known in the field of engineering methods. Such methods include, but are not limited to, the use of bifunctional conjugating drugs (see, for example, U.S. patents№№ 5756065; 5714631; 5696239; 5652361; 5505931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; and 5808003; the contents of each of which are hereby incorporated by reference in full.

Diagnosis of a disease or disorder associated with expression or abnormal expression of VEGF and/or alfaretta an animal, preferably a mammal and most preferably a human, may include the discovery stage molecules alfaretta and/or VEGF in macpicasso. In one embodiment, after administration of the VEGF antagonist diagnosis includes (a) introduction (for example, parenterally, subcutaneously or intraperitoneally) to a mammal an effective amount of labeled antibodies against alpha ETA; (b) waiting for a time interval after injection to allow the labeled molecules mainly focus on sites in the subject where the molecule is expressed alfaretta (and allow the concentration of unbound labeled molecule to be reduced to background level); (c) determining background level; and (d) determining the labeled molecule in the subject, and the detection of labeled molecule in the subject above the background level indicates that the subject had a disease or disorder associated with expression or aberrant expression alfaretta. The background level can be determined using various methods, including comparing the number of detected labeled molecule with a standard value previously determined for a particular system.

In accordance with one specific embodiment, expression or overexpression of a polypeptide Alfama is determined in a diagnostic or prognostic studies after administration of a therapeutic compound is an antagonist of VEGF by assessing levels alfaretta present on the cell surface (for example, by immunochemical studies using antibodies against alfaretta). Alternative or additionally, it is possible to measure the levels encoding the polypeptide alfaretta nucleic acid or mRNA in principle this is Kyo, for example, using fluorescence in situ hybridization using based on the nucleic acid probe corresponding to the coding alfaretta nucleic acid or the complement her (FISH; see WO98/45479 published October, 1998), southern blot, Northern blot, or methods polymerase chain reaction (PCR), such as quantitative real-time PCR (RT-PCR). You can also explore an excessive expression alfaretta by measuring the "exfoliating" antigen in biological fluids such as serum, e.g, using assays based on antibodies (see also, for example, U.S. patent No. 4933294, issued June 12, 1990; WO91/05264 published April 18, 1991; U.S. patent 5401638, issued March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above analyses, a professional practitioner available in various in vivo studies. For example, you can expose cells to a mammal processing antibodies, which probably marked detectable label, e.g. a radioactive isotope, then the binding of the antibody with the cells of a mammal can be assessed, for example, by an external scan of radioactivity or by analyzing a biopsy taken from a mammal previously treated with antibodies.

All publications (including patents and patent applications)cited here, put this mod is contained in its entirety by reference, including in particular, the provisional application U.S. No. 60/784704, filed on March 21, 2006, provisional application U.S. No. 60/785330 filed March 22, 2006, and provisional application U.S. No. 60/871743, filed December 22, 2006

The following DNA sequences were deposited on the terms of the Budapest Agreement in the American type culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA, as described below:

MaterialRoom DepositDate Deposit

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

The deposition was carried out on the conditions of the Budapest agreement on the international recognition of deposits of microorganisms for purposes of patent procedure and Regulations on the basis of this agreement (the Budapest Treaty). This assures maintenance of a viable culture of the Deposit for 30 years from the date of Deposit. The deposits were made available by the ATCC on the terms of the Budapest agreement and shall be subject to agreement between Genentech, Inc. and ATCC, to the / establishment, which assures permanent and unrestricted availability of the descendants of the culture of the Deposit to the company after the issue of the relevant U.S. patent or since the publication of any patent application, U.S. or foreign, depending on what comes first, and assures availability of the progeny to have the right of access defined by the Commissioner of patents and trademarks in accordance with 35 U.S.C. 122 and its corresponding regulations of the Commissioner (including 37 C.F.R. 1.14 with special reference to 886 OG 638).

Authorized by representing this application has agreed that if a culture or material Deposit will die or be lost or destroyed in the process of cultivation under reasonable conditions, the materials will be quickly replaced by notice to the other the same. Availability of the deposited material should not be construed as a license to use the invention in violation of the rights given by the authority of any government in accordance with its patent laws.

Commercially available reagents referred to in the Examples were used according to manufacturer's instructions, unless otherwise indicated. Source of cells indicated in the following examples and in the specification of the access numbers ATCC is the American type culture Collection, Manassas, VA. Unless otherwise stated, the present invention uses standard procedures of recombinant DNA technology, such as those described herein above and in the following manuals: Sambrook et al., above; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiey 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 the claims the word "include" or variations such as "includes" or "including"shall be understood to mean the inclusion of the specified integer or group of integers but not the exclusion of any other integer or group of integers.

The preceding text description is sufficient to enable a person skilled in the art to carry out the invention in practice. The following Examples are offered only for illustrative purposes and not meant limiting the scope of the presented invention in any way. Moreover, various modifications of the invention in addition to the shown and described it will be obvious to experts in the art from the preceding description and fall within the scope of the attached claims.

Examples

Example 1. Restore populations expressing alfaretta stroma cells after therapy against VEGF

Sections of xenografts of human colorectal carcinoma HT-29, which were subjected to monotherapy with anti-VEGF antibody B20-4.1 in Nude mice,were stained for the expression of antibodies against alfaretta. Compared with the control group treated with the control antibody (antibody against ragweed), in this study, monotherapy B20-4.1 gave median time to endpoint (TTE), which corresponded to absent or minimal activity. Tumors were measured twice a week for 58 days. Animals were euthanized when their tumors reached a volume of endpoints 1000 mm3or on day 58, depending on what happened before, then (TTE) was calculated for each mouse. Treatment outcome was determined by the percentage of delay of tumor growth (%TGD), defined as the percentage increase in average TTE in the treated mice compared with the control, differences were considered significant at the 0.01 ≤ P ≤ 0,05, and highly significant at P ≤ 0.01 by analysis of Lagrange. The average value of the TTE of the control group was 20.6 days. Treatment monotherapy B20-4.1 gave the average value of TTE 20.1 days, which corresponds to no activity.

The figure 1 shows a section of the tumor, stained with antibodies against alfaretta. Observed increased recovery of populations of stromal cells after treatment against VEGF. These stromal cells were positive for the integrin alfaretta (light green staining).

Example 2. Antibodies against al abeta

Mice were introduced purified human alfaretta (Chemicon CC1027). Were selected cancer cells expressing antibodies against alfaretta and transformed cell lines hybridoma. Two cell lines, hybridomas, designated 7H5.4.2.8 and 7H12.5.1.4, were placed in the ATCC, see above. Antibodies obtained from hybridoma 7H5.4.2.8 was mIgG2a antibody Kappa (also referred to here as "antibody 7H5"). Antibodies obtained from hybridoma 7H12.5.1.4 was mIgG2b antibody Kappa (also referred to here as "antibodies 7H12").

Example 3. Studies on direct binding to HUVEC cells

Tissue culture containing growing human cells of the umbilical vein endothelium (HUVEC)were washed twice FSB. Cells were separated from the culture vial using 3-4 ml of 5 mm EDTA/FSB. Cells were mixed with fresh medium for cultivation. It was determined the number of cells in the aliquot of the mixture. 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 seeded in 25 µl per well on 96-well tablets MSD high bind in the amount of 25,000 cells per well or 4000 cells per well in 384-well plate (Cat #L11XB-1 or #L11XB-2, respectively, Meso Scale Diagnostics, LLC). Cells were incubated for 1 hour at room the th temperature for to capture. To block the wells in the well was added 25 μl of the underlying buffer (30% fetal calf serum (FBS) in TBS (50 mm Tris, 150 mm NaCl +1 mm CaCl2/1 mm MgCl2, pH 7.5), after which the plate was incubated at room temperature for 30 minutes to 1 hour.

There was prepared a series of dilutions of antibodies against alfaretta buffer for analysis (TBS with 1 mm CaCl2/1 mm MgCl2pH of 7.2 + 2-4% FSB) for various concentrations of antibodies. The wells were washed twice with buffer for washing, after which the liquid residue removed using absorbent material. Well was added 25 μl of the dilutions of antibodies, after which the tablet incubated on ice for one hour. Then the wells were three times washed with TBS.

25 μl of the solution xmuFc-sulfo-tag with a concentration of 0.5 μg/ml was added to each well, after which the tablet incubated on ice for 45 minutes to one hour (xmuFc-sulfo-tag is an antibody goat against mouse IgG, catalog number R23-AC-5). Added MSD-SA-tag (catalog number R32-21-AD-5) and incurable on ice for 45 minutes to 1 hour. The wells were three times washed with TBS. To each well was added 150 μl of 2x buffer to read (4x buffer for reading MSD, divorced dH2O to 2, No. R92TD-1 (without surfactants)). Subsequent electroluminescent (ECL) signal was measured with photoshopadobe and expressed in relative light units using a scanner MSD Protocol (default 6000). The figure 2 shows the results of the research on direct binding to HUVEC cells. EC50 antibody 7H5 amounted to 0.22 nm. EC50 antibodies 7H12 amounted to 0.38 nm.

Example 4. Research FACS antibodies against alfaretta

Antibodies 7H12 or 7H5 were or incubated with RAJI cells (cell line, which does not expresses mRNA alfaretta)or HUVEC cells (cell line, which expresses high levels of mRNA alfaretta) in 100 μl. The bound cells were detected using conjugated secondary antibodies. On figure 3 is shown by FACS analysis that 7H12 and 7H5 contact HUVEC cells, but not with RAJI cells. Using the same methods synoviocyte rabbit (HIG-82) or cells of the macaque-rhesus (CL-160 fibroblasts Macaca mulatta or CRL-1780 from endothelial cells of the retina), we observed binding 7H12 and 7H5 with cells of the rabbit or monkey.

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

Fibronectin (Sigma F1141 (bullish) or Roche 1080938 (human)) was diluted to 1 μg/ml in sodium carbonate buffer. In 96-well plates NUNC maxisorp was added 100 μl of a solution of fibronectin per well, then the plate was left to bind at 4°C over night ((96-hole immunological tablets NUNC MaxiSorp N/Ster 439454 (VWR 62409-002)). Then the wells were washed with phosphate-saline buffer (FSB) and zablokirovany% BSA (Sigma A9418) for at least 30 minutes. Then the tablets were rinsed three times the FSB. To each well was added 20,000 HUVEC cells, followed by incubation with different concentrations 7H5 or in 7H12 medium for growth, containing 1.4 mm MgCl2and 1.4 mm CaCl2. Then the incubation mixture was added to coated with fibronectin tablet. Approximately 20,000 cells in the same environment for growth were added to each control well without adding inhibitory antibodies.

The tablets were centrifuged for 5 min at 140 g for synchronization contact of cells with the substrate. Cells were incubated in CO2incubator during different periods of time (from 0 to 120 min). The incubation time varied for each cell line. Then the tablets were washed FSB three times. All liquid was removed from the cells, after which the plate was frozen at -80°C. the Tablets were ottani at room temperature. To the wells was added to the buffer CyQuant (Molecular Probes CyQuant C7026), after which the plate was incubated at room temperature for 10 minutes Was measured OD. The figure 4 shows that the IC50 antibody 7H5 0.85 µg/ml (3,44 nm)and IC50 antibody 7H12 was 0.7 µg/ml (of 4.38 nm).

Example 6. Studies of reproduction using HUVEC cells

96-well tablets were coated with fibronectin (1 μg/ml) over night. Then the plate was washed FSB. In each of the 6 holes were added 3000-5000 endothelial cells (EC), then the plate was left to flow full attachment of cells to the wells. Were added antibodies against alpha (including control isotypes). For each condition was used 3 holes. Then cells were incubated with antibodies for 1-24 hours. Antibodies against integrin alfaretta were tested at several concentrations (for example, 0 μg/ml, 4 μg/ml 16 μg/ml, 60 μg/ml to 120 mcg/ml).

Then cells were BrdU labeled by incubation with 2 ál of BrdU solution (25 mg/ml in the FSB) in 1 ml of medium for tissue culture (EGM2 + all the extra nutrients from Clonetics (catalog No. CC-4176). After this incubation, the cells were fixed with 4% PFA, treated with a 1 N. HCl for 20 min, washed several times the FSB, and then blocked with 10% goat serum (FSB with 0.2% Triton) for 1-2 hours. Then cells were stained with monoclonal antibodies against BrdU (catalog No. BD 347580, 1:40) FSB with 0.2% Triton and 5% goat serum), and then incubated overnight at 44°C. the next day cells were washed FSB 3 times and incubated with conjugated with Alexa-594 secondary antibodies against rabbit (1:800) at room temperature in the dark for 4 hours. The wells were again washed and incubated with DAPI (1:10000 in the FSB) within 10 minutes After the last washing of the FSB, the total number of cells per well was counted potentiative staining with DAPI 5x. Cells that were positive for BrdU, the same sections were photographed using a red filter. Reproduction was assessed as the percentage of cells positive for BrdU on the site. The results were then analyzed using the Excel program. Figure 5A shows the total number of HUVEC cells 32 hours when the initial amount of 5000 cells. Figure 5B shows the total number of cells HUVEC after 24 hours when the antibody concentration of 20 μg/ml

Example 7. The study Protocol migration

The HUVEC cells were grown to the formation of a solid layer in EGM2 with the addition of all the extra nutrients from Clonetics (catalog No. CC-4176) on 24-hole tablets coated with 5 μg/ml fibronectin. Cells in the center of each well were erased using 2 ál pipette tip, removed by scraping cells were washed away. In the different wells was added to the medium for culturing the cells with a control antibody 7H5 or 7H12. All tested antibodies were used with a concentration of 20 µg/ml Then the cells were allowed to grow for 1 to 2 days. For the traumatized area observed. The figure 6 shows the photograph of the migration of HUVEC cells at 5 µg/ml fibronectin with 20 μg/ml of antibodies against alpha (7H5) ECM-2 at 0 hours and 30 hours. Figure 7 is a diagram % migrat and 30 hours for cells, treated with antibody 7H5 or 7H12.

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

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

The next day in one set of holes, the medium was replaced by serum-free and conducted by incubation of cells for 4-6 hours to deprive cells of power and bring them in non-breeding condition. Another set of holes were maintained in complete medium and were actively proliferating cells. After 4-6 hours antibodies were added (including the control isotypes). Usually, for each state were used 3 holes. Then the wells were incubated with antibodies for 1-48 hours. Antibodies against integrin alfaretta usually were tested at the following concentrations: 0 mg/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 (FSB with 0.2% Triton) for 1-2 hours, then painted with a monoclonal antibody which is specific races will osnet the activated form of Caspase-3 (for example, rabbit antibody against active caspase-3 from Bio Vision, diluted 1:50 in the FSB with 0.2% Triton and 5% goat serum). Antibodies against caspase-3 and the fixed cells were incubated at 4°C over night. The next day cells were washed FSB 3 times and incubated with conjugated with Alexa-594 secondary antibody against rabbit (1:800) for 4 hours at room temperature in a dark place. The wells were again washed and incubated with DAPI (1:10000 in the FSB) within 10 minutes After the last washing of the FSB, the total number of cells per well was estimated by photographing DAPI staining at 5x. Cells that gave a positive response in the study on active caspase-3 on the same plots were photographed using a red filter. Reproduction was assessed as the percentage of cells positive in the study on active caspase-3 at the site. The results were then analyzed using the Excel program. The figure 8 shows that 7H5 and 7H12 not actively induce apoptosis.

Example 9. Coulometrically study caspase 3/7 HUVEC

Studies have been conducted activity of caspase 3/7 using antibody 7H5 and 7H12 (Analysis caspase/7 Apo-One from Promega, see Technical Bulletin No. 295 for instructions on how to carry out routine analysis in 96 wells).

Usually, 96-well tablets were coated with 1 μg/ml fibranet is on during the night. The tablets were washed FSB. Then were sown 3000-5000 cells HUVEC on each of the 96 wells, and then were grown overnight in complete medium (medium EBM-2 (Cambrex CC-3156) with EGM-2 SingleQuots (Cambrex CC-4176)). If for studies of apoptosis will be used murine endothelial cells 2H-11, Wednesday will be the environment 50/50 with 10% FBS.

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

After incubation each well was added 100 μl of reagent on caspase 3/7 Apo-One, after which the contents of the tablet was subjected to careful mixing using a flatbed shaker at 300 rpm for 30 seconds. Then the plate was incubated at room temperature for 1 to 8 hours, after which data were removed using a flatbed scanner. Was measured fluorescence in to the each hole when the excitation light with a wavelength of 485 nm and emission wavelength 530 nm.

Fluorescent signal resulting from cleavage of the substrate by caspase 3/7 meant apoptosis. The figure 9 shows that 7H5 and 7H12 not causing apoptosis.

Example 10. The study of education tubes

Can be investigated the ability of antibodies against alfaretta suppress the formation of tubes. The following is an example for the study of pipes based on the growth of HUVEC cells and studies of the formation of the tubes described Nakatsu et al. (2003) Microvascular Research 66 (2003) 102-112.

Usually HUVEC cells can be mixed with covered detrano Mironosetsky Cytodex 3 (Amersham Pharmacia Biogech, Piscataway, NJ) at a concentration of 400 cells on microchart in 1 ml of medium EGF-2. The beads with cells may be subjected to careful shaking every 20 minutes for 4 hours at 37°C and 5% CO2. After incubation, the beads can be transferred into a culture flask 25 cm2(BD Biosciences, Bedford, MA) and left for 12-16 h in 5 ml of medium of the DOM-2 at 37°Media 5% CO2. The next day the beads from the cells can be washed three times with 1 ml of the DOM-2 and resuspendable at a concentration of 200 is covered by cells of the particles/ml 2.5 mg/ml fibrinogen (Sigma, St. Louis, MO). Five hundred microlitres solution of fibrinogen with beads was added to of 0.625 units of thrombin (Sigma) in each well of a 24-hole culture plate. Fibrinogen with beads can form gustke for 5 minutes at room temperature, and then at 37°C and 5% CO2within 20 minutes. In each well can be added to one ml of EGM-2 containing 2% FBS), and then balanced with the fibrin clot for 30 minutes at 37°C and 5% CO2. The medium was removed from cells and replaced with 1 ml of fresh medium. Approximately twelve thousand cells, skin fibroblasts (Detroit 551, ATCC, Rockville, MD) can be sown on the surface of the clot. The environment can be replaced every day. Samples with beads can be tracked within 7 days.

Covered HUVEC particles can be cultured in fibrin gels in the presence or absence of 500 μl of antibodies against alfaretta (7H5 and 7H12) on the surface of the gel for 2-3 days, then transferred to the platform Nicon Eclipse TE300 with axes in several dimensions and maintained at 37°C and 5% CO2within 72 hours. The final concentration of the antibodies used can be calculated, taking into account the volume of the fibrin gel, i.e. the final concentration of antibody = total weight of the antibody/volume environment + the volume of the fibrin gel. Using the software Metamorph you can take pictures of many particles every 20 minutes. Quantification of blood vessels in vitro can be made using images of particles high resolution (for example, Microcom Olympus IX70 lens 4x). May be determined by the number for Atkov of each particle in comparison with the untreated control, the rudiment of the vessel can be defined as a vessel with a length equivalent to the diameter of the particles. The length of the rudiment of the vessel can be measured in arbitrary units.

Example 11. Studies combining models of tumors on the basis of xenografts and allografts.

Competitive and consistent destination therapy antagonists alfaretta and VEGF may be Centeno on xenotransplantation/allotransplantation models of tumors. Preferably, the models have low or absent response to monotherapy with VEGF antagonist. The following are examples of models that can be used: (a) allograft Fo5 in Nude naked mice (tumor of the breast obtained from transgenic mice with mmtv-Her2) (Finkle, D., et al., (2004) Clin. Cancer Res. 10:2499-2511); (b) xenotransplant HT29 in Nude naked mice (human colorectal line); and (c) RIP-TbAg (pancreatic tumor model Tg). Treatment can be assigned intraperitoneally, subcutaneously or intravenously. For example, antibodies against VEGF can be assigned at a dose of 10 mg/kg once a week or 5 mg/kg twice a week. The number of input antagonist alfaretta, such as an antibody, may be determined based on affinity and activity. In one experiment, the VEGF antagonist and the antagonist alfaretta could be entered on a joint schedule during the 5-6 weeks. Alternative or additionally, the VEGF antagonist and the antagonist alfaretta could be introduced sequentially (for example, antibodies against VEGF for three weeks followed by a dosage of antibodies against alfaretta within three weeks).

The effectiveness of treatment can be estimated based, inter alia, on the progression of the tumor, blood tumor, the density of tumor vessels, morphology and/or survival. The progression of the tumor can be measured, for example, tumor volume and/or weight of the tumor. To assess vascular changes associated neoplastic progression, can be used perfusion FITZ-lecithin, as well as staining of vascular markers.

Example 12. Model of breast cancer human MDA-MB231

Females naked mice HRLN were in the side introduced subcutaneously with 5×106breast cancer cells human (HRLN is the name of the line). Tumors were allowed to grow until they reach the average size of 80-120 mm3. Then tumor bearing mice were divided into 4 groups, treatment was initiated when the average tumor volume in the group amounted to approximately 100 mm3.

The volumes of the tumors were measured during the study twice a week. The measurement of the volume of the tumors was performed using the standard method with the use of calipers. Monoc the regional hamster antibodies against murine integrin alpha, known as 10E7, were obtained from Genentech. The endpoint of the experiment was achieved when the tumor weight was 1.5 grams or after 60 days, depending on what happened before. In some cases, monitoring response to therapy was carried out longer. Upon reaching the end point the animals were euthanized.

Treatment details are described below.

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

(2) Group single drug against VEGF: entered monoclonal antibodies against VEGF B20.4.1 (10 mg/kg, WB, once a week).

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

(4) Group single drug against integrin alpha: monoclonal hamster antibodies against murine integrin alpha E (10 mg/kg, WB, twice a week).

Data control group:

Study day149131620237
the animal numberTV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
163751964055504866001080
26375126196320320446527
37512628866666693610802048
475126196 320320446527936
5751262884464867879081764
688144221288288405550550
788144320550726100813522025
888144144446600126812681913
9108144245 4866507009081437
1014416232052752784713522138
average86,5126,6234,4432,8513,2720,2898,91441,6
SEM7,79,32243,549,796,6112,3198,2
N10101010101010 10

These groups of single drug against VEGF:

tr>
Study day1491316202327
the animal numberTV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
163108144320405550256500
26363100162221288288550
375196365405500320500550
475751962562885005501099
575126196320500500550787
688144221320352446446600
788196365405405666726 864
888196288320352384288365
9108172256500405320288320
1014424541656775096812961296
average86,5152254,6357,5417,8494,1518,8693
SEM7,718,732,636,9 45,864,5of 99.1100
N1010101010101010

These groups of drug against VEGF and alfaretta:

Study day1491316202327
the animal numberTV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
1636363 6363108126108
263108172256288288288288
375126126221245245320320
4757575126196288245446
575108172405352650650908
688196221 320320288196196
78875196256196288288446
88888144320320288320405
9108126144196256320320486
10144221270446600650600787
average86,5118,5 158,1260,8283,6341,3335,3438,8
SEM7,716,519,937,34454,752,278,1
N1010101010101010

These groups of single drug against integrin alpha:

Study day1491316202327
the animal numberTV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
TV
(mm3)
1637519632048678710082025
26310812636536572610081352
375751442882885506001008
4751081443203208471152 1960
5751081723653655504861152
68819635265078790813521666
7881001622453524861764Endpoint 12.08.06, the tumor exceeded 1500 mm3
888126162320446486650650
91082884466001008 13529363179
10144162245384352486486288
average86,5134,6214,8385,6476,7717,7944,21475,6
SEM7,720,733,14274,286,7129,7286,4
N101010101010109

These preliminary Yes the nye show early signs of the combined activity of antagonists alpha and VEGF.

After the end of the study were computed average tumor volumes for each group (figure 11A). There were also diagrams Kaplan-Meier, showing the percentage of animals remaining in the experiment as a function of time (figure 11B). Data show that antibodies against integrin α5βl increase the effectiveness of the antagonist of VEGF in a model of breast cancer.

Example 13. 7H12 and bevacizumab on the model of wound healing rabbit ear

New Zealand white rabbits were weighed and anastasiaruby isofluorane. Each rabbit with the inner surface and along the edges of both ears was shorn wool. The remaining hair was removed from the operating field depilarsi lotion. Operating margins were cleared betulinum scrub, followed by wetting with alcohol. Round 8 mm connectors tool for biopsy was used to obtain in each ear wounds depth of the cartilage. Below perichondrium was removed using a periosteal lifts and fine scissors. Each wound was placed adhesive dressings Opsite®. Then the rabbit was left to come out of the anesthesia. Daily dressings Opsite®was removed, the wound looked, locally applied treatment, and superimposed fresh dressings. The size of the wound was determined by measuring the diameter is tra wounds at 0 (immediately after surgery), 7, 10, 14 and 18 days.

Groups of treatment were as follows:

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

7H12 (antibody against alfaretta) 100 μg in 30 μl into each wound daily (n=4)

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

Trastuzumab (antibody against HER2) 100 μg in 30 μl into each wound daily (n=3)

Data show that combination therapy with a VEGF antagonist and an antagonist alfaretta had a striking effect on this model of angiogenesis compared with the drugs one by one (Fig. 10).

Example 14. Combination therapy with antibodies against alfaretta and antibodies against VEGF in colorectal cancer

The HRLN female mice nu/nu was introduced 1 mm3fragments of HT29 tumor (tumor of the intestine) subcutaneously in the flank. Tumors were allowed to grow until they reach the average size of 80-120 mm3after the treatment. Then tumor bearing mice were divided into 4 groups:

GroupNumber
mice
Treatment 1Treatment 2
Medicationmg/kgPathGraph Medicationmg/kgPathSchedule
110Control10WB1 x weeks x 7FSB-WB2 x weeks x 7
210B20-4.110WB1 x weekFSB-WB2 weeks
310B20-4.110WB1 x week10E710WB2 weeks
410FSB-WB-10E710 WB2 weeks

Measurement of tumor volume were held twice a week using the standard method of measurement with a caliper. Monoclonal hamster antibodies against murine alpha known as 10E7, were obtained from Genentech. As a control IgG were used monoclonal antibodies against ragweed. Body weight was measured 5 times during the first 2 days, then twice a week until the end of the research. The experiment ended when the tumour volume 1 gram or 90 days, depending on what came before. Some respond to treatment the animals were observed for longer. Upon reaching the end point the animals were euthanized. The dosage was 10 ml/kg (or 200 ál per 20 gram mouse), the volume was adjusted according to body weight. For animals showing complete regression (CR)at the end point of the tissue from the site of implantation of the tumor were collected and fixed with formalin and then stored in 70% EtOH for further study. All samples for freezing were placed in crimond, wrapped in foil and quickly frozen in liquid nitrogen.

After the end point of the study, were calculated the average tumor volumes for each group (figure 12A). There were also diagrams Kaplan-Meier, showing the percentage of animals, the OS is audacia in the experiment as a function of time (figure 13B). The data show that the antibody against integrin α5βl increases the efficiency of VEGF in a model of intestinal cancer.

Example 15. Antibodies against alfaretta and chemotherapy in cancer of the intestines.

The HRLN female mice nu/nu was introduced 5×106of HCT116 tumor cells (tumor of the intestine) subcutaneously in the flank. Tumors were allowed to grow until they reach the average size of 80-120 mm3after what was started therapy. Then tumor bearing mice were divided into 4 groups:

GroupNumber
mice
Treatment 1Treatment 2
Medicationmg/kgPathScheduleMedicationmg/kgPathSchedule
110FSB-WB2 x weeks x 7--WB-
21010E710WB2 x weeks x 7--WB-
310FSB-WB2 x weeks x 7irinotecan100WB1 x weeks x 3
41010E7-WB2 x weeks x 7irinotecan100WB1 x weeks x 3

Measurement of tumor volume were held twice a week using the standard method of measurement with a caliper. Monoclonal hamster antibodies against murine alpha 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 research. The experiment ended when the tumor volume 1.5 grams or 60 day, depending what from whichever comes first. Some respond to treatment the animals were observed for longer. Upon reaching the end point the animals were euthanized. The dosage was 10 ml/kg (or 200 ál per 20 gram mouse), the volume was adjusted according to body weight. 10E7 was administered 30 minutes prior to administration of irinotecan. For animals showing complete regression (CR)at the end point of the tissue from the site of implantation of the tumor were collected and fixed with formalin and then stored in 70% EtOH for further study. All samples for freezing were placed in crimond, wrapped in foil and quickly frozen in liquid nitrogen.

After the end point of the study, were calculated the average tumor volumes for each group (figure 13A). There were also diagrams Kaplan-Meier, showing the percentage of animals remaining in the experiment as a function of time (figure 13B). Data show that antibodies against integrin alfaretta not increase the effectiveness of chemotherapeutic drug (irinotecan) on the model of bowel cancer, but also does not prevent the action of chemotherapeutic drug. The observations were consistent with our assumption that the vascular damage occurring prior to treatment with the antagonist alfaretta, can be very useful when antiangiogenesis, and in particular when antiangiogenic shall see in the Oncology setting. Such damage to the blood vessels can be caused by a VEGF antagonist, such as an antibody AVASTIN®. By itself, a chemotherapy drug in this model does not cause noticeable damage to the vessels. Should assume the use of these drugs (VEGF antagonist/antagonist alfaretta/a chemotherapy drug) simultaneously or sequentially in such a way that the VEGF antagonist is present and causes vascular damage.

Example 16. Graphics of Scatchard for alfaretta

Antibodies against alfaretta were iodised using the lodogen method, radioactively labeled antibodies were purified from free125I-Na using gel filtration using a column PD-10. Cells R9ab, cell line fibroblasts rabbit (purchased from ATS, No.CCL-193) were sown in the amount of approximately 50,000 per well in 24-hole plates and incubated for 48 hours in 5% CO2at 37°C. Cells were washed three times with buffer for binding (50:50 DMEM/F12 medium containing 2% FBS and 50 mm HEPES, pH of 7.2), and then incubated on ice for 15 minutes. The washed cells were incubated for 4 hours on ice with approximately 50 PM125I-monoclonal antibodies against alfaretta containing decreasing concentrations of unlabeled monoclonal antibodies against alfaretta, serially diluted to 0.5 μm in a buffer the La binding 13 concentrations, selected in three replications. Cells were washed three times with buffer for binding, then solubilisation 200 ál lyse buffer with SDS (1% SDS, 8 M urea, 100 mm glycine, pH 3.0). Was defined as the radioactivity of the lysate of cells with gamma counter Wallac Wizard 1470. Data binding were estimated using the program Genentech "NewLigand", which uses the algorithm of approximation of the curve of Munson and Roberta (Munson, P. and Robard, D. (1980) Anal. Biochem. 107: 220-239) to determine the affinity of binding of the antibody and the concentration of binding sites. In figures 14 and 15 shows that in these studies, the binding of the antibody 7H5 have a Kd of 0.10 nm, and antibodies 7H12 have a Kd of 0.30 nm, respectively.

Example 17. Research epitope mapping/competitive binding of IgG against integrin alfaretta

First, a series of three-fold dilutions of IgG against integrin α5β1 was incubated in 96-well tablet Nunc Maxisorp antigen coated - human integrin α5β1 (1 μg/ml, R&D) in PBST buffer (FSB and 0.5% (wt/vol) BSA, and 0.05% (volume) tween-20) for 1-2 hours at room temperature, followed by addition of 0.3 nm biotinylated hIgG1 h7H5.vl (variant antibody 7H5 received Genentech, Inc.), which was first defined by submaximal signal binding (50-70%), for 15 minutes. Then the plate was washed 5 times with PBT buffer (FSB and 0.05% (volume) tween-20). the knitted biotinylated h7H5.vl hIgG1 were detected using a conjugate with streptavidin horseradish peroxidase (Pierce), diluted 1:2500 in PBST buffer, shown using the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Kirkegaard &Perry Labs, Gaithersburg, MD) for approximately 5 minutes, the reaction was terminated with 1.0 M H3PO4after which measurements were made on a spectrophotometer at 450 nm. Curves were selected using the program selection curves with nonlinear regression and four parameters (Kaleidagraph, Synergy Software).

The figure 16 shows that the bound h7H5.v1 compete with increasing amounts of cold m7H5. In fact, the curve of competition m7H5 was almost identical to the curve of competition h7H5.v1 (data not shown). Cold m7H12 also competed with Biotin-h7H5.vl when linking with alfaretta, demonstrating that the epitope binding alfaretta h7H5.v1 and m7H12 overlap. On the other hand, the control antibody did not compete with the associated h7H5.vl.

1. Antibody against alfaretta produced by hybridomas, deposited under number access ATSS no MOUTH-7421, where the antibody is an IgG2 and has a Kd of 0.10 nm, measured using cells R9ab with the access number of ATSS No. CCL-193 according to the method of Scatchard.

2. Antibody against alfaretta produced by hybridomas deposited by Panamera access ATSS no MOUTH-7420, where the antibody is an IgG2 and has a Kd value of 0.30 nm, measured using cells R9ab with the access number of ATSS No. CCL-193 according to the method of Scatchard.

3. Antibody conjugated with a therapeutic tool for the inhibition of angiogenesis and/or vascular permeability, where the antibody is produced by hybridomas, deposited under number access ATSS no MOUTH-7421, or hybridomas, deposited under number access ATSS no MOUTH-7420, where the antibody is an IgG2, where the antibody produced by hybridomas ATSS no MOUTH-7421, has a Kd of 0.10 nm, and the antibody produced by hybridomas ATSS no MOUTH-7420 has a Kd value of 0.30 nm, which is measured using cells R9ab with the access number of ATSS No. CCL-193 according to the method Scatchard.

4. The antibody according to claim 3, where therapeutic agent is selected from the group consisting of cytotoxic tools, radioactive, and chemotherapeutic agents.

5. Antibody conjugated with a label, for use in the diagnosis of disease associated with expression of Alfama, where the antibody is produced by hybridomas, deposited under number access ATSS no MOUTH-7421, or hybridomas, deposited under number access ATSS no MOUTH-7420, where the antibody is an IgG2, where the antibody produced by hybridomas ATSS no MOUTH-7421, has a Kd of 0.10 nm, and the antibody produced hybridomas no MOUTH-7420, has a Kd value of 0.30 nm, which is measured using cells R9ab with the access number of ATSS No. CCL-193 according to the method of Scatchard.

6. The antibody according to claim 5, where the label is selected from the group consisting of a radioisotope, a fluorescent dye or an enzyme.

7. An isolated nucleic acid molecule encoding the antibody according to claim 1 or 2.

8. Expression vector encoding a nucleic acid molecule according to claim 7.

9. Recombinant cell containing the nucleic acid molecule according to claim 7.

10. The cell according to claim 9, where the cell is hybridomas, deposited under number access ATSS no MOUTH-7421, or hybridomas, deposited under number access ATSS no MOUTH-7420.

11. A method of producing an antibody comprising culturing cells according to claim 9 or 10, and the selection of antibodies from cell culture.

12. Pharmaceutical composition for inhibiting angiogenesis and/or vascular permeability comprising an effective amount of the antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

13. The method for detecting protein alfaretta in the sample from the patient in vitro by bringing the sample into contact with an antibody according to claim 1 or 2, and the detection of antibodies against alfaretta associated with protein alfaretta.

14. The method according to item 13, where the antibody is used in immunohistochemical studies (tissues) or in studies using ELISA.

15. The application is the development of antibodies according to claim 1 or 2 in the production of pharmaceuticals for inhibition of angiogenesis and/or vascular permeability in a subject.

16. The use of antibodies according to claim 1 or 2 in the manufacture of a medicinal product for the treatment of the disease in the subject where the disease is accompanied by pathological angiogenesis or pathological vascular permeability.

17. The use of antibodies against alfaretta according to claim 1 or 2 in the production of pharmaceuticals for inhibition of angiogenesis and/or vascular permeability in a subject suffering from diseases involving pathological angiogenesis or pathological vascular permeability, where the antibody against alfaretta according to claim 1 or 2 enter a specified subject in combination with a VEGF antagonist.

18. The application 17, where the disease is selected from cancer, eye diseases and autoimmune diseases.

19. The application 17, where the subject is administered the VEGF antagonist, and then the antibody against alfaretta.

20. The application 17, where the VEGF antagonist and an antibody against alfaretta administered to the subject simultaneously.

21. The application 17, where the subject is undergoing treatment with a VEGF antagonist to until the entity ceases to respond to treatment with a VEGF antagonist, after which the subject is subjected to treatment with antibody against alfaretta.

22. The application 17, where the disease is a cancer, and the subject being treated with the VEGF antagonist when the cancer is not invasive, and treatment of antibody protevangel, when the cancer is invasive.

23. The application 17, where the subject has elevated levels alfaretta in diseased tissue compared to tissue of a subject not suffering from the disease.

24. The application 17, where the subject impose additional therapeutic agent selected from the group consisting of anti-neoplastic means, a chemotherapeutic drug that inhibits the growth of money and cytotoxic tools.

25. The application 17, where the VEGF antagonist is an bevacizumab.

26. The application 17, where the antibody against alfaretta conjugated with a cytotoxic agent.

27. Use p, where the cytotoxic agent is a radioactive isotope, a chemotherapeutic agent or a toxin.

28. Pharmaceutical composition for inhibiting angiogenesis and/or vascular permeability, containing an effective amount of a VEGF antagonist, an effective amount of antibodies against alfaretta according to claim 1 or 2 and a pharmaceutically acceptable carrier.

29. Kit for detection alfaretta the subject, which were subjected to treatment with the VEGF antagonist containing
antibody against alfaretta according to claim 1 or 2, and
instructions for detecting alfaretta the subject, which were subjected to treatment with the VEGF antagonist.

30. The use of a composition according p during the production of medicines is about the means for inhibiting angiogenesis and/or vascular permeability in a subject, suffering from disease.

31. The application of article 30, where the disease is selected from the group consisting of cancer, eye diseases and autoimmune diseases.

32. The application of article 30, where the disease is selected from the group consisting of solid tumors, metastatic tumors, soft tissue tumors, diseases associated with neovascularization, inflammatory eye diseases accompanied by abnormal angiogenesis, diseases that occur after transplantation to a subject, and diseases involving pathological development of vascular fibrous tissue.

33. Use p where the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, lung cancer, nehodgkinski lymphoma (NHL), pochernkletocny cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, cancer of the soft tissues and multiple myeloma.

34. The application of article 30, where the disease is selected from the group consisting of retinopathy, caused by age related macular degeneration, rubeosis, psoriasis, psoriatic arthritis and inflammatory kidney disease, haemolytic uraemic syndrome, diabetic nephropathy, arthritis, inflammatory bowel disease, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitrite, exclusion Tr is spuntata of the cornea, the corneal neovascularization, neovascularization of corneal transplant, Crohn's disease, myopia, ocular neovascular disease, osteoarthritis, Paget's disease, pemphigoid, polyarthritis, postlesional radial keratotomy, neovascularization of the retina, Sjogren syndrome, ulcerative colitis, transplant rejection, lung inflammation, nephrotic syndrome, edema, ascites associated with malignancy, stroke, angiofibromas and neovascular glaucoma.

35. The use of antibodies according to claim 1 or 2 in the manufacture of a medicinal product for the treatment of a subject suffering from a disease where the subject responded to the treatment with the VEGF antagonist, but partially or completely ceased to respond to VEGF antagonist.

36. Use p where the subject has elevated levels alfaretta in diseased tissue compared to a subject not suffering from the disease.

37. Use p, where the subject impose additional therapeutic agent selected from the group consisting of antineoplastics means, a chemotherapeutic drug that inhibits the growth of money and cytotoxic funds.



 

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SUBSTANCE: antibodies are characterised by amino acid sequences of VH and VL chains. There are also presented: nucleic acid coding the antibody; an antibody expression vector; a recombinant cell for antibody expression. There are offered: a method for producing a humanised antibody; as well as a pharmaceutical composition and a kit for treating or preventing hepatitis C; a method of treating or preventing an infection caused by hepatitis C virus; an analytic method of identifying the agent increasing or enhancing the efficacy of neutralising activity of the humanised antibody; a method of detecting the presence or absence of hepatitis C virus in a sample on the basis of the use of the antibody.

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30 cl, 6 ex, 32 dwg, 39 tbl

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