Alpha5-beta 1 antibodies and using them

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a recovered human integrin α5β1 monoclonal antibody. The antibody is characterised by the fact that it contains 6 CDR, 3 CDR from a light chain and 3 CDR from a heavy chain. A nucleic acid (NA) coding the antibody according to the invention, an expression vector containing a NA molecule, a host cell containing the vector, and a method for preparing the antibody on the basis of the cell are described. There are disclosed: a composition and a method for growth inhibition of the tumour cells expressing human integrin α5β1 on the basis of the antibody. What is described is a version of the method for growth inhibition of the tumour cells expressing human integrin α5β1 using the composition.

EFFECT: invention provides the new antibodies with high (approximately nm, as measured by FACS) binding affinity for human integrin α5β1 that can find application in medicine in therapy of the tumours mediated by integrin α5β1 expression.

13 cl, 36 dwg, 3 tbl, 11 ex

 

This application claims the priority of provisional application U.S. No. 61/026027, filed February 5, 2008, and provisional application U.S. No. 61/095429, filed September 9, 2008, both of which are given in this document as a reference in full.

The technical field to which the invention relates

The present description relates to antibodies and their antigennegative areas that are associated with α5β1. Description also applies to the molecules of nucleic acids encoding such antibodies and antigennegative areas, methods of generating antibodies and antigenspecific plots against α5β1, compositions containing these antibodies and antigennegative areas, and to methods of using the antibodies, antigenspecific plots and compositions.

The level of technology

Integrin α5β1 is a heterodimeric protein on the cell surface that binds to fibronectin and involved in the attachment of cells and angiogenesis. This heterodimer composed of subunits α5 and β1 subunit. Integrin α5β1 is designated as "classical receptor for fibronectin, which is of key importance in adhesion to the matrix, migration, proliferation, differentiation and survival. Despite the fact that fibronectin (FN) binds several integrins, the election in respect of FN is the integrin α5β1, as detected is of the ligand and the optimal interaction requires both the peptide sequence on the ninth (PHSRN) and tenth (RGDS) repetition of type III fibronectin FN (Danen et al. J. Biol. Chem. 270(37):21612-21618 (1995); Redisk et al.J. Cell Biol.149(2):521-527 (2000); Takagi et al.EMBO J. 22:4607-4615 (2003)). Integrin-mediated adhesion of cells to FN can run the influx of calcium, activate tyrosine - and serine/threonine protein kinase and Inositol-lipid metabolism and regulate the activity of a family of small GTP-AZ Rho, which controls the actin cytoskeleton and cell cycle.

The expression of α5β1 observed in most embryonic tissues, but after birth the level drops in accordance with the terminal differentiation of the cells (Muschler & HorwitzDevelopment113(1):327-337 (1991)). In adult wild-type mice expression occurs mainly in the vascular and connective tissue, although low levels of the receptor are widely distributed. Expression as α5β1, and FN is significantly and consistently increased in blood vessels of human tumors and in tissues stimulated by growth factors and cytokines.

Angiogenic cytokines such as bFGF, VEGF, IL-8, TGF-beta and TNF-α increases the expression of α5β1 on endothelial cells in-vitro and in-vivo, while in normal vessels and tissues these molecules are expressed in a minimal amount (Kim et al.Am. J. Path. 156(4):1345-62 (2000); Enaida et al.Fukushima J. Med. Sci. 44(1):43-52 (1998); Klein et al.Mol. Biol. Cell4(10):973-982 (1993)).

High levels of expression of α5β1 not limited to the vascular system, as well as often on neugebaur, that Express α5β1 tumor cells in many types of cancer. Hypoxia of the tumor was associated with increased expression of α5β1 tumor (Mousa et al.J. Cell. Biochem. 74:135-143 (1999)). I believe that integrins are important for intranasal tumors in newly formed capillaries and extravasation in remote areas, leading to tumor spread and metastasis. In General, the pattern of expression of α5β1 is consistent with a large number of values in the cancer progression by mediating activities as stromal and tumor cells. In a large number of clinical trials increased α5β1 on tumor cells was associated with progression of melanoma, oral squamous cell carcinoma and b-cell leukemias man (Jin & Varner,Br. J. Cancer90:561-565 (2004); Danen et al.Histopathology24(3):249-256 (1994); Shinohara et al.,Am. J. Clin. Pathol. 111(1):75-88 (1999)).

The invention

This description applies to the selection of monoclonal antibodies, in particular monoclonal antibodies man who exhibit high affinity binding to integrin α5β1 person. Antibodies described in the present description, as a rule, are human antibodies, although in alternative cases, the antibody may be an antibody mouse, chimeric antibodies or humanized antibodies.

In one aspect of the description relates the I to the selected monoclonal antibody or its antigennegative section, where the antibody: (a) binds to integrin α5β1 person with KD1×10-7M or less; and (b) is able to induce antibody-dependent cellular cytotoxicity. For example, in one case, the antibody belongs to the subclass, which is able to induce ADCC, such as IgG1 or IgG3. In another case, the ADCC activity is a result of the introduction in the Fc-region of carbohydrate modifications, such as altered patterns of glycosylation compared with native carbohydrate pattern.

In some cases, the antibody binds to integrin α5β1 person with KD5×10-8M or less, 2×10-8M or less, 1×10-8M or less, 5×10-9M or less, 4×10-9M or less, 3×10-9M or less, or 2.7×10-9M or less.

In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative the site where the antibody: (a) binds to integrin α5β1 person with KD1×10-7M or less; and (b) has enhanced ADCC activity relative to antibodies comparison. For example, in one case, the antibody with enhanced ADCC activity contains at least one mutation in the Fc region compared to the Fc-region of a wild type and an increased ADCC activity is relative to the same antibody, but containing the Fc-region of the wild type. In some ol the measures antibody binds to integrin α5β1 person with K D5×10-8M or less, 2×10-8M or less, 1×10-8M or less, 5×10-9M or less, 4×10-9M or less, 3×10-9M or less, or 2.7×10-9M or less. In additional examples, the antibody has an increased ADCC activity relative to antibodies comparison, which raised at least 1.1 fold, at least 1.2 fold, at least 1.3 times, at least 1.5 times, at least 2 times, at least 3 fold, at least 5-fold, at least 10 times, at least 20 times, at least 30 times, at least, 40-fold, at least 50-fold, at least 100 times, at least 200 times, at least 500-fold, or at least 1000 times with respect to the antibody comparison, where the antibody comparison represents the same antibody, but with the Fc-region of the wild type.

In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative the site where the antibody: (a) binds to integrin α5β1 person with KD1×10-7M or less; and (b) contains at least one mutation in the Fc region compared to the Fc-region of the wild type. For example, in one case, the antibody belongs to the IgG1 subclass, and at least one of the amino acids in the Fc region of the IgG1 subclass metirovan. In an additional example, by at least one mutation is at position serine 247, alanine 338 or isoleucine 340 in the Fc region of the IgG1 subclass. In an additional example, at least one mutation selected from the group consisting of S247D, A338L and I340E. In yet another additional example, the antibody contains mutations S247D, A338L and I340E.

In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative the site where the antibody cross-competes or competes for binding to the integrin α5β1 person with an antibody that contains: (a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:7, or conservative modifications; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:8, or conservative modifications.

An additional aspect of the present description represents a selected monoclonal antibody or its antigennegative plot containing the variable region of the heavy chain, which is a product of or derived human gene VH4-39, where the antibody specifically binds to the integrin α5β1 person.

An additional aspect of the present description represents a selected monoclonal antibody or its antigennegative plot containing the variable region of light chain, to ora is a product of or derived human gene V KL6, where the antibody specifically binds to the integrin α5β1 person.

In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR1-containing sequence of SEQ ID NO:1, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR2, containing SEQ ID NO:2, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR3 containing SEQ ID NO:3, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain CDR1, containing SEQ ID NO:4, or a conservative modification. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR2, containing SEQ ID NO:5, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative the area is containing a series of variable region light chain CDR3, containing SEQ ID NO:6, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative site that contains:

(a) the variable region of the heavy chain CDR1, containing SEQ ID NO:1, or conservative modifications;

(b) the variable region of the heavy chain CDR2, containing SEQ ID NO:2, or conservative modifications;

(c) variable region of the heavy chain CDR3 containing SEQ ID NO:3, or conservative modifications;

(d) variable region light chain CDR1, containing SEQ ID NO:4, or conservative modifications;

(e) variable region light chain CDR2, containing SEQ ID NO:5, or conservative modifications; and

(f) variable region light chain CDR3 containing SEQ ID NO:6, or conservative modifications.

In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:7, or conservative modifications. For example, the description refers to the antibody or antigennegative section containing the variable region of the heavy chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least, is 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:7. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain containing the amino acid sequence of SEQ ID NO:8, or conservative modifications. For example, the description refers to the antibody or antigennegative section containing the variable region of light chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%or at least 99% identical to the amino acid sequence shown in SEQ ID NO:8. In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:7, or conservative modifications, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:8, or conservative modifications. For example, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain that is at least 80%, at IU is e, 85%, at least 90%, at least 95%, at least 97%, at least 98%or at least 99% identical to the amino acid sequence shown in SEQ ID NO:7, and the variable region of light chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least at 97%, at least 98%or at least 99% identical to the amino acid sequence shown in SEQ ID NO:8.

In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR1-containing sequence of SEQ ID NO:13, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR2-containing sequence of SEQ ID NO:14, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR3-containing sequence of SEQ ID NO:15, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing rebellow region light chain CDR1, contains the sequence of SEQ ID NO:16, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR2-containing sequence of SEQ ID NO:17, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR3-containing sequence of SEQ ID NO:18, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative site that contains:

(a) the variable region of the heavy chain CDR1, containing SEQ ID NO:13, or conservative modifications;

(b) the variable region of the heavy chain CDR2, containing SEQ ID NO:14, or conservative modifications;

(c) variable region of the heavy chain CDR3 containing SEQ ID NO:15, or conservative modifications;

(d) variable region light chain CDR1, containing SEQ ID NO:16, or conservative modifications;

(e) variable region light chain CDR2, containing SEQ ID NO:17, or conservative modifications; and

(f) variable region light chain CDR3 containing SEQ ID NO:18, or conservative modifications.

In an additional aspect, the crust is ASEE description refers to the selected monoclonal antibody or its antigennegative section, containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:19, or conservative modifications. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain containing the amino acid sequence of SEQ ID NO:20, or conservative modifications. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:19, or conservative modifications, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:20, or conservative modifications.

In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR1, containing SEQ ID NO:23, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR2, containing SEQ ID NO:24, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody is whether it antigennegative section, containing the variable region of the heavy chain CDR3 containing SEQ ID NO:25, or a conservative modification. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain CDR1, containing SEQ ID NO:26, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR2, containing SEQ ID NO:27, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR3 containing SEQ ID NO:28, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative site that contains:

(a) the variable region of the heavy chain CDR1, containing SEQ ID NO:23, or conservative modifications;

(b) the variable region of the heavy chain CDR2, containing SEQ ID NO:24, or conservative modifications;

(c) variable region of the heavy chain CDR3 containing SEQ ID NO:25, or conservative modifications;

(d) variable region light chain CDR1, containing SEQ ID NO:26, or conservative modifications;

(e) variable region is here light chain CDR2, containing SEQ ID NO:27, or conservative modifications; and

(f) variable region light chain CDR3 containing SEQ ID NO:28, or conservative modifications.

In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:29, or conservative modifications. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain containing the amino acid sequence of SEQ ID NO:30, or conservative modifications. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:29, or conservative modifications, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:30, or conservative modifications.

In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR1, containing SEQ ID NO:33, or conservative modifications. In other the second aspect of the description relates to the selected monoclonal antibody or its antigennegative section, containing the variable region of the heavy chain CDR2, containing SEQ ID NO:34, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain CDR3 containing SEQ ID NO:35, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain CDR1, containing SEQ ID NO:36, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR2, containing SEQ ID NO:37, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative section containing the variable region light chain CDR3 containing SEQ ID NO:38, or conservative modifications. In another aspect of the description relates to the selected monoclonal antibody or its antigennegative site that contains: (a) the variable region of the heavy chain CDR1, containing SEQ ID NO:33, or conservative modifications; (b) the variable region of the heavy chain CDR2, containing SEQ ID NO:34, or conservative modifications; (c) variable is inuu region of the heavy chain CDR3, containing SEQ ID NO:35, or conservative modifications; (d) variable region light chain CDR1, containing SEQ ID NO:36, or conservative modifications; (e) variable region light chain CDR2, containing SEQ ID NO:37, or conservative modifications; and (f) variable region light chain CDR3 containing SEQ ID NO:38, or conservative modifications.

In an additional aspect, the present description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:39, or conservative modifications. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the light chain containing the amino acid sequence of SEQ ID NO:40, or conservative modifications. In an additional aspect, the description refers to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:39, or conservative modifications, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:40, or conservative modifications.

In an additional aspect offers you elenoa antibody or antigennegative plot, which bind to the same epitope on integrin α5β1 person, and any antibody described herein, and/or compete for binding to the integrin α5β1 person with such an antibody.

In one of the embodiments, the description refers to the material deposited in ATSS under access number of MOUTH-9377. In another embodiment, the description refers to the material deposited in ATSS under access number of MOUTH-9378. In another embodiment, the description refers to the selected antibody that contains the variable region of the heavy chain, deposited in ATSS under access number of MOUTH-9377. In another embodiment, the description refers to the selected antibody that contains a variable region light chain, deposited in ATSS under access number of MOUTH-9378. In another embodiment, the description refers to the selected antibody containing the variable region of the heavy chain, deposited in ATSS under access number of MOUTH-9377, but which in the VH-region derived mutations germline I30S and N33S. In an additional embodiment, the description refers to the selected antibody that contains the variable regions of the heavy chain and light chain, deposited in ATSS under the access numbers of the MOUTH-9377 and MOUTH-9378, respectively, or the specified antibody in which VH-region POPs the Ana mutations germline I30S and N33S. In an additional embodiment, the description refers to the selected antibody that contains regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the heavy chain, deposited in ATSS under access number of MOUTH-9377, or contains the specified region CDR1, CDR2 and CDR3, if the specified variable region of the heavy chain contains mutations in the germ line I30S and N33S. In an additional embodiment, the description refers to the selected antibody that contains regions CDR1, CDR2 and CDR3 light chain variable region of the light chains are deposited in ATSS under access number of MOUTH-9378. In an additional embodiment, the description refers to the selected antibody that contains regions CDR1, CDR2 and CDR3 light chain and the region CDR1, CDR2 and CDR3 of the heavy chain variable regions of the heavy and light chains are deposited in ATSS under the access numbers of the MOUTH-9378 and MOUTH-9378, respectively, or contains the specified region CDR1, CDR2 and CDR3, if the specified variable region of the heavy chain contains mutations in the germ line I30S and N33S.

Antibodies according to the invention may constitute, for example, full-length antibodies, for example, subclass IgG1 or IgG4. Alternatively, antibodies may represent fragments of antibodies, such as Fab or Fab'2 fragments, or single-chain antibodies. In one case, this description applies to any anti is Elo, described above, which is a full-sized antibody of human subclass IgG1, in which at least one amino acid in the Fc region of the IgG1 subclass metirovan. In an additional case, at least one mutation occurs at position serine 247, alanine 338 or isoleucine 340. In an additional case, at least one mutation selected from the group consisting of S247D, A338L and I340E. In one additional case, the antibody contains mutations S247D, A338L and I340E.

In an additional aspect, the description refers to the selected monoclonal antibody containing heavy chain, represented in SEQ ID NO:9, or a conservative modification. For example, the antibody contains a heavy chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%or at least 99% identical to the amino acid sequence shown in SEQ ID NO:9. In an additional aspect, the description refers to the selected monoclonal antibody containing light chain, represented in SEQ ID NO:10, or conservative modifications. For example, the antibody includes a light chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%or at least 99% identical linakis is now sequence, shown in SEQ ID NO:10. In an additional aspect, the description refers to the selected monoclonal antibody containing heavy chain, represented in SEQ ID NO:9, or a conservative modification and light chain, represented in SEQ ID NO:10, or conservative modifications. For example, the antibody contains a heavy chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%or at least 99% identical to the amino acid sequence shown in SEQ ID NO:9 and a light chain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%or at least 99% identical to the amino acid sequence shown in SEQ ID NO:10.

In some embodiments, the implementation of the C-terminal lysine of the heavy chain of any of the described antibodies against α5β1 according to the invention decompose and, therefore, he is not represented. For example, in some embodiments, the implementation of the antibodies of the present invention contain a constant region of the heavy chain, represented in SEQ ID NO:43, but where the C-terminal lysine is not represented. In a large number of cases of heavy and light chains of antibodies against α5β1 may not necessarily include the signal sequence.

In additional is the SPECTA description relates to compositions, containing any of the antibodies or their antigenspecific sites described herein, and a pharmaceutically acceptable carrier.

In an additional aspect, the description refers to immunoconjugate containing any of the antibodies or their antigenspecific areas described in this document, associated with a therapeutic agent. In one case, therapeutic agent is a cytotoxin or a radioactive isotope. In an additional aspect of the description relates to compositions containing any immunoconjugate described herein, and a pharmaceutically acceptable carrier. The description also refers to bespecifically molecule containing an antibody or antigennegative area associated with the second functional fragment having binding specificity that is distinct from the binding specificity of the indicated antibodies or antigennegative plot.

Also provides compositions containing the antibody or antigennegative plot or immunoconjugate, or bespecifically molecule according to the invention and a pharmaceutically acceptable carrier.

The invention also covers a molecule of nucleic acids encoding antibodies or their antigennegative areas, as well as expressing vectors containing such nucleic acids, and cells of the host, the soda is containing such expressing vectors. One aspect, for example, represents an isolated nucleic acid molecule or expressing a vector containing the sequence represented in SEQ ID NO:11, or conservative modifications. An additional aspect is an isolated nucleic acid molecule or expressing a vector containing the sequence represented in SEQ ID NO:12, or conservative modifications. An additional aspect is an isolated nucleic acid molecule or expressing a vector containing a sequence selected from the group consisting of SEQ ID NO:21, 22, 31, 32, 41 and 42, or conservative modifications. The description also relates to transgenic mice containing the transgene of the heavy and light chains of human immunoglobulin, where the mouse expresses an antibody according to the invention, as well as hybridomas obtained from the mouse, where hybridoma produces the antibody according to the invention.

Description additionally refers to the cell host containing any of expressing the vectors described in this document.

In an additional aspect of the description relates to a method for obtaining antibodies against integrin α5β1, which includes the expression of antibodies in any cell, the owner described in this document and isolating the antibody from the host cell.

In an additional aspect, the OPI is the W relates to a method of inhibiting the growth of tumor cells, expressing the integrin α5β1, comprising bringing the cells into contact with the antibody or antigennegative area that are associated with integrin α5β1 person with KD1×10-7M or less and is able to induce antibody-dependent cellular cytotoxicity. In one case, the antibody is a fully human antibody. In an additional case, the antibody or antigennegative plot create to enhance its ability to induce antibody-dependent cellular cytotoxicity. In one additional case the amplification is achieved by mutation of at least one amino acid residue in the Fc-region.

In an additional aspect, the invention relates to a method of inhibiting the growth of tumor cells expressing the integrin α5β1, comprising bringing the cells into contact with any of the antibodies or their antigenspecific areas described in this document, in an amount effective for inhibiting the growth of tumor cells.

In an additional aspect, the invention relates to the use of any of the antibodies or their antigenspecific areas described in this document, for producing a medicinal product for the treatment of abnormal cell growth. In yet another additional aspect of the invention relates to any of the antibodies or the Academy of Sciences of evansvasilesco plots described in this document, for use in the treatment and/or diagnosis of abnormal cell growth. In one embodiment, the implementation of this method, the abnormal cell growth is a cancer, including but not limited to, mesothelioma, cancer of the hepatobiliary system (liver and biliary tract), primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, cancer of the gastrointestinal tract (stomach, colorectal and 12 duodenal ulcer), breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma the renal pelvis, neoplasms of the Central nervous system (CNS), pervichnuyu CNS lymphoma, nahodkinskuju lymphoma, spinal cord tumours, glioma, brain stem, pituitary adenoma, adrenocortical cancer, gall bladder, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above malignant tumors.

The description also relates to a method of creating a "second generation" of antibodies against α5β1 on the basis of the sequences of antibodies against α5β1 proposed in this document. For example, the description relates to a method for obtaining antibodies against α5β1, containing: (a) obtaining: (i) sequence variable regions of heavy chain antibodies containing the CDR1 sequence presented in SEQ ID NO:1, 13, 23 or 33, a CDR2 sequence presented in SEQ ID NO:2, 14, 24 or 34, and/or CDR3 sequence presented in SEQ ID NO:3, 15, 25 or 35; and/or (ii) the sequence of the variable region of the light chain antibodies containing the sequence of CDR1, presented in SEQ ID NO:4, 16, 26 or 36, a CDR2 sequence presented in SEQ ID NO:5, 17, 27 or 37, and/or CDR3 sequence presented in SEQ ID NO:6, 18, 28 or 38; (b) modifying at least one amino acid residue sequences of variable regions of heavy chain antibodies and/or sequence of the variable region of the light chain of the antibody to create, less is th least one of the modified sequence of the antibody; and (C) the expression of the modified sequence of the antibody in the form of protein.

Other features and advantages of the present description will be understood from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank, patents and published patent applications cited in this description of the invention, the right provided in this document as a reference.

Brief description of drawings

On figa presents the DNA sequence of the variable region of the heavy chain of the antibody V (SEQ ID NO:11); figv presents the amino acid sequence of the variable region of the heavy chain of the antibody V (SEQ ID NO:7) - CDR region are underlined; figs presents the DNA sequence of the variable region of the light chain antibodies V (SEQ ID NO:12); fig.1D presents the amino acid sequence of the variable region of the light chain antibodies V (SEQ ID NO:8) - CDR region are underlined; five presents the DNA sequence of the variable region of the heavy chain of the antibody S (SEQ ID NO:21); fig.1F presents the amino acid sequence of the variable region of the heavy chain of the antibody S (SEQ ID NO:19) - CDR region are underlined; fig.1G presents the DNA sequence of the variable region l is gcoi chain antibodies S (SEQ ID NO:22); on fign presents the amino acid sequence of the variable region of the light chain antibodies S (SEQ ID NO:20) - CDR region are underlined; Fig presents the DNA sequence of the variable region of the heavy chain of the antibody 1D9 (SEQ ID NO:31); fig.1J presents the amino acid sequence of the variable region of the heavy chain of the antibody 1D9 (SEQ ID NO:29) - CDR region are underlined. On FIGC presents the DNA sequence of the variable region of the light chain of the antibody 1D9 (SEQ ID NO:32); fig.1L presents the amino acid sequence of the variable region of the light chain of the antibody 1D9 (SEQ ID NO:30) - CDR region are underlined; film presents the DNA sequence of the variable region of the heavy chain of the antibody 2D2 (SEQ ID NO:41); fig.1N presents the amino acid sequence of the variable region of the heavy chain of the antibody 2D2 (SEQ ID NO:39) - CDR region are underlined; Figo presents the DNA sequence of the variable region of the light chain antibodies 2D2 (SEQ ID NO:42); figr presents the amino acid sequence of the variable region of the light chain antibodies 2D2 (SEQ ID NO:40) - CDR region are underlined; fig.1Q presents the amino acid sequence of the constant region of the heavy chain IgG1 with the underlined mutations S247D, A338L and I340E; and fig.1R presents the amino acid sequence of the constant region of the light C is PI IgG1.

Figure 2 presents an alignment of the variable domain of the heavy chain (VH) antibodies V with the corresponding germline sequence. In addition, the alignment of the variable domain of the light chain (VK) antibodies V with the corresponding germline sequence. CDR region are underlined, identical residues are represented by the dotted line, and the points indicate deletions.

Figure 3 presents the imposition sensogram obtained by the introduction of a large number of concentrations of recombinant extracellular domain α5β1 over immobilized W/DLE. These data were obtained in the presence of 4.0 mm CaCl2. The order of injections was from low to high concentration.

In figure 4 by FACS shows dose-dependent binding of antibodies W/DLE with HUVEC cells.

Figure 5 presents a comparison of the equilibrium dissociation constant for Fcγ-receptors in human and mouse. "wt" indicates IgG1 wild-type 22B5; "DLE" means W/DLE.

Figure 6 presents the results of the analysis of the blockade of adhesion of HUVEC cells. The results indicate the level of inhibition of adhesion of HUVEC cells to fibronectin for antibodies V and a large number of variants of the subclass, as well as negative control (VNA IgG1). Also presents the calculated values IC50.

Figure 7 presents the expression of α5 cells HUVEC and 20 tumor cell the lines, measured by Western blot.

On Fig presents in-vitro ADCC induced by antibody W/DLE compared to wt IgG1 22B5. On figa presents the analysis based on the detection of LDH measuring ADCC U87MG cells in the presence of cells RVMS through W/DLE and V wt IgG1. On FIGU presents the analysis on the basis of the detection ToxiLight measuring ADCC cells HUVEC in the presence of cells RVMS through W/DLE and V wt IgG1.

Figure 9 presents the analyses based on the determination of LDH, which indicate a considerable strengthening of the ADCC using antibodies W/DLE compared with wt antibody V IgG1 across a wide range of expression levels of the antigen.

Figure 10 presents inhibitory activity of antibodies W/DLE on the model of experimental metastasis A-Luc. Figa: the size of the metastasis in the lung, as measured by BLI for 8 week (n=11 for control group, n=14 for group V IgG2 and n=12 for group W/DLE). Figw: resumption of growth of lung tumors in the group treated W IgG2, after stopping treatment. For comparison, in the group treated W/DLE, there was a slight resumption of growth. Figs: Graph of Kaplan-Meier estimates of the degree of survival of animals from each treated group (endpoint = BLI 1×108photons/second), p<0,0001 when comparing the control group, treated by the media, with all other groups, p<0.05 using cf the ranking between groups W/DLE and V IgG2.

Figure 11 through FACS analysis shows the dose-dependent binding of the antibody 1D9, 1D9/DLE, 24C7/DLE, 2D2/DLE, and 22B5 W/DLE with HUVEC cells.

On Fig presents in-vitro ADCC induced by antibody 1D9, 1D9/DLE, 2D2/DLE, W/DLE and S/ DLE.

On Fig presents ADCC-dependent antitumor efficacy of antibody 1D9/DLE on syngeneic model of metastatic melanoma. Figa: Macroscopic picture of lung resected from all groups. Figw: Quantitative assessment of the weight of the lung (*p<0,05 when compared 1D9 IgG1 DLE with 1D9 IgG2). Figs: a Quantitative estimate of the number of metastatic colonies that are visible on the surface of the lung. Statistical analysis by analysis of variance ANOVA test and multiple comparisons of Bonferroni (*p<0,05, 1D9 IgG1 DLE compared with 1D9 IgG2; *p<0,05, 1D9 IgG1 DLE compared to IgG2 against KLH).

Detailed description

This description applies to the selection of monoclonal antibodies, in particular monoclonal antibodies person who specifically associated with α5β1 with high affinity. In some cases, the antibodies according to the invention originate from the specific sequences of the heavy and light chain germline and/or contain certain structural features, such as a CDR region containing a specific amino acid sequence. The description refers to the selected antibodies, methods for the teaching of such antibodies, immunoconjugates and bespecifically molecules containing such antibodies, and pharmaceutical compositions containing the antibodies, immunoconjugates and bespecifically molecules according to the invention. The description also relates to methods of using the antibodies, such as detection α5β1, and treatment of diseases associated with expression of α5β1, such as abnormal cell growth (e.g., cancer). Accordingly, the description also relates to methods of using antibodies against α5β1 or antigenspecific plots for treatment of a large number of types of abnormal cell growth, such as cancer.

To facilitate understanding of the present description of certain terms of the first definition. Additional definitions are given for a detailed description.

Unless otherwise defined herein, scientific and technical terms used in connection with the present description, must have values that are commonly understood by experts in the field. In addition, unless the context implies otherwise, the terms in the singular shall include the plural number and terms in the plural shall include the singular. In General, the terminology used for the cultivation of cells and tissues, molecular biology, immunology, Microbiology, genetics and hee the AI proteins and nucleic acids and hybridization, described herein, and methods of their implementation are well known and widely used in this field.

Unless otherwise specified, the methods and techniques according to the present description, as a rule, is carried out in accordance with methods well known in the field and described in a large number of General and more specific references that are cited and discussed in the present description of the invention. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring HarborLaboratoryPress,Cold Spring Harbor, NY (1990). Enzymatic reactions and methods purification is carried out in accordance with the manufacturer's specifications, as usually carried out in the field or as described in this document. The terminology used in relation to analytical chemistry, chemistry of organic synthesis and medicinal and pharmaceutical chemistry described in this document, and laboratory methods and techniques are well known and widely used in this field. For chemical synthesis, chemical analysis, receiving, mixing and delivery of medicines and treatment of patients using standard methods.

Used in this document each of the following terms has the meaning associated with it in this section.

As used in this document of the twenty standard amino acids and their abbreviations follow the accepted use. SeeImmunology-A Synthesis(2ndEdition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

The terms "α5β1 and integrin α5β1" are used interchangeably, and include variants, isoforms and species homologs of integrin α5β1 person. Native α5β1 person, for example, composed of subunits α5 (which is a sequence of predecessor, later splitting into two chains connected by a disulfide bridge (access number in GenBank R) and β1 subunit (which is a sequence of predecessor, subsequently processorsa in Mature form) (access number in GenBank R-1). It is known that the β1 subunit exists as multiple isoforms produced through alternative splicing (see, for example, access numbers in GenBank R-2, R-3, R-4 and R-5). Human antibodies against α5β1 according to the invention may in some cases cross-interact with α5β1 species other than human. In other cases, the antibodies may be completely specific in relation to human α5β1, and may not be observed interspecies cross-reactivity or cross-reactivity of other types.

By "immune response", as should be clear to experts who have in this area, include, but are not limited to, any detected antigen-specific or allogeneic activation of T-helper or cytotoxic T-cell response, production of antibody-mediated T-cell activation in allergic reactions and the like. The term includes the effect of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement)that causes selective damage to, destruction of, or elimination from the human body introduced pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmune response or pathological inflammation, normal cells or tissues.

Under "by signal transduction" understand the biochemical interaction between a large number of signal transduction molecules that are important in the transfer of signal from one part of cells in another part of the cell. It is used in this document, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules able to receive the signal and transfer the signal across the plasma membrane of cells. An example of a "cell surface receptor" of the present invention is αβ1 integrin.

The term "antibody", as set out in this document, understand whole antibodies and any of their antigennegative fragment (i.e. "antigennegative site") or single chains. By "antibody" is understood glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigennegative plot. Each heavy chain contains a variable region heavy chain (referred to in this document as VH) and the constant region of the heavy chain. The constant region of the heavy chain consists of three domains WithH1WithH2and CH3. Each light chain consists of a variable region light chain (referred to in this document as VL) and the constant region of the light chain. The constant region of the light chain consists of a single domain CL. Region VHand VLcan be further subdivided into the field of hypervariability, called scopes, complementarity determining (CDR), interspersed with regions that are more conservative, called frame regions (FR). Each VHand VLcomposed of the three CDR regions and four regions FR, located from amino end to the carboxy-end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable regions of the heavy and light chains contain binding on the men, which interacts with the antigen. The constant region of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including a large number of cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Within light and heavy chain variable and constant region are connected by a region "J", consisting of about 12 or more amino acids, with the heavy chain also includes the region "D", consisting of about 10 or more amino acids. Cm. in General, the manual Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)).

The term "antigennegative plot" of an antibody (or simply "part of the antibodies used in this document, understand one or more fragments of an antibody that retain the ability to specifically bind to the antigen (for example, integrin α5β1). It was shown that antigennegative function antibodies can perform fragments of full-length antibodies. Examples of binding fragments encompassed by the term "antigennegative plot" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of domains VLVHCLand CH1; (ii) F(ab')2-fragment, a bivalent fragment comprising two Fab fragments, connected by a disulfide bridge at the hinge region; (iii) Fd-f is agment, consisting of domains VHand CH1; (iv) an Fv fragment consisting of domains VLand VHone shoulder antibodies; (v) a dAb fragment (Wardet al., (1989)Nature341:544-546), which consists of domain VH; and (vi) the allocated region complementarity determining (CDR). Moreover, although the two domains of Fv-fragment, VLand VHthat are encoded by separate genes, they can be joined, using recombinant techniques, using a synthetic linker that will allow you to create them as a single protein chain in which the region VLand VHconstitute a pair with the formation of monovalent molecules (known as single-chain Fv (scFv); see, for example, article Birdet al.(1988)Science242:423-426; and Hustonet al. (1988)Proc. Natl. Acad. Sci. USA85:5879-5883). Such single-chain antibodies are also covered by the term "antigennegative plot" antibodies. These fragments of antibodies may be obtained using any suitable technique, including those techniques known to experts in this field, and the fragments can be skanirovaniya for suitability in the same manner as are intact antibodies.

Under "isolated antibody", as used in this document, see antibody, which is essentially free of other antibodies having excellent antigenic specificnosti (for example, the selected antibody that specifically binds to α5β1, almost free of antibodies that specifically bind to the antigens other than α5β1). The selected antibody that specifically binds to α5β1 may, however, have cross-reactivity with other antigens, such as molecules α5β1 other species. Moreover, the selected antibody can be essentially free of other cellular material and/or chemicals.

The terms "monoclonal antibody" or "composition of monoclonal antibodies", as used in this document, understand the preparation of antibody molecules of the same molecular composition. The compositions of monoclonal antibodies observed a single binding specificity and affinity for a particular epitope.

Under the term "human antibody" or "fully human antibody", as used in this document, understand antibodies having variable regions in which both the frame region, and the CDR region come from immunoglobulin sequences of the germline of the person. Furthermore, if the antibody contains a constant region, the constant region also comes from immunoglobulin sequences of the germline of the person. Human antibodies of the invention or their antigennegative areas may include amino acid residues not encoded by immuno oblikovani sequences of the germline of the person (for example, mutations introduced by random or site-specific mutagenesisin vitroor by somatic mutationin vivo). However, the term "human antibody", as used herein, does not include antibodies in which frame in the sequence of transplanted human CDR sequence derived from the germline of another mammal, such as a mouse.

The terms "monoclonal antibody human" or "fully human monoclonal antibody" refer to antibodies that have been the only binding specificity, having variable regions in which both the frame and the CDR region come from immunoglobulin sequences of the germline of the person. In one embodiment, the implementation of a monoclonal human antibodies are produced by hybridomas, which includes In-cell obtained from a transgenic animal other than human, such as a transgenic mouse, having a genome containing the heavy chain transgene and a light chain transgene, human In-cell merge with immortalizing cell.

The term "recombinant human antibody", as used herein, is understood by all human antibodies that receive, Express, create, or produce by recombinant methods, such as (a) antibodies, is dedicated from the animal (e.g., mouse)that is transgenic or transpromotional in terms of genes of human immunoglobulins, or from hybridoma obtained on the basis of this (additionally described below), (b) antibodies isolated from a host cell transformed to Express the human antibody, for example, from transfection, (C) antibodies isolated from a recombinant, combinatorial libraries of human antibodies, and (d) antibodies obtained, expressed, created or selected by any other means that involve splicing of the gene sequences of human immunoglobulins with other DNA sequences. Such recombinant human antibodies have variable regions in which the frame and the CDR region come from immunoglobulin sequences of the germline of the person. In some embodiments, implementation, however, such recombinant human antibodies can be subjected toin vitromutagenesis (or, when using animal, transgenic sequences Ig man,in vivosomatic mutagenesis) and thus the amino acid sequence of the VLand VH-regions of the recombinant antibodies are sequences that, despite the origin and relationship with VLand VHsequences of the germline of theh the rights, may not exist in nature in the germinal set of antibodies manin vivo.

Under used in this document the term "isotype" or "class" means the class of antibodies (e.g., IgM or IgG), which is encoded by the genes of the constant regions of the heavy chains. The constant domains of antibodies are not involved in binding to antigen, but they have a large number of effector functions. Depending on the amino acid sequence of the constant region of the heavy chain of the human antibody or immunoglobulin can be attributed to one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Structure and three-dimensional configurations of different classes of immunoglobulins are well known. It is known that a large number of classes of human immunoglobulins only IgG1, IgG2, IgG3, IgG4 and IgM person activate complement. It is known that people ADCC mediated by IgG1 and IgG3 human.

Under used in this document, the term "subclass" see more granularity within isotype gene constant region of the heavy chain, such as, for example, the subclasses IgG1, IgG2, IgG3 or IgG4 in the IgG isotype.

It is used in this document the term "connection" or "pharmaceutical compound" include antibodies, their antigennegative areas, immunoconjugate and bespecifically molecule.

The phrase "antibody, resposne the relevant antigen and antibody specific for an antigen" are used herein interchangeably with the term "antibody that specifically binds to the antigen".

The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" understand cell-mediated reaction in which nonspecific cytotoxic cells (e.g., NK cells, neutrophils, macrophages, etc.) recognize the antibody associated with the target cell, and after that cause lysis of the target cells. These cytotoxic cells that mediate ADCC, as a rule, Express Fc receptors (FcR). Basic cells mediating ADCC, (NK-cells) Express FcγRIII, whereas monocytes Express FcγRI, FcγRII, FcγRIII and/or FcγRIV. The FcR expression on hematopoietic cells is summarized in the article Ravetch and Kinet,Annu. Rev. Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule can be madein vitrothe ADCC analysis, such as analysis, described in U.S. patent 5500362 or 5821337. To be used for such analyses effector cells are mononuclear cells of peripheral blood (RVMS) and natural killer cells (NK-cells). Alternative or additionally, ADCC activity of the interesting molecules can be evaluated byin vivofor example, in animal models, such as the model described in article Clynes et al.,Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).

The terms "Fc receptor" or "FcR" usage is used to describe a receptor, which binds to Fc region of antibodies, where the Fc-region contains the hinge region and domains WithN2, and CN3 heavy chain. For example, FcR can be a FcR person with a native sequence. FcR may be a receptor that binds to IgG-antibody (a gamma receptor)and includes receptors of the subclasses of the FcγRI, FcγRII, FcγRIII and FcγRIV, including allelic variants and alternative splanirowannya forms of these receptors. To 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 in its cytoplasmic domain immunoreceptor the tyrosine activation motif (ITAM). Inhibiting receptor FcγRIIB contains in its cytoplasmic domain immunoreceptor the tyrosine inhibitory motif (ITIM) (see Daeron,Annu. Rev. Immunol., 15:203-234 (1997)). The FcR receptors are considered in the articles of 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). The term "FcR" in this document are covered by other FcR, including not identified currently receptors. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer IgG the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)). The main FcR-binding site on the Fc-fragments of immunoglobulins is located in the hinge region between domains WithN1 and CN2. This hinge region interacts with FcR1-3 different leukocytes and starts the attack of these cells to the target (Wines et al.,J. Immunol., 164:5313-5318 (2000)). The hinge area encompasses, but is not limited to the sequence described in U.S. patent 6165476.

The term "ability to induce antibody-dependent cellular cytotoxicity" means the ability of an agent, such as an antibody, to demonstrate ADCC, measured through analysis of(s)known(s) are specialists in this field. This activity is usually different binding Fc-region with a large number of receptors FcR. Not limited to any particular mechanism, the person skilled in the art should understand that the ability of antibodies to demonstrate ADCC may occur, for example, in accordance with its subclass such as IgG1 or IgG3), due to mutations introduced in the Fc region, or in accordance with modifications of the carbohydrate patterns in the Fc region of antibodies. Such modifications are described, for example, in patent publication U.S. 2007-0092521.

The term "derived human antibodies," you see any modified form of human antibodies, for example, conjugate antibodies and other agent or antibody.

The term "humanitariannet antibody" refer to antibodies in which the frame sequence transplanted human CDR sequences derived from the germline of another mammal, such as a mouse. In frame sequences person can be made an additional modification of the frame region.

The term "chimeric antibody" refer to antibodies in which the sequence of the variable regions are derived from the same species, and sequences of constant regions originate from other species, such as antibody, in which the sequence of the variable regions are from antibodies mouse, and sequences of constant regions originate from human antibodies.

The phrase "specifically binds", as used in this document, understand the connection, e.g., protein, nucleic acid, antibody and the like, which recognizes and binds to a specific molecule, but almost does not recognize or is not associated with other molecules in the sample. For example, an antibody or a peptide inhibitor, which recognizes and binds to cognatum ligand (e.g. antibody against α5β1, which is associated with his cognatum antigen α5β1) in the sample, but almost does not recognize or is not associated with other molecules in the about is the style. Thus, under specified conditions analysis specified binding fragment (e.g., antibody or antigennegative site) binds preferentially to a particular target molecule, for example, α5β1, and does not bind in a significant amount to other components present in the tested sample. For selection of an antibody that specifically binds with a target molecule, can be used a large number of formats analyses. For example, among the many studies that can be used to identify antibodies that specifically interacts with α5β1 include solid-phase immunoassay ELISA, immunoprecipitation, BIAcore, FACS and analysis of Western blot. Typically a specific or selective reaction should be at least twice background signal or noise and better more than 10 times above background, even more precisely, say that an antibody "specifically binds" to an antigen when the equilibrium dissociation constant (KD) ≤1 μm, for example, ≤100 nm, and for example, ≤10 nm.

Under used in this document, an antibody that "specifically binds to the integrin α5β1 person" refers to an antibody which binds to the integrin α5β1 person with KD1×10-7M or less, 5×10-8M or less, 3×10-8M or less, 1×10-8 M or less, or 5×10-9M or less.

The term "kon"used in this document, understand the rate of direct reaction, or the rate of Association of a specific interaction of the antibody-antigen, whereas the term "koff"used in this document, understand the rate of reverse reaction or dissociation rate of the specific interaction of antibody-antigen. The term "KD"used in this document, understand the dissociation constant, which is obtained from the ratio of koffkon(i.e. koff/kon) and is expressed as a molar concentration (M). Values of KDfor antibodies can be determined using methods standard in this area. One way to determine KDantibodies is the use of surface plasmon resonance, typically using a biosensor system such as the Biacore system®.

Under used in this document, the term "high affinity" in relation to IgG-antibodies antibody understand, with KD1×10-7M or less, 5×10-8M or less, or 5×10-9M or less for a target antigen. However, tying with "high affinity" may vary for other isotypes of antibodies. For example, binding with high affinity for IgM isotype antibody understand, with KD106 M or less, 10-7M or less, or 10-8M or less.

The term "compete", as used herein with respect to the antibody, understand that if the first antibody or antigennegative plot competes for binding with a second antibody or antigennegative plot, the binding of the first antibody with its cognatum epitope is reduced detektivami way in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Opposite, the binding of the second antibody to its epitope also detektivami reduces in the presence of the first antibody may be true, but not necessarily. That is, the first antibody can inhibit the binding of the second antibody to its epitope, but the second antibody may inhibit the binding of the first antibody with its corresponding epitope. However, if each antibody detektivami way inhibits the binding of another antibody with his cognatum epitope or ligand, or to the same, higher or lesser extent, say, that the antibody cross-competes" with each other for binding to the corresponding(them) they epitope(s). For example, cross-competing antibodies can contact the epitope or site of the epitope is bound to an the body, described in this document. The present invention encompasses the use of both competing and cross-competing antibodies. Regardless of the mechanism, which manifests such competition or cross-compete (e.g., steric mismatch, conformational change, or linking with the shared epitope or any part thereof, and the like), the person skilled in the art should understand, based on the ideas proposed in this document that such competing and/or cross-competing antibodies are encompassed by the invention and can be used for the methods described in this document.

The term "epitope" refers to any protein determinant capable of specific contact immunoglobulin or T-cell receptor. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or side chains of sugars, and usually have the characteristics of a specific three-dimensional structure, as well as specific charge characteristics. Conformational and conformational epitopes differ in that the binding with the first, but not last, decreases in the presence of denaturing solvents.

Under the "glycoforms" understand complex oligosaccharide structure that contains the concatenation of a large number uglev is the breaking units. Such structures are described, for example, the manualEssentials of GlycobiologyVarki et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), which also features an overview of the standard glycobiological signs. Such glycoforms include, but are not limited to, G2, G1, G0, G-1 and G-2 (see, for example, international patent publication WO 99/22764).

"Glycosylation pattern" is defined as a pattern of carbohydrate units that are covalently linked to protein (e.g., glycoforms), as well as the website(s)for which glycoform(s) covalently attached to the peptide backbone of a protein, more specifically to a protein of the immunoglobulin.

Apparently, antibodies expressed by different cell lines or in transgenic animals will have different glycoforms and/or patterns of glycosylation compared with each other. However, all antibodies encoded by nucleic acid molecules according to this description, or containing the amino acid sequence according to this description, are part of the present description, regardless of the glycosylation of these antibodies.

Under used in this document, the term "individual" understand any person or animal. The term "animal" see all vertebrates, for example, mammals and animals other than mammals, such as primates, sheep, dogs, Ko is Ki, horses, cows, chickens, amphibians, reptiles, etc.

Under used in this document, the term "treat" refers to reducing the frequency with which the patient experiences symptoms (i.e. tumor growth and/or metastasis, or other effect that is mediated through the number and/or activity of immune cells, and the like). The term refers to the introduction of the compounds or agents of the present invention to prevent or delay the onset of symptoms, complications, or biochemical evidence of disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition or disorder. The treatment may be prophylactic (to prevent or delay the onset of the disease or to prevent the manifestation of clinical or subclinical symptoms) or therapeutic suppression or alleviation of symptoms after the detection of the disease.

In more detail, a large number of aspects of the invention described in the following subsections.

Antibodies against α5β1

Antibodies according to the invention has certain functional features or properties of the antibodies. For example, antibodies specifically associated with α5β1 person. Preferably, the antibody according to the invention associated with α5β1 with high affinity, for example, with KD1 the 10 -7M or less.

Preferably, the antibody was associated with α5β1 person with KD5×10-8M or less, 2×10-8M or less, 5×10-9M or less, 4×10-9M or less, 3×10-9M or less, or 2.7×10-9M or less. In this area known assays to evaluate the binding ability of the antibodies to α5β1, these include, for example, ELISA, Western blot, RIA and analysis of flow cytometry. Suitable assays are described in detail in the examples. Kinetic analysis of binding (e.g., affinity of binding of the antibodies also can be assessed using assays known in this field, such as Biacore analysis.

Antibodies against α5β1 of the present invention is also able to induce antibody-dependent cellular cytotoxicity (ADCC). Such functionality can be achieved, for example, by applying specific subclasses (e.g., IgG1, IgG2, IgG3), including mutations in the Fc domain, which can further increase the level of ADCC activity of the antibodies. Such mutations and ways to measure ADCC are further outlined in the examples.

Monoclonal antibody V

One of the obvious antibodies according to the invention is a monoclonal antibody human V described in examples 1 and 2. Amino acid sequence of VHantibodies V presented on figv and is contained in SQ ID NO:7. Amino acid sequence of VLantibodies V presented on fig.1D and is given in SEQ ID NO:8. As shown in figv and figure 2, variable region heavy chain antibodies V contains two mutations compared with the gene sequence of the germline of the person. Namely, V contains a single mutation of isoleucine to serine at amino acid residue number 30 (I30S) and one mutation of asparagine to serine at amino acid residue number 33 (N33S). Under used in this document, marked "V" understand antibody, which were made by these mutations (I30S and N33S) variable regions of the heavy chain germ line.

Given that U can communicate with α5β1, the sequence VHand VLcan be "mixed and combined with other antibodies against α5β1 to create additional binding molecules against α5β1 according to the invention. Binding to α5β1 such "mixed and combined" antibodies can be tested using the assays of the binding described above and in the examples (e.g., ELISA assays). In one case, the mixing ratio of the combination of VHand VL-chain VHsequence specific pair of VH/VLreplaced similar structurally VH-sequence. Similarly, in another case VLsequence specific pair of VH/VL replaced similar structurally VLsequence.

In another aspect the invention relates to antibodies that contain region CDR1, CDR2 and CDR3 of the heavy and light chains of antibodies B. Amino acid sequence of CDR1 region region VHantibodies V presented in SEQ ID NO:1. Amino acid sequence of CDR2 region region VHantibodies V presented in SEQ ID NO:2. Amino acid sequence of CDR3-region region VHantibodies V presented in SEQ ID NO:3. Amino acid sequence of CDR1 region VL-region antibody V presented in SEQ ID NO:4. Amino acid sequence of CDR2 region VL-region antibody V presented in SEQ ID NO:5. Amino acid sequence of CDR3-region VL-region antibody V presented in SEQ ID NO:6. CDR-area is defined using the Kabat system (Kabat E.A.et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

Given that the antibody V binds to α5β1 and that antigennegative specificity is provided primarily by the regions CDR1, CDR2 and CDR3, the sequence of CDR1, CDR2 and CDR3 VH-scope and sequence of the CDR1, CDR2 and CDR3 VL-the field can be "mixed and combined" (i.e., CDR-various antibodies against α5β1 can be mixed and combined, although each antibody which they contain, as expected, CDR1, CDR2 and CDR3 region VHand CDR1, CDR2 and CDR3 region VLto create additional binding molecules against α5β1 according to the invention. Binding to α5β1 such "mixed and combined" antibodies can be tested using the assays of the binding described above and in the examples (e.g., ELISA assays, Biacore analysis). In one case, the mixing ratio of the combination of CDR sequences for the region VHthe sequence of CDR1, CDR2 and/or CDR3 of sequence VHis replaced with a structurally similar(s) sequence(s) CDR. Similarly, when mixing and combining sequences of CDR region VLthe sequence of CDR1, CDR2 and/or CDR3 of sequence VLas a rule, is replaced with a structurally similar(s) sequence(s) CDR. The person skilled in the art will easily understand that the new sequence VHand VLcan be created by replacing one or more sequences of CDR-areas VHand/or VLstructurally similar sequences from the CDR sequences described in this document.

Accordingly, in another aspect, the invention relates to the selected monoclonal antibody or its antigennegative site that contains: (a) variable region heavy the Oh chain CDR1, containing the amino acid sequence of SEQ ID NO:1; (b) the variable region of the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:2; (c) variable region of the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:3; (d) variable region light chain CDR1, containing the amino acid sequence of SEQ ID NO:4; (e) variable region light chain CDR2 containing the amino acid sequence of SEQ ID NO:5; and/or (f) variable region light chain CDR3 containing the amino acid sequence of SEQ ID NO:6, where the antibody specifically binds to α5β1, preferably with α5β1 person.

Monoclonal antibodies 2D2, 24C7 and 1D9

Another illustrative antibody according to the invention is a monoclonal antibody human 2D2 described in examples 1 and 8. Amino acid sequence region VHantibodies 2D2 presented in SEQ ID NO:39. Amino acid sequence region VLantibodies 2D2 presented in SEQ ID NO:40.

Another illustrative antibody according to the invention is a monoclonal antibody human S described in examples 1 and 8. Amino acid sequence region VHantibodies IS presented in SEQ ID NO:19. Amino acid sequence region VLantibodies IS presented in SEQ ID NO:20.

Another illustrative antibody according to the invention Ave is dstanley a monoclonal human antibody 1D9, described in examples 1 and 8. Amino acid sequence region VHantibody 1D9 presented in SEQ ID NO:29. Amino acid sequence region VLantibody 1D9 presented in SEQ ID NO:30.

Given that antibodies 2D2, S and 1D9 can contact α5β1, the sequence VHand VLthese antibodies can be mixed and combined with other antibodies against α5β1 to create additional binding molecules against α5β1 according to the invention. Binding to α5β1 such "mixed and combined" antibodies can be tested using the assays of the binding described above and in the examples (e.g., ELISA assays,). In one case when mixing and matching circuits VHand VLthe sequence VHa specific pair of VH/VLis replaced with a structurally similar sequence VH. Similarly, in another case, the sequence VLa specific pair of VH/VLis replaced with a structurally similar sequence VL.

In another aspect the invention relates to antibodies that contain region CDR1, CDR2 and CDR3 of the heavy and light chains of antibodies 2D2, S and 1D9. The corresponding amino acid sequences of these CDR regions are indicated below.

CDR-area is defined using the Kabat system (Kabat E.A.et al. (1991) Sequences of Proteins of Imunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

Given that antibodies 2D2, S and 1D9 associated with α5β1 and that antigennegative specificity is provided primarily by the regions CDR1, CDR2 and CDR3, the sequence of CDR1, CDR2 and CDR3 region VHand the sequence of CDR1, CDR2 and CDR3 region VLcan be "mixed and combined" (i.e., CDR-various antibodies against α5β1 can be mixed and combined, although each antibody will contain, as expected, CDR1, CDR2 and CDR3-region region VHand CDR1, CDR2 and CDR3-region region VLto create additional binding molecules against α5β1 according to the invention. Binding to α5β1 such "mixed and combined" antibodies can be tested using research associate, described above and in the examples (e.g., ELISA assays, Biacore analysis). In one case, the mixing ratio of the combination of CDR sequences for the region VHthe sequence of CDR1, CDR2 and/or CDR3 sequence VHis replaced with a structurally similar(s) sequence(s) CDR. Similarly, when the mixing and combination of CDR sequences for the region VLthe sequence of CDR1, CDR2 and/or CDR3 sequence VLas a rule, is replaced with a structurally similar(s) sequence(s) CDR. The person skilled in the art will easily understand, Thu the new sequence V Hand VLcan be created by substituting one or more sequences of CDR region VHand/or VLstructurally similar sequences from the CDR sequences described in this document.

Accordingly, in another aspect, the invention relates to the selected monoclonal antibody or its antigennegative site that contains: (a) the variable region of the heavy chain CDR1, containing the amino acid sequence of SEQ ID NO:33; (b) the variable region of the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:34; (c) variable region of the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:35; (d) variable region light chain CDR1, containing the amino acid sequence of SEQ ID NO:36; (e) variable region light chain CDR2, containing amino acid sequence of SEQ ID NO:37; and/or (f) variable region light chain CDR3 containing the amino acid sequence of SEQ ID NO:38; where the antibody specifically binds to α5β1, preferably α5β1 person.

In another aspect the invention relates to the selected monoclonal antibody or its antigennegative site that contains: (a) the variable region of the heavy chain CDR1, containing the amino acid sequence of SEQ ID NO:13; (b) the variable region of the heavy chain CDR2, sotiriadou the amino acid sequence of SEQ ID NO:14; (c) variable region of the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:15; (d) variable region light chain CDR1, containing the amino acid sequence of SEQ ID NO:16; (e) variable region light chain CDR2 containing the amino acid sequence of SEQ ID NO:17; and/or (f) variable region light chain CDR3 containing the amino acid sequence of SEQ ID NO:18; where the antibody specifically binds to α5β1, preferably α5β1 person.

In another aspect the invention relates to the selected monoclonal antibody or its antigennegative site that contains: (a) the variable region of the heavy chain CDR1, containing the amino acid sequence of SEQ ID NO:23; (b) the variable region of the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:24; (c) variable region of the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:25; (d) variable region light chain CDR1, containing the amino acid sequence of SEQ ID NO:26; (e) variable region light chain CDR2 containing the amino acid sequence SEQ ID NO:27; and/or (f) variable region light chain CDR3 containing the amino acid sequence of SEQ ID NO:28; where the antibody specifically binds to α5β1, preferably α5β1 person.

Antibodies having specific sequences ZAR is disavow line

In some aspects, the antibody according to the invention contains the variable region of the heavy chain of a particular gene heavy chain immunoglobulin germ line and/or the variable region of the light chain of a particular gene light chain immunoglobulin germ line.

For example, in one aspect the invention relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain, which is a product of or derived human gene VH4-39, where the antibody specifically binds to α5β1. In another aspect the invention relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain, which is a product of or derived human gene VH3-30 .3, where the antibody specifically binds to α5β1. In another aspect the invention relates to the selected monoclonal antibody or its antigennegative section containing the variable region of light chain, which is a product of or derived human gene VKL6, where the antibody specifically binds to α5β1. In yet another illustrative aspect of the invention relates to the selected monoclonal antibody or its antigennegative the site where the antibody:

(a) contains the variable region of the heavy chain, which is a product of or derived human gene VH4-39 (this gene encodes the amino acid sequence shown in SEQ ID NO:7) or gene human 3-30 .3 (this gene encodes the amino acid sequence shown in SEQ ID NO:19, 29 or 39);

(b) contains a variable region light chain, which is a product of or derived human gene VKL6 (this gene encodes the amino acid sequence shown in SEQ ID NO:8, 20, 30 or 40); and

(c) specifically binds to α5β1, preferably α5β1 person.

An example of an antibody having the VHand VLsequences of genes VH4-39 and VKL6, respectively, is V. Examples of antibodies having VHand VLsequencegene VH3-30 .3 and VKL6, respectively, are S, 2D2 and 1D9.

Used in this document, the human antibody contains a variable heavy or light chain, which are the "product" or "derived" a particular germline sequence if the variable regions of antibodies derived from the system, which uses immunoglobulin germline genes of the person. This includes immunizing a transgenic mouse carrying the genes of immunoglobulins che is oweka, using the antigen of interest or screening libraries of human immunoglobulin genes in the format of phage display using the antigen of interest. The antibody of the person, which is a "product" or "derived" immunoglobulin sequence to the germline of the person can be identified into force of this by comparing the amino acid sequences of human antibodies with amino acid sequences of immunoglobulin germline of man and selection immunoglobulin sequence to the germline of the person that is closest in sequence (i.e. has the highest % identity) to the sequence of human antibodies. The antibody of the person, which is a "product" or "derivative" of a specific immunoglobulin sequence to the germline of the person, may contain amino acid differences compared to the germline sequence, due to, for example, natural somatic mutations or intentional introduction of site-specific mutations. However, the selected human antibody, typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by immunoglobulin gene germline human, and content of the it amino acid residues, to identify the antibody of man as a human when compared with the amino acid sequences of immunoglobulin germline of other species (e.g., sequences of the germline of the mouse). In some cases, the antibody may be at least 95% or even at least 96%, 97%, 98% or 99% identical in amino acid sequence to the amino acid sequence encoded by immunoglobulin gene germ line. In some cases, the antibody man identical in amino acid sequence to the amino acid sequence encoded Ig genome of the germ line. As a rule, human antibodies derived from a particular germline sequence of a person, there will be no more than 10 amino acid differences from the amino acid sequence encoded by immunoglobulin gene germline person. In some cases, human antibodies may be no more than 5 or even no more than 4, 3, 2 or 1 amino acid differences from the amino acid sequence encoded by immunoglobulin gene germ line.

Homologous antibodies

In yet another aspect, the antibody according to the invention contains variable regions of the heavy and light chains containing amino acid posledovatel the activity, which homologous amino acid sequences of visual antibodies described in this document, and where the antibodies retain the desired functional properties of antibodies against α5β1 according to the invention.

For example, the invention relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain and the variable region of light chain, where:

(a) the variable region of the heavy chain contains an amino acid sequence that is at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO:7, 19, 29 and 39;

(b) variable region light chain contains an amino acid sequence that is at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO:8, 20, 30 and 40; and antibodies are observed in one or more of the following properties:

(i) the antibody binds to human α5β1 with KD1×10-7M or less;

(ii) the antibody is able to induce antibody-dependent cellular cytotoxicity.

In a large number of examples, the antibody may represent, for example, a human antibody, humanitariannet antibody or chimeric antibody.

In other examples, the amino acid sequence of VHand/or VLcan be 85%, 90%, 95%, 6%, 97%, 98% or 99% homologous to the sequences above. The antibody with the region VHand VLhaving high (i.e. 80% or greater) homology to the regions VHand VLsequences above can be obtained by mutagenesis (e.g., site-specific or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NO:7, 19, 29, 39, 8, 20, 30 and/or 40, followed by testing of the encoded altered antibody against stored functions (i.e. the properties listed in (i) and/or (ii) above)using the functional assays described in this document.

Used in this document, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions belonging to the sequence (ie,% homology = # of identical positions/total # of positions × 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, described in neogranichena what were the examples below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)), which is built into the ALIGN program (version 2.0), using a table of residues weights RAM, the penalty for continuing gap 12 and the penalty for gap 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which is embedded in the GAP program in the GCG software package (available at ), using either a matrix Blossum 62 or matrix RAM, and the penalty for opening a gap 16, 14, 12, 10, 8, 6 or 4, and for his continued 1, 2, 3, 4, 5 or 6.

Additionally or alternatively, the protein sequence of the present invention may additionally be used as a "search sequence" to perform a search in the published databases, for example, identify related sequences. The search can be carried out using the XBLAST program (version 2.0) authors Altschulet al. (1990)J. Mol. Biol.215:403-10. Search proteins by BLAST can be carried out using the XBLAST program, weight align = 50, word length = 3 to obtain amino acid sequences homologous to the antibody molecules according to the invention. To obtain alignments with what asriani with the purposes of comparison may be used by the program Gapped BLAST, described in article Altschulet al.(1997)Nucleic Acids Res.25(17):3389-3402. When using the programs BLAST and Gapped BLAST can be used the default parameters of the respective programs (e.g., XBLAST and NBLAST). Cm. http://www.ncbi.nlm.nih.gov.

Antibodies with conservative modifications

In some cases, the antibody according to the invention contains a variable region heavy chain comprising sequences of CDR1, CDR2 and CDR3, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3, where one or more of these CDR sequences contain certain amino acid sequences on the basis of demonstrable antibodies described herein (e.g., V, 1D9, 24C7 and 2D2), or conservative modifications, and where the antibodies retain one or more desired functional properties of antibodies against α5β1 according to the invention. Accordingly, the invention relates to the selected monoclonal antibody or its antigennegative section containing the variable region of the heavy chain comprising sequences of CDR1, CDR2 and CDR3, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3, where:

(a) sequence of the variable region of the heavy chain CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO:3, 15, 25 and 35 and their conservative modificati is;

(b) sequence of the variable region of the light chain CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO:6, 18, 28 and 38 and their conservative modifications; and antibodies are observed in one or more of the following properties:

(i) the antibody binds to human α5β1 with KD1×10-7M or less;

(ii) the antibody is able to induce antibody-dependent cellular cytotoxicity.

In other cases, the sequence of the variable region of the heavy chain CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO:2, 14, 24 and 34 and their conservative modifications; and the sequence of the variable region of the light chain CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO:5, 17, 27 and 37 and conservative modifications. In another case, the sequence of the variable region of the heavy chain CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NO:1, 13, 23 and 33 and their conservative modifications; and the sequence of the variable region of the light chain CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NO:4, 16, 26 and 36 and their conservative modifications.

Under used in this document, the term "conservative modifications" understand amino acid modified the paths which does not substantially affect or do not alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include substitutions, additions and deletions of amino acids. Modifications can be introduced into the antibody according to the invention, standard techniques known in this field, such as site-specific mutagenesis and PCR-mediated mutagenesis. Conservative modification of this sequence may include sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the sequence. Conservative substitutions of amino acids are replaced, in which amino acid residue is substituted with amino acid residue having a similar side chain. In this area identified a family of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side C is pami (for example, alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies according to the invention can be substituted by other amino acid residues from the same family of the side chains and the altered antibody can be tested against stored functions (i.e. the properties listed in (i) and (ii) above)using the functional assays described herein. Another type of amino acid modification is the elimination of pairs of asparagine-glycine, which form the sites of potential desametasone, by changing one or both residues. In another example, splintered the C-terminal lysine of the heavy chain of antibodies against α5β1 according to the invention and it is, therefore, not presented. The cleavage of C-terminal lysine can be developed in advance or may be the result of conditions used for the expression and purification of antibodies. In many cases, the heavy and light chains of antibodies against α5β1 may optionally include a signal sequence.

Antibodies that bind to the same epitope as an illustrative antibodies against α5β1 according to the invention

the other aspect of the invention relates to antibodies, which bind to the same epitope on human α5β1, and that any visual monoclonal antibody against α5β1 according to the invention (i.e. antibodies that are able to cross-compete for binding to α5β1 with any monoclonal antibody according to the invention). For example, the reference antibody for studies of cross-compete can be a monoclonal antibody V (having the sequence VHand VLpresented in SEQ ID NO:7 and 8, respectively) or the monoclonal antibody S (having the sequence VHand VLpresented in SEQ ID NO:19 and 20, respectively), or monoclonal antibody 1D9 (having the sequence VHand VLpresented in SEQ ID NO:29 and 30, respectively), or monoclonal antibody 2D2 (having the sequence VHand VLpresented in SEQ ID NO:39 and 40, respectively). Such cross-competing antibodies can be identified based on their ability to cross-compete with antibodies V, S, 1D9, or 2D2 in standard assays of binding to α5β1. For example, to demonstrate cross-competition with visual antibodies of the present invention can be used BIAcore analysis, ELISA assays or flow cytometry. The ability of a test antibody to inhibit the binding of, for example, antibodies V, S, 1D9, or 2D2 with α5β1 person indicates that the test antibody can compete with antibodies V, S, 1D9, or 2D2 for binding to α5β1 person and thus binds to the same epitope on human α5β1, and antibodies V, S, 1D9, or 2D2. In one case, the antibody that binds to the same epitope on human α5β1, and antibodies V, S, 1D9, or 2D2, is a monoclonal antibody man. Such monoclonal antibodies can be obtained and allocated as described, for example, in the examples.

Created and modified antibodies

The antibody or antigennegative plot according to the invention can additionally be obtained using an antibody having one or more sequences of VHand/or VLdescribed in this document as source material to create a modified antibodies, and modified antibody may have altered properties with respect to the original antibody. The antibody may be generated by modifying one or more residues within one or both variable regions (i.e., VHand/or VL), for example, in one or more CDR regions and/or in one or more frame regions. Additionally or alternatively, an antibody can be generated by modification of residues in the constant(s) about the Asti(s), for example, to change the effector(s) function(s) of the antibody.

One of the types create a variable regions, which may be implemented, is a transplant CDR. Antibodies interact with target antigens predominantly through amino acid residues that are located in six regions, determining complementarity, heavy and light chain (CDR fields). Therefore, the amino acid sequence of the CDR regions differ more between individual antibodies than the sequence outside the CDR regions. Because CDR sequences are responsible for most interactions, antibody-antigen, it is possible to Express recombinant antibodies that mimic the properties of specific natural antibodies by design expressing vectors that include CDR sequences from the specific natural antibodies transplanted to a frame sequence excellent antibody with different properties (see, for example, articles Riechmann L.et al.(1998)Nature332:323-327; Jones, P.et al. (1986)Nature321:522-525; Queen, C.et al.(1989)Proc. Natl. Acad. Sci. U.S.A.86:10029-10033; U.S. patent 5225539 author Winter, and U.S. patent 5530101, 5585089, 5693762 and 6180370 authors Queenet al.)

Accordingly, another aspect of the invention relates to the selected monoclonal antibody or its antigennegative is costco, containing the variable region of the heavy chain comprising sequences of CDR1, CDR2 and CDR3 containing amino acid sequence selected from the group consisting of SEQ ID NO:1, 13, 23 and 33, SEQ ID NO:2, 14, 24 and 34 and SEQ ID NO:3, 15, 25 and 35, respectively, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3 containing amino acid sequence selected from the group consisting of SEQ ID NO:4, 16, 26 and 36, SEQ ID NO:5, 17, 27 and 37, and SEQ ID NO:6, 18, 28 and 38, respectively. Thus, such antibodies contain the CDR sequences of the regions VHand VLmonoclonal antibodies V, S, 1D9, or 2D2, but may contain the frame sequence that is different from the frame sequences of these antibodies.

These frame sequences can be obtained from published DNA database or published references, which include gene sequences of antibody germline. For example, the DNA sequence of a germ-line genes for the variable regions of the heavy and light chains can be found in the database sequences of the germline of the person "VBase" (available online ), as well as in the manual Kabat E.A.et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; articles I.M. Tomlinsonet al. (1992),J. Mol. Biol.227:776-798; and J.P.L. Coxet al. (1994),Eur. J. mmunol .24:827-836; the contents of which are expressly contained in this document by reference). As another example, the DNA sequence of the germline gene variable regions of the heavy and light chains can be found in Genbank. For example, the following sequence of heavy chain germline found in mouse HCo7 HuMAb available in the accompanying non-access database Genbank: 1-69 (NG_0010109, NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637). As another example, the following sequence of heavy chain germline found in mouse HCo12 HuMAb available in the accompanying non-access database Genbank: 1-69 (NG_0010109, NT_024637 and BC070333), 5-51 (NG_0010109 and NT_024637) and 4-34 (NG_0010109 and NT_024637), 3-30 .3 (H) and 3-23 (AJ406678).

To frame sequences used antibodies according to the invention, include, but are not limited to, sequences that are structurally similar frame sequences used by selected antibodies according to the invention, for example, a similar frame sequences VH4-39 and/or VH3-30 .3 and/or frame sequences VKL6 used in visual monoclonal antibodies according to the invention. For example, the sequence of CDR1, CDR2 and CDR3 region VHand the sequence of CDR1, CDR2 and CDR3 region VLmouthbut transplanted in the frame region, which have a sequence identical to the sequence found in immunoglobulin gene germ line, which is a frame sequence, or the CDR sequences can be grafted in a frame region, which contain one or more mutations compared to the germline sequences. For example, it was found that in some cases it is useful to mutate residues in frame areas to preserve or enhance antigennegative the ability of antibodies (see, for example, U.S. patents 5530101; 5585089; 5693762 and 6180370 authors Queenet al.).

Another type of modification of the variable regions is matirovanie amino acid residues in regions CDR1, CDR2 and/or CDR3 regions VHand/or VLto improve one or more binding properties (e.g., affinity) of interest antibodies. To introduce the mutation(s) can be made site-specific mutagenesis or PCR-mediated mutagenesis, and the effect on the binding of an antibody, or other interesting functional property can be evaluated in the analysis ofin vitroorin vivodescribed in this document and proposed examples. As a rule, impose conservative modifications (described above). Mutations may represent amino acid substitutions, additions or deletions. Balayage, as a rule, change no more than one, two, three, four or five residues in the CDR field.

Accordingly, in another embodiment, the invention relates to the selection of monoclonal antibodies against α5β1 or antigennegative areas containing the variable region of the heavy chain, comprising: (a) a CDR1 region region VHcontaining the amino acid sequence selected from the group consisting of sequences SEQ ID NO:1, 13, 23 and 33, and amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additives compared with sequences SEQ ID NO:1, 13, 23 or 33; (b) CDR2 region region VHcontaining the amino acid sequence selected from the group consisting of sequences SEQ ID NO:2, 14, 24 and 34, and amino acid sequence having one, two, three, four or five aminokislotnykh substitutions, deletions or additives compared with sequences SEQ ID NO:2, 14, 24 or 34; (c) CDR3-region region VHcontaining the amino acid sequence selected from the group consisting of sequences SEQ ID NO:3, 15, 25 and 35, and amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additives compared with sequences SEQ ID NO:3, 15, 25 or 35; (d) CDR1 region region VLthe content of asuu amino acid sequence, selected from the group consisting of sequences SEQ ID NO:4, 16, 26 and 36, and amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additives compared with sequences SEQ ID NO:4, 16, 26 or 36; (e) a CDR2 region region VLcontaining the amino acid sequence selected from the group consisting of sequences SEQ ID NO:5, 17, 27 and 37, and the amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additives compared with sequences SEQ ID NO:5, 17, 27 or 37; and (f) CDR3-region region VLcontaining the amino acid sequence selected from the group consisting of sequences SEQ ID NO:6, 18, 28 and 38, and amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additives compared with sequences SEQ ID NO:6, 18, 28 or 38.

To the antibodies of the invention include antibodies in which modifications are carried by the frame remains in the region VHand/or VLfor example, to improve properties of antibodies. Typically, such modifications of the frame regions is performed with the purpose of reducing the immunogenicity of antibody. For example, one way is a "return matirovanie" one or more residues frame to fit the existing sequence germ line. More specifically, the antibody that has undergone somatic mutation may contain frame residues, which differ from the germline sequence from which the antibody occurs. These residues can be identified by comparison of frame sequences of the antibodies with the germline sequences from which the antibody occurs. To return sequences of frame area to their germline configuration, the somatic mutations can be "back motivovany" to the germline sequence by, for example, site-specific mutagenesis or PCR-mediated mutagenesis.

Another type of modification of the frame region includes matirovanie one or more residues in the framework region, or even within one or more CDR regions to remove T-cell epitopes, so as to reduce the potential immunogenicity of the antibody. This approach is also referred to as reimmunization", and he further described in patent publication U.S. 20030153043 authors Carret al.

In addition to or in contrast to the modifications created in the framework or CDR regions, antibodies of the invention can be created by incorporating modifications in the Fc region, typically to modify one or more functional properties of antibodies, such as peri is on the half-life of serum, fixation of complement, binding to Fc receptor and/or an antigen of cellular cytotoxicity. Moreover, the antibody according to the invention may be chemically modified (for example, the antibody can be attached to one or more fragments or modified to change its glycosylation, again to change one or more functional properties of the antibody. Each of these aspects is described in more detail below. The numbering of the residues in the Fc-region corresponds to the EU index in Kabat.

In one case, the hinge region domain SN modify in such a way that change the number of cysteine residues in the hinge region, for example, increase or decrease. This approach is additionally described in U.S. patent 5677425 authors Bodmeret al. The number of cysteine residues in the hinge region IN change, for example, to facilitate Assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another case are matirovanie hinge region of Fc fragment of antibodies to reduce the biological half-life of the antibody. More specifically, in the edge region domains CH2-CH3 Fc-hinge fragment is administered one or more amino acid mutations, so that the binding of an antibody with staphylococcal protein A (SpA) is weakened compared to hydrogen bonds is the W SpA native Fc-hinge domain. This approach is further described in U.S. patent 6165745 authors Wardet al.

In another case, the antibody is modified to increase its biological half-life. Perhaps a large number of approaches. For example, can be integrated with one or more of the following mutations: T252L, T254S, T256F, as described in U.S. patent 6277375 author Ward. Alternatively, to increase the biological half-life of the antibody may be modified in CN domain or CL-region in such a way as to contain the epitope binding receptor of salvation taken from two loops of a CH2 domain of the Fc region of immunoglobulin IgG, as described in U.S. patent 5869046 and 6121022 authors Prestaet al.

In other cases, to change the effector(s) function(s) of antibodies alter Fc-region by replacing at least one amino acid residue is an excellent amino acid balance. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a good amino acid balance, so that the antibody will have an altered affinity in relation to the effector ligand, but it will keep antigennegative the ability of the parent antibody. Effector ligand, the affinity to which change can be a Fc-receptor or the C1 component of complement. This approach is described in more detail is highlighted in U.S. patent 5624821 and 5648260 authors Winter et al.

In another case, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a good amino acid balance, so that the antibody will have altered C1q binding and/or reduced or absent complementability cytotoxicity (CDC). This approach is further described in U.S. patent 6194551 authors Idusogieet al.

In another example, alter one or more amino acid residues in the positions of the amino acids 231 and 239, thus altering the ability of the antibody to fix complement. This advanced approach is described in PCT publication WO 94/29351 authors Bodmeret al.

In another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of antibodies to Fcγ-receptor by modifying one or more amino acids at the following positions: 238, 239, 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, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398. 414, 416, 419, 430, 434, 435, 437, 438 or 439.

This advanced approach is described in PCT publication WO 00/42072 author Presta. Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and described variants with improved swazilan the eat (see Shields R.L.et al. (2001)J. Biol. Chem.276:6591-6604). It is shown that specific mutations at positions 256, 290, 298, 333, 334 and 339 improve binding to FcγRIII. In addition, it is shown that these combined mutations improve binding to FcγRIII: TA/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. For example, the antibody V/DLE described in this document, is a subclass IgG1 isotype IgG, but introduced by site-specific mutagenesis of the following mutations in comparison with the subclass IgG1 wild type): S247D; A338L and I340E. Similarly, as described in more detail below, these mutations S247D, A338L and I340E were introduced in monoclonal antibodies S, 1D9 and 2D2. As further described in the examples, these mutations may increase the affinity of antibodies to Fcγ-receptors and, thus, increase its effector function. Therefore, the invention relates to an antibody that contains at least one mutation in the Fc-region and has detektirano large ADCC response than otherwise identical antibody that does not contain at least one mutation.

In another example, modify the glycosylation of antibodies. For example, can be created glycosylamine antibody (i.e. the antibody with the lack of glycosylation). Glycosylation may be amended, for example, increasing the affinity of the antibody to the antigen. Such carbohydrate modifications can be accomplished, in the example, by changing one or more glycosylation sites in the sequence of the antibody. For example, can be carried out one or more amino acid substitutions that lead to the elimination of one or more sites of glycosylation frame region variable region, precluding glycosylation at this site. This glycosylamine may increase the affinity of the antibody to the antigen. This approach is described in more detail in U.S. patent 5714350 and 6350861 authors Coet al.

Additionally, or alternatively, may be generated antibody that has an altered type of glycosylation, such as hepatocanalicular antibody having reduced amounts fucosamine residues or an antibody having increased the number of bisecting GlcNac structures. It is shown that such altered patterns of glycosylation increase ADCC-the ability of the antibodies. Such carbohydrate modifications can be accomplished, for example, by expression of antibodies in the cell host with a modified mechanism of glycosylation. In this area were described cells with a modified mechanism of glycosylation, and they can be used as host cells that Express recombinant antibodies according to the invention, thus producing an antibody with altered glycosylation. Nab is emer, cell lines Ms704, Ms705, and Ms709 missing gene fucosyltransferase FUT8 (alpha (1,6) fucosyltransferase), so the antibodies expressed on cell lines Ms704, Ms705, and Ms709, no fucose their carbohydrates. Cell lines Ms704, Ms705, and Ms709 FUT8-/-created by targeted destruction of the FUT8 gene in cells of Cho/DG44, using two replacement vectors (see patent publication U.S. 20040110704 authors Yamaneet al. and article Yamane-Ohnukiet al. (2004)Biotechnol. Bioeng87:614-22). As another example, in EP 1176195 authors Hanaiet al. describes a cell line with a functionally destroyed FUT8 gene, which encodes fucosyltransferase, so that antibodies expressed in this cell line, observed gipoholesterinova due to the reduction or elimination of the enzyme, the corresponding alpha-1,6-linkages. The authors Hanaiet al. also describes cell lines that have low enzymatic activity against adding fucose to N-acetylglucosamine which binds to Fc region of the antibody, or do not have the enzymatic activity, e.g., a myeloma cell line rat YB2/0 (ATCC CRL 1662). In PCT publication WO 03/035835 author Presta describes a variant Cho cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hepatocanalicular the antibodies is, expressed in a cage-the master (see also Shields R.L.et al. (2002)J. Biol. Chem.277:26733-26740). In PCT publication WO 99/54342 authors Umanaet al. describes cell lines created for the expression of glycoprotein-modified glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), so that antibodies expressed in established cell lines, the observed increased bisectorial structure GlcNac, which leads to an increased ADCC activity of the antibodies (see also Umanaet al. (1999)Nat. Biotech.17:176-180). Alternatively, pokosnye remains antibodies can be derived using the enzyme fucosidase. For example, fucosidase alpha-L-fucosidase removes antibodies fucosamine residues (Tarentino A.L.et al. (1975)Biochem.14:5516-23).

Another modification of the antibodies according to the invention is tahilramani. The antibody can be pagalilauan order, for example, to increase biological (e.g., serum) half-life of the antibody. For tahilramani antibodies antibody or its fragment, as a rule, lead in contact with the polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to antibody or antibody fragment. As a rule, tahilramani carried out through the reaction allerban the I or the alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Under used in this document, the term "polyethylene glycol" understand any form of PEG, which was used to obtain the derivatives of other proteins, such as mono (C1-C10) alkoxy - or aryloxypropanolamine or polietilenglikolya. In some cases bagilishema antibody is glycosylamine antibody. In this area there are ways to tahilramani proteins, and they can be applied to the antibodies according to the invention. See, for example, EP 0154316 authors Nishimuraet al. and ER 0401384 authors Ishikawaet al.

How to create antibodies

As described above, antibodies against α5β1, having the sequence VHand VLdescribed in this document can be used to create new antibodies against α5β1 by modifying the sequences VHand/or VLor constant(s) region(s), connected(s) with them. Thus, in another aspect of the invention, the structural features of antibodies against α5β1 according to the invention, such as antibodies V, S, 1D9, or 2D2, used to create related structurally antibodies against α5β1, which retains at least one functional property of the antibodies according to the invention, such as binding to α5β1 person. For example, one or more CDR regions of antibodies V, S, 1D9, or 2D2 or mutations can be volun who inany using recombinant techniques known frame areas and/or other CDR regions to create additional, created using recombinant techniques, the antibody against α5β1 according to the invention described above. Other types of modifications include modifications described in the previous section. The source material for the method of creation represents one or more sequences of VHand/or VLaccording to this invention, or one or more of their CDR regions. To create a new antibodies is not necessary actually to get (i.e. to Express in the form of protein) antibody having one or more sequences of VHand/or VLaccording to this invention, or one or more of their CDR regions. Enough information contained in the sequence(s), can be used as source material to create the sequence(s) "second generation"received(s) from the source(s) sequence(s), and then the sequence(s) "second generation" receive and Express in the form of protein.

Accordingly, in another aspect the invention relates to a method for producing antibodies against α5β1, containing:

(a) obtaining: (i) sequence variable regions of the heavy chain of the antibody containing a CDR1 sequence selected from the group consisting of SEQ ID NO:1, 13, 23 and 33, a CDR2 sequence selected from the group consisting of SEQ ID NO:2, 14, 24 and 34, and/or posledovatelno the ü CDR3, selected from the group consisting of SEQ ID NO:3, 15, 25 and 35; and/or (ii) the sequence of the variable region of the light chain of the antibody containing a CDR1 sequence selected from the group consisting of SEQ ID NO:4, 16, 26 and 36, a CDR2 sequence selected from the group consisting of SEQ ID NO:5, 17, 27 and 37, and/or a CDR3 sequence selected from the group consisting of SEQ ID NO:6, 18, 28 and 38;

(b) modifying at least one amino acid residue sequences of variable regions of heavy chain antibodies and/or sequence of the variable region of the light chain of the antibody to create at least one altered sequences of the antibodies; and

(c) the expression of the modified sequence of the antibody in the form of protein.

To obtain and expression of the modified sequence of the antibody can be used standard molecular biology techniques.

Preferably, the antibody encoded by the altered(s) sequence(s) of antibodies, represented the antibody that retains one, some or all of the functional properties of antibodies against α5β1 described in this document, these functional properties include, but are not limited to:

(i) binding to α5β1 person with KD1×10-7M or less;

(ii) the ability to induce ADCC.

The functional properties of the ISM the United antibodies can be assessed, using standard assays available in this area and/or described herein, such as the assays provided in the examples (e.g., flow cytometry, analysis of binding).

In some aspects, methods generate antibodies according to the invention, mutations can be introduced randomly or selectively along all or part of the sequence that encodes the antibody against α5β1, and the resulting modified antibodies against α5β1 can be skanirovaniya against binding activity and/or other functional properties as described herein. Methods mutation engine described in this field. For example, in PCT publication WO 02/092780 the author of the Short describes how to create and screening of mutations antibodies, using saturating mutagenesis, Assembly, artificial legirovaniem or a combination thereof. Alternatively, in PCT publication WO 03/074679 authors Lazaret al.describes ways of using computer methods of screening to optimize the physico-chemical properties of antibodies.

Molecules of nucleic acids encoding antibodies of the invention

Another aspect of the invention relates to nucleic acid molecules that encode the antibodies according to the invention. The nucleic acid may be present in whole cells, cell lysate, or in a partially purified or substantially pure FD is IU. Nucleic acid "allocate" or "do practically pure, cleansing from other cellular components or other contaminants, for example, other nucleic acids or proteins, by using any suitable techniques, including alkaline/SDS, CsCl-banding, column chromatography, agarose gel electrophoresis, and others. Cm. manual F. Ausubelet al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acid according to the invention may constitute, for example, DNA or RNA, and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

Nucleic acids according to the invention can be obtained using any suitable techniques of molecular biology. In respect of the antibodies expressed by hybridomas (e.g., hybridomas derived from transgenic mice carrying the genes of human immunoglobulin, further described below), the cDNA molecule encoding the light and heavy chains of the antibodies created by hybridomas, can be obtained by PCR amplification or cDNA cloning techniques. In respect of the antibodies obtained from the library of immunoglobulin genes (e.g., using the technique of phage display), nucleic acid encoding the antibody can be obtained from the library.

The molecules nuclei the OIC acid according to the invention include, for example, molecules encoding sequence VHand VLmonoclonal antibodies B. The DNA sequence, the coding sequence of the VHantibodies V presented in SEQ ID NO:11. The DNA sequence, the coding sequence of the VLantibodies V presented in SEQ ID NO:12. Other illustrative nucleic acid molecule described herein, are molecules encoding sequence VHand VLmonoclonal antibodies S, 1D9, or 2D2. The DNA sequence, the coding sequence of the VHantibodies IS presented in SEQ ID NO:21. The DNA sequence, the coding sequence of the VLantibodies IS presented in SEQ ID NO:22. The DNA sequence, the coding sequence of the VHantibody 1D9 presented in SEQ ID NO:31. The DNA sequence, the coding sequence of the VLantibody 1D9 presented in SEQ ID NO:32. The DNA sequence, the coding sequence of the VHantibodies 2D2 presented in SEQ ID NO:41. The DNA sequence, the coding sequence of the VLantibodies 2D2 presented in SEQ ID NO:42.

After obtaining DNA fragments coding segments VHand VLthese DNA fragments can be further processed using any suitable techniques of recombinant DNA, for example, for prevrasheniya variable regions genes in chains full-size antibodies, in genes Fab fragments or scFv gene. In these manipulations, a DNA fragment encoding the VLor VHfunctionally linked with another DNA fragment encoding another protein, such as a constant region of the antibody or a flexible linker. The term "functionally linked"as used in this context, I understand that two of the DNA fragment are combined in such a way that the amino acid sequence encoded by the two DNA fragments remain in the frame are read.

The selected DNA encoding the VHthe area can be turned into full gene heavy chain by functional binding VH-encoding DNA to another DNA molecule that encodes a constant region of the heavy chain (CN, CH2 and CH3). Gene-sequences of the constant regions of the heavy chain of the man known in the art (see, for example, the guide Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The constant region of the heavy chain may be a constant region of immunoglobulins IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but most preferably, if is a constant region IgG1 or IgG4. The sequence of the constant region IgG1 could be any and the large number of alleles or allotypes, as is known, detectable among different individuals, such as Gm(1), Gm(2)Gm(3) Gm(17). These allotype represent natural amino acid substitution in the constant regions of IgG1. In relation to gene heavy chain Fab fragment, a DNA encoding the VHmay be functionally linked to another DNA molecule encoding only the constant region of the heavy chain SN.

The selected DNA encoding the VLthe area can be turned into full gene light chain (as well as the gene for the light chain of the Fab-fragment) by functional binding VL-encoding DNA to another DNA molecule that encodes a constant region of light chain CL. Gene-sequences of constant regions of the light chain of the man known in the art (see, for example, the guide Kabat E.A.et al. (1991) Sequences of Proteins of Immunological Interest, Fifth edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The constant region of the light chain may be a constant region light chain Kappa or lambda.

To create a scFv gene VHand VL-encoding DNA fragments are functionally linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3so that the sequence VHand Vsub> Lcan be expressed as a contiguous single-chain protein, with VLand VHthe region is connected by a flexible linker (see, for example, article Birdet al. (1988)Science242:423-426; Hustonet al. (1988)Proc. Natl. Acad. Sci. USA85:5879-5883; McCaffertyet al., (1990)Nature348:552-554).

Production of monoclonal antibodies according to the invention

Monoclonal antibodies (mAb) of the present invention can be obtained by a large number of techniques, including the methodology adopted for monoclonal antibodies, for example, the standard technique of hybridization of somatic cells authors Kohler and Milstein (1975)Nature256:495. For the production of monoclonal antibodies can also be used and other techniques, e.g., viral or oncogenic transformation of b lymphocytes.

The preferred system of the animal to obtain a hybrid system is a system in mice. Products hybridoma a mouse is a common procedure. The immunization protocols and equipment selection immunized splenocytes for fusion are known in this field. The partners in the merger (for example, myeloma cells mouse) and merging is also known.

Chimeric or humanized antibodies of the present invention can be obtained based on the sequence of mouse monoclonal antibodies, obtained as described above. NAM is, encoding the immunoglobulins with heavy and light chain, can be obtained from the relevant hybridoma mouse and designed to contain immunoglobulin sequence that is different from the mouse (e.g., human), using appropriate molecular biological techniques. For example, to create a chimeric antibody variable regions of the mouse may be linked to constant regions of a human, using methods known in the art (see, for example, U.S. patent 4816567 authors Cabillyet al.). To create gumanitarnogo antibody CDR region of the mouse can be embedded in a frame region of a person using methods known in the art (see, for example, U.S. patent 5225539 author Winter, and U.S. patent 5530101; 5585089; 5693762 and 6180370 authors Queenet al.).

In some cases, the antibodies according to the invention are monoclonal antibodies person. Such monoclonal human antibodies against α5β1, can be obtained using transgenic or transamazonic mice carrying parts of the human immune system instead of the immune system of the mouse. These transgenic or transpromotional mice include mice, referred to in this document as the HuMAb Mouse® and KM Mouse®, respectively, and together are referred to in this document as "mouse Ig man."

The HuMAb mouse®(the product is Medarex®, Inc.) contains minilogue human immunoglobulin genes that encode nepristoinye sequence heavy (μ and γ) and κ light chain immunoglobulin along with targeted mutations that inactivate the endogenous loci µ and κ-chains (see, for example, article Lonberget al. (1994) Nature368(6474):856-859). Accordingly, in mice observed reduced expression of IgM or κ-chain of the mouse, and in response to immunization integrated transgenes heavy and light chains of human experience switching class and somatic mutation that results in high-affinity monoclonal human IgGk (Lonberg N.et al. (1994),above; considered in the review Lonberg, N. (1994)Handbook of Experimental Pharmacology113:49-101; Lonberg, N. and Huszar, D. (1995)Intern. Rev. Immunol.13:65-93, and Harding F. and Lonberg, N. (1995)Ann. N.Y. Acad. Sci.764:536-546). Receipt and application of the HuMAb mouse®and the genomic modifications carried by such mice, is additionally described in articles L. Tayloret al.(1992)Nucleic Acids Research20:6287-6295; Chen, J.et al.(1993)International Immunology5:647-656; Tuaillonet al.(1993)Proc. Natl. Acad. Sci. USA90:3720-3724; Choiet al.(1993)Nature Genetics4:117-123; Chen, J.et al.(1993)EMBO J.12:821-830; Tuaillonet al.(1994) J.Immunol.152:2912-2920; Taylor L.et al.(1994)International Immunology6:579-591; and Fishwild, D. et al. (1996)Nature Biotechnology14;845-851. Cm. additional U.S. patents 5545806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429 authors Lonberg and Kay; paten the U.S. 5545807 authors Surani et al.; PCT publication WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962 authors Lonberg and Kay; and PCT publication WO 01/14424 authors Kormanet al.

In another case, the human antibodies of the invention can be obtained using the mouse, which carries an immunoglobulin sequence of the human transgenes and transpromotional, such as a mouse that carries the transgene, the heavy chain of human and transfromation light chain of a human. Such mice, referred to in this document as "mouse KMTM"that is described in detail in PCT publication WO 02/43478 authors Ishidaet al.

In addition, in this area available alternative systems of transgenic animals expressing the genes of human immunoglobulins, and they can be used to generate antibodies against α5β1 according to the invention. For example, there may be used an alternative transgenic system, denoted as xenomai (Abgenix, Inc.); such mice are described, for example, in U.S. patents 5939598; 6075181; 6114598; 6150584 and 6162963 authors Kucherlapatiet al.

Moreover, in this area available alternative systems transamazonic animals expressing the genes of human immunoglobulins, and they can be used to generate antibodies against α5β1 according to the invention. For example, can be used mice carrying both transfromation the heavy chain of human and transfromation light chain h the rights, referred to as "mouse TC"; such mice are described in article Tomizukaet al. (2000)Proc. Natl. Acad. Sci. USA97:722-727. Moreover, in this area described the cows, carrying transhumanity heavy and light chains of the person (Kuroiwaet al. (2002)Nature Biotechnology20:889-894)and can be used to generate antibodies against α5β1 according to the invention.

Monoclonal human antibodies according to the invention can also be obtained using the methods of phage display for screening libraries of human immunoglobulin genes. Such methods of phage display for selection of human antibodies are well known in this field. See, for example, the U.S. patents 5223409; 5403484 and 5571698 authors Ladneret al.; U.S. patents 5427908 and 5580717 authors Doweret al.; U.S. patents 5969108 and 6172197 authors McCaffertyet al.; and U.S. patents 5885793; 6521404; 6544731; 6555313; 6582915 and 6593081 authors Griffithset al.

Monoclonal human antibodies according to the invention can also be obtained using the SCID mice, which were restored immune cells, so that the immunization may be obtained or antibody-based test answer man. Such mice are described, for example, in U.S. patents 5476996 and 5698767 authors Wilsonet al.

Immunization of mice with human Ig

Using mice with human Ig for obtaining human antibodies according to the invention, such mice can be immunitary using purified or about Gasanova drug antigen α5β1 and/or recombinant α5β1 or fused protein α5β1, as described in the articles Lonberg N.et al. (1994)Nature368(6474):856-859; Fishwild, D.et al. (1996)Nature Biotechnology14:845-851; and PCT publications WO 98/24884 and WO 01/14424. Preferably, if the first infusion was taken of the mouse at the age of 6-16 weeks. For example, a purified or recombinant preparation (5-50 μg) of antigen α5β1 can be used for intraperitoneal immunization of mice with human Ig. Moreover, immunization of mice can be used polypeptide fragments corresponding proteins, for example, α5 and/or β1. For example, polypeptide fragments can be conjugated to a molecule with the media to enhance their immunogenicity. Such molecules media are well known in this area, and these include among others hemocyanin lymph snails, bovine serum albumin, thyroglobulin, diphtheria toxoid and tetanus toxoid.

Detailed procedures for obtaining a fully human monoclonal antibody against α5β1 described in example 1 below. Summarizing the experiment with a large number of antigens have shown that transgenic mice were answered during the initial intraperitoneal immunization (IP) with antigen in complete Freund's adjuvant followed IP immunizations every two weeks (up to 6) with antigen in incomplete Freund's adjuvant. However, it also found that effective and other fillers other than adjuvant's adjuvant. In addition to the CSO, found that highly immunogenic and whole cells in the absence of filler. The immune response can be monitored during the course of the immunization Protocol using plasma samples obtained using retroorbital bleeding. The plasma can be skanirovana using ELISA (as described above), and mice with sufficient titers of immunoglobulin against α5β1 can be used for fusions. The mouse can be induced by the intravenous injection of antigen for 3 days before slaughter and removal of the spleen. It is assumed that it may take 2-3 merge for each immunization. Each antigen is usually subjected to immunization from 6 to 24 mice. Usually use both strain NSO and NSO. In addition, both the transgene and NSO, and So can be combined together in one mouse having two different heavy chain transgene person (NSO/NSO). Alternative or additionally may be used strain KM Mouse®, as described in example 1.

Development of hybridomas producing monoclonal antibodies person

To obtain hybridomas producing monoclonal human antibodies according to the invention, splenocytes and/or cells of lymph nodes of immunized mice can be isolated and fused with a suitable immortalizing cell line such as a myeloma cell line mouse. Received hybridoma can be skanirovaniya in relation to the production of antigen specific antibodies. For example, a suspension of single cells of spleen lymphocytes immunogenic mice can be fused with cells " non-secretory myeloma mouse RJ-Ag8.653 (ATCC, CRL 1580) in the amount of one to six with 50% PEG. Cells placed in a quantity of approximately 2×105in flat-bottomed tablet for micrometrology followed by incubation for two weeks in selective medium containing 20% serum FetalClone, 18% air-conditioned environment "653", 5% origen (the company's products IGEN), 4 mm L-glutamine, 1 mm sodium pyruvate, 5 mm HEPES, 0.055 mm 2-mercaptoethanol, penicillin at a concentration of 50 U/ml, streptomycin at a concentration of 50 mg/ml, gentamicin at a concentration of 50 mg/ml and 1-fold the nut Supplement (production company Sigma; NAT add 24 hours after the merger). About two weeks cells can be cultured in an environment in which the nut is replaced by NT. Individual wells can then be skanirovaniya by ELISA against monoclonal antibodies to human IgM and IgG. After the advent of the widespread growth of hybridoma environment, you can monitor typically 10-14 days. Hybridoma, secreting the antibody can be re-placed again skanirovaniya, and if still positive in relation to human IgG, monoclonal antibodies, can be subcloned at least twice by limiting dilution. Stable subclones can then b is to be cultivated in vitroto obtain small amounts of antibody in tissue culture medium for features.

For purification of monoclonal antibodies person selected hybridoma can be grown in two-liter roller flasks for the purification of monoclonal antibodies. Supernatant can be filtered and concentrated before affinity chromatography with protein a-sepharose (production company Pharmacia, Piscataway, N.J.). To guarantee purity suirvey IgG can be checked using gel electrophoresis and high performance liquid chromatography. The buffer solution can be replaced with PBS, and the concentration can be determined by OD280 using a damping coefficient of 1.43. Monoclonal antibodies can be glassed aliquot and stored at -80°C.

Getting transfection producing monoclonal antibodies

Antibodies according to the invention can also be obtained in transfectant host cell, using, for example, a combination of the techniques of recombinant DNA and methods transfection of genes is well known in this field (e.g., Morrison, S. (1985) Science 229:1202).

For example, for the expression of antibodies or their fragments of the DNA molecule encoding fragmented or full light and heavy chains, can be obtained by standard molecular biological techniques (e.g., PCR signal amplification for the or cDNA cloning, using hybridoma or phage that Express the desired antibody), and DNA molecules can be embedded in expressing vectors, so that the genes are functionally associated with sequences that control transcription and translation. In this context, the term "functionally linked" is understood that the antibody gene are ligated in the vector, so that the sequence controlling transcription and translation in the vector, perform the intended function regulation of transcription and translation of the antibody gene. Expressing vector and a sequence controlling the expression, are chosen so that they are consistent with the expressing cell host. Gene light chain antibody gene and the heavy chain of the antibody can be built into separate vectors, or more frequently both genes are built in the same expressing vector. Genes antibodies embed in expressing vector by any appropriate means (for example, legirovaniem of complementary restriction sites on the gene fragment of the antibody and the vector or legirovaniem blunt ends in the absence of restriction sites). Variable region light and heavy chains of the antibodies described herein can be used to create genes with full-length antibodies of any isotype and subclass of antibodies by embedding them in expresser is the following vectors, already encoding the constant region of the heavy and light chains of the desired isotype and subclass, so that VHsegment functionally associated withNthe segment(s) in the vector and VKsegment functionally associated with the CL-the segment within the vector. Additionally or alternatively expressing recombinant vector can encode a signal peptide that facilitates secretion of the chain of the antibody from the host cell. Gene chain antibodies can be cloned into a vector such that the signal peptide is bound inside reading frames with aminocom.com gene chain antibodies. The signal peptide may be a signal peptide immunoglobulin or a heterologous signal peptide (i.e. the signal peptide of the protein, other than immunoglobulin).

In addition to genes chains of the antibody expressing recombinant vectors according to the invention generally have regulatory sequences that control expression of genes chains of the antibody in the cell host. The term "regulatory sequence" refers to promoters, enhancers and other elements controlling the expression (e.g., polyadenylation signals)that control the transcription or translation of genes chains of antibodies. Such regulatory sequences are described, for example, the manual Goedel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, san Diego, CA (1990)). The person skilled in the art will understand that the creation of expressing the vector, including the selection of regulatory sequences may depend on such factors as the selection of transformed host cells, the level of expression of the desired protein, etc. To the preferred regulatory sequences for expression in the cell of the host mammal include viral elements that lead to high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), monkey virus 40 (SV40), adenovirus (e.g., major late promoter of adenovirus (AdMLP)) and virus polyoma. Alternatively, it may be used non-viral regulatory sequences, such as ubiquitine promoter or β-glubinoy promoter. Further, regulatory elements composed of sequences from different sources, such as promotor SR system, which contains sequences from the early SV40 promoter and the long terminal repeat of the virus T-cell leukemia human type 1 (Takebe Y.et al. (1988)Mol. Cell. Biol.8:466-472).

In addition to the genes of the antibody chains and regulatory sequences expressing recombinant vectors according to the invention may carry additional sequences, such to the to the sequence, that regulate replication of the vector in the cells of the host (for example, the starting point of replication and genes selectivity markers. Gene selectivity marker facilitates the selection of host cells into which the vector has been integrated (see, for example, U.S. patents 4399216, 4634665 and 5179017 authors Axelet al.). For example, a typical gene selectivity marker confers resistance to drugs, such as G418, hygromycin or methotrexate, cell host into which the vector has been incorporated. To genes selectivity markers are gene digidrofolatreduktazy (DHFR) (for use in cell-hosts dhfr - selection/amplification on background methotrexate) and the neo gene (for selection on the background of G418).

For the expression of light and heavy chains expressing(e) vector(s)encoding the heavy and light chain, transferout in cell host using any suitable techniques. Under a large number of forms of the term "transfection" understand a wide range of techniques commonly used for integration of exogenous DNA into a prokaryotic or eukaryotic cell host, for example, electroporation, precipitation with calcium phosphate, transfection of DEAE-dextran, and the like. While it is possible to Express the antibodies according to the invention in either prokaryotic or eukaryotic cells-the masters, the most common expression of antibodies in eukaryotic who ledah and usually in the cells of the host mammal.

The cells of the host mammal for expression of recombinant antibodies according to the invention include, for example, cells of the Chinese hamster ovary (Cho) (including cells of the Cho dhfr is described in the article Urlaub and Chasin, (1980)Proc. Natl. Acad. Sci. USA77:4216-4220, used with a selectivity marker DHFR, for example, as described in article R.J. Kaufman and P.A. Sharp (1982)Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and Sp2 cells. In particular, for use with NS0 myeloma cells or cells SNO another expressing the system represents expressing the gene system GS (glutamine synthase), described in WO 87/04462, WO 89/01036 and EP 333841. When embedding expressing recombinant vectors encoding antibody genes, in cells of the host mammal produced antibodies by culturing the host cells for a period of time sufficient for expression of the antibody in the cells of the host or the secretion of antibodies into the culture medium in which cells-the hosts are grown. Antibodies can be extracted from the culture medium using any suitable methods protein purification.

Characterization of the binding of an antibody to an antigen

Antibodies or their antigennegative plots according to the invention can be tested for binding to α5β1, for example, using a standard ELISA. In short, die for micrometrology dormancy is to see purified α5β1 at a concentration of 0.25 μg/ml in PBS and then blocked with 5% bovine serum albumin in PBS. To each well add breeding antibodies (for example, dilution of plasma obtained from α5β1-immunized mice) and incubated for 1-2 hours at 37°C. the Plate is washed with PBS/Tween and then incubated with secondary reagent (e.g., for human antibodies with Fc-specific polyclonal reagent Ig goats against a person), conjugated with alkaline phosphatase for 1 hour at 37°C. After washing the plates work with pNPP substrate (1 mg/ml) and analyzed at OD 405-650. Preferably, for m should be used mice that have created the most high titers.

The ELISA described above can also be used for screening hybridomas have been positive reactivity with the immunogen α5β1. Hybridoma that are associated with α5β1 with high avidity, subcloning and further characterize. One clone of each hybridoma, which retains the reactivity of the parent cells (using ELISA), can be selected to create a cell Bank in the amount of 5-10 vials, stored at -140°C, and for the purification of antibodies.

For the purification of antibodies against α5β1 selected hybridoma can be grown in two-liter roller flasks for the purification of monoclonal antibodies. Supernatant can be filtered and concentrated before affinity chromatography with protein a-with Feroz (production company Pharmacia, Piscataway, N.J.). To ensure purity, suirvey IgG can be checked using gel electrophoresis and high performance liquid chromatography. The buffer solution can be replaced with PBS, and the concentration can be determined by OD280 using a damping coefficient of 1.43. Monoclonal antibodies can be glassed aliquot and stored at -80°C.

Additionally, the epitope to bind to the antibody can be characterized by standard methods known in this field. Such methods include the production of a set of overlapping peptide fragments α5 and/or β1 and evaluation of antibody binding sites with a large number of fragments. Alternatively, the peptide α5 and/or β1 can be introduced by mutation, for example, using alanine-scanning mutagenesis, in which each amino acid is substituted by alanine residue and the binding of an antibody to a mutant peptide can be compared with the binding of an antibody to a protein of the wild type, thus, identifying the sites where the mutation(s) effect(s) on the binding. See, for example, article Cunninghamet al., (1989)Science244:1081-1085.

To determine contacted if selected monoclonal antibodies against α5β1 or antigennegative plot with unique epitopes, each antibody can be biotinylation using commercially available reagents (p is aboutinduction firm Pierce, Rockford, IL). Can be carried out comparative studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies using tablets for conducting ELISA covered α5β1, as described above. Binding of biotinylated mAb can be detected by a probe streptavidin-alkaline phosphatase.

To determine the isotype of purified antibodies can be performed ELISA assays to determine the isotypes using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of monoclonal human antibodies holes tablets for micrometrology can be coated with immunoglobulin against a person in a concentration of 1 μg/ml for at 4°C. After blocking with 1% BSA tablets lead in contact with the tested monoclonal antibodies or controls treated isotypes in a concentration of 1 μg/ml or less at ambient temperature for one to two hours. The wells can then be brought into contact with either IgG1-or IgM human-specific probes conjugated with alkaline phosphatase. The tablets are treated and analyzed as described above.

IgG-antibodies against α5β1 can be further tested against the interaction with antigen α5β1 using Western blot. Briefly, and tegrin α5β1 can be obtained and subjected to electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. After electrophoresis the separated antigens transferred to nitrocellulose membranes, blocked with 10% fetal calf serum and treated with the tested monoclonal antibodies. The binding of human IgG can be detected using IgG against human, conjugated with alkaline phosphatase, and treating tablets substrate BCIP/NBT (production company Sigma Chem. Co., St. Louis, Mo.).

Immunoconjugate

In another aspect, the present invention relates to an antibody against α5β1 or its fragment, anywherefrom with a therapeutic fragment, such as a cytotoxin, a drug (e.g., immunosuppressant) or radiotoxins. Such conjugates are referred to herein as "immunoconjugate". Immunoconjugate that include one or more cytotoxins, are referred to as "immunotoxins". To a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells (for example, destroys them). Examples include Taxol, cytochalasin B, gramicidin D, ethidiumbromid, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. To therape the political agents also include, for example, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-ferrallitization), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustin (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and CIS-dichlorodiammine platinum (II) (DDP) (cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and astromicin (AWSs)and antimitoticescoe agents (e.g. vincristine and vinblastine).

Other examples of therapeutic cytotoxins that can be conjugated with the antibody or antigennegative plot according to the invention are duocarmycin, calicheamicin, maytansine and auristatin and their derivatives. One example of a conjugate antibodies with calicheamicin commercially available (MylotargTM; Wyeth-Ayerst).

Cytotoxins can be conjugated with antibodies according to the invention or their antihistamine plots, using a number of technologies with the use of linkers. Examples of the types of linkers that were used for cytotoxin conjugation with the antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptideatlas linkers. The linker may be chosen in the way, so, for example, he was sensitive to the splitting of the low pH values in the lysosomal compartment or sensitive to cleavage by proteases such as protease predominantly expressed in tumor tissue, such as cathepsins (such as cathepsins b, C, D).

For additional discussion on the types of cytotoxins, linkers and methods of conjugating therapeutic agents to antibodies, see also article Saito G.et al.(2003)Adv. Drug Deliv. Rev.55:199-215; Trail P.A.et al.(2003)Cancer Immunol. Immunother.52:328-337; Payne, G. (2003)Cancer Cell3:207-212; Allen T.M. (2002)Nat. Rev. Cancer2:750-763; Pastan I. and Kreitman R.J. (2002)Curr. Opin. Investig. Drugs3:1089-1091; P.D. Senter and Springer, C.J. (2001)Adv. Drug Deliv. Rev.53:247-264.

Antibodies or their antigennegative plots according to the present invention can also be conjugated to a radioactive isotope to obtain cytotoxic radioactive drugs, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated with antibodies for diagnostic or therapeutic applications include, but are not limited to, iodine131, indium111, yttrium90and Lu177. Methods of obtaining radioimmunoconjugates known in this field. Some radioimmunoconjugates commercially available, including ZevalinTM(about ukcia company IDEC Pharmaceuticals) and Bexxar TM(production company Corixa Pharmaceuticals), and to obtain radioimmunoconjugates can be used similar methods, using antibodies according to the invention.

Conjugates of the antibody according to the invention can be used for modifying a given biological response, and a fragment of a medicinal product should not be construed as limited to classical chemical therapeutic agents. For example, a fragment of a medicinal product may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, enzymatically active toxin, or active fragment such as abrin, ricin A, pseudomonades exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocytopenia-macrophage colony-stimulating factor ("GM-CSF"), colony-stimulating granulocytopenia factor ("G-CSF"), or other growth factors.

Methods of conjugating such therapeutic fragment of the antibody is known, see, e.g., Arnonetal.,"Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, Reisfeldet al.(eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstromet al,"Antibodies for Drug Delivery", in Cotrolled Drug Delivery (2nd Ed.), Robinsonet al.(eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications, Pincheraet al.(eds.), pp.475-506 (1985); "Analysis, Results, and Future Prospective of therapeutic Use of Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwinet al.(eds.), pp.303-16 (Academic Press 1985), and Thorpeet al."The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

Bespecifically molecules

In another aspect, the present invention relates to bespecifically molecules containing antibody against α5β1 or its fragment according to the invention. The antibody according to the invention or its antigennegative areas can form the derived or to contact with another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) with the formation of bespecifically molecule that binds at least two different binding sites or target molecules. The antibody according to the invention can in fact be derived form, or contact with more than one other functional molecule with the formation of multispecificity molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also covered by the term "bespecifically molecule", as used in this documentedly create bespecifically molecules according to the invention, the antibody according to the invention can be functionally linked (e.g., using chemical combinations, genetic fusion, noncovalent Association or otherwise) with one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, resulting in bespecifically molecule.

Accordingly, the present invention relates to bespecifically molecules containing at least one first binding specificity for α5β1 and a second binding specificity for a second target epitope. In a particular aspect of the invention, the second target epitope is an Fc receptor, e.g., human FcγRI (CD64) or Fcγ-receptor (CD89). Thus, the invention relates to bespecifically molecules, able to communicate both with FcγR or effector cells expressing FcγR (e.g., monocytes, macrophages or polymorphonuclear cells (PMN), and to target cells expressing α5β1. These bespecifically molecules target α5β1-expressing cells to effector cell and trigger the activity of effector cells, mediated by Fc receptor, such as phagocytosis α5β1-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.

In one aspect of the invention, in which bis is elficiency molecule is multispecific, the molecule can further include a third binding specificity, in addition to the binding specificity against Fc and binding specificity against α5β1. In one case, a third binding specificity is a plot against the increase factor (EF), for example, a molecule that binds to a surface protein involved in cytotoxic activity, and enhances the immune response against target cells. "The plot against the increase factor" can be an antibody, a functional fragment of the antibody or ligand that binds to the molecule, for example, antigen or receptor, and thus leads to increased influence of the determinants of binding to Fc receptor or antigen of the target cell. "The plot against the increase factor" can communicate with the Fc-receptor or antigen of the target cell. Alternatively, the plot against factor gain can contact the object, which is different from the object, which contact the first and second binding specificity. For example, the plot against factor gain can contact cytotoxic T-cell (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1) or other immune cell, which leads to increased immune response against target cells).

In one case bespecifically molecules according to the invention the content is t as binding specificity, at least one antibody or a fragment of this antibody, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv. The antibody may also be a dimer of light or heavy chains, or any minimal fragment such as Fv or single-chain design, as described in U.S. patent 4946778 authors Ladneret al.

In one case, the binding specificity Fcγ-receptor is provided a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). Under used in this document, the term "IgG receptor" understand any of the eight genes chains γ, localized on chromosome 1. These genes encode in General twelve transmembrane or soluble receptor isoforms which are grouped in three classes of Fcγ receptors: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). In one case, the Fcγ-receptor is a high-affinity FcγRI person. FcγRI person is a molecule size of 72 kDa, which has a high affinity for Monomeric IgG (108-109M-1).

Production and characterization of some monoclonal antibodies against the Fcγ described by the authors Fangeret al. in PCT publication WO 88/00052 and in U.S. patent 4954617. These antibodies bind to an epitope FcγRI, FcγRII or FcγRIII in the website, which is different from the Fcγ-binding site of the receptor and thus binding on things the TSS is not blocked by physiological levels of IgG. Specific antibodies against FcγRI used in this description represent a mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Hybridoma producing mAb 32, available from the American Type Culture Collection, ATCC under access number NV. In other cases, the antibody against the Fcγ-receptor is humanitarian form of monoclonal antibody 22 (n). Production and characterization of antibodies n described in article Graziano R.F.et al. (1995)J. Immunol.155(10):4996-5002 and PCT publication WO 94/10332. The cell line producing the antibody n, deposited in the American Type Culture Collection under the name HA022CL1 and assigned access number CRL 11177.

In other cases, the specificity of binding of the Fc receptor is provided by an antibody that binds to IgA-receptor human, for example, an Fc-alpha receptor (FcαRI (CD89)), the binding of which is usually not blocked immunoglobulin And human (IgA). The term "IgA-receptor" understand gene product of one α-gene (FcαRI), localized on chromosome 19. It is known that this gene encodes several alternative splaisiruemym transmembrane isoforms ranging in size from 55 to 110 kDa. FcαRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophils and neutrophils, but not on the cell populations that are not effector. FcαRI has medium affinity (≈ 5×107M-1as IgA1 and IgA2, which is increased when Expo is icii with cytokines, such as G-CSF or GM-CSF (Morton H.C.et al. (1996)Critical Reviews in Immunology16:423-440). Described four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62 and A that are associated with FcαRI outside the domain, a ligand-binding IgA (Monteiro R.C.et al. (1992)J. Immunol.148:1764).

FcαRI and FcγRI are clear trigger receptors for use in bespecifically molecules according to the invention because they are (1) expressed primarily on immune effector cells, such as monocytes, PMN cells, macrophages and dendritic cells; (2) expressed at high levels (for example, 5000-100000 per cell); (3) are mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including antigens, target for them.

Although the preferred monoclonal human antibodies other antibodies that can be used in bespecifically molecules according to the invention are murine, chimeric and humanized monoclonal antibodies.

Bespecifically molecules according to the present description can be obtained by conjugation of the components of specificdate binding, for example, specificdate binding to FcR and α5β1, using any suitable methods. For example, each binding specificity of bispecific the Oh molecules can be obtained separately and then conjugated to one another. If the binding specificity are proteins or peptides, for covalent conjugation can be used a large number of combining or cross-linking agents. Examples of cross-linking agents include protein a, carbodiimide, N-Succinimidyl-S-acetylthiourea (SATA), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), ortho-phenylenedimaleimide (oPDM), N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, articles Karpovskyet al.(1984)J. Exp. Med.160:1686; Liu, MAet al. (1985)Proc. Natl. Acad. Sci. USA82:8648). Other methods include the methods described in articles Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennanet al. (1985)Science229:81-83) and Glennieet al. (1987)J. Immunol. 139:2367-2375). Suitable conjugating agents are SATA, sulfo-SMCC, both are products of the firm Pierce Chemical Co. (Rockford, IL).

If the binding specificity are antibodies, they can be conjugated via a sulfhydryl connection between the two heavy chains in the area With all the hinge areas. In one case, the hinge region is altered so that she would before conjugation contained an odd number of sulfhydryl residues, such as one residue.

Alternatively, both the binding specificity can be encoded in one and the same vector and expressed and collected in the same cell as the host. This method can be used, in particular, if bespecifically molecule is a protein mAb × mAb, mAb × Fab, Fab × F(ab')2or ligand × Fab. Bespecifically molecule according to the invention can be a single-stranded molecule containing a single-chain antibody and the determinants of binding or single-stranded bespecifically molecule containing two determinants of binding. Bespecifically molecule may contain at least two single-stranded molecules. Methods of obtaining bespecifically molecules described, for example, in U.S. patent 5260203; U.S. patent 5455030; U.S. patent 4881175; U.S. patent 5132405; U.S. patent 5091513; U.S. patent 5476786; U.S. patent 5013653; U.S. patent 5258498 and U.S. patent 5482858.

Linking bespecifically molecules to their specific targets can be confirmed by using, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioanalysis (e.g., growth inhibition) or analysis of Western blot. In each of these analyses, as a rule, detected the presence of interesting complexes, protein-antibody, using a labeled reagent (e.g., antibody)that is specific for the complex. For example, complexes FcR-antibody can be detected using, for example, the antibody bound to the enzyme, or fragments the antibodies, which recognizes and specifically binds to complexes of antibody-FcR. Alternatively, the complexes can be detected using any other immunoassay. For example, the antibody can be radioactively observed and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such methods as the use of γ-counter or a scintillation counter or by autoradiography.

The pharmaceutical composition

In another aspect, the present description relates to compositions, for example, pharmaceutical compositions containing one or a combination of monoclonal antibodies or their antigennegative(s) parcel(s) of the present invention, present in the mixture along with a pharmaceutically acceptable carrier. Such compositions may include one or a combination (for example, two or more different antibodies, or immunoconjugates or bespecifically molecules according to the invention. For example, the pharmaceutical composition according to the invention may contain a combination of antibodies (or immunoconjugates or bespecifically molecules)that bind to different epitopes on the target antigen or that have complementary activities.

The pharmaceutical compositions according to the invention so the e can be used in combination therapy, for example, in combination with other agents. For example, the combination therapy can include an antibody against α5β1 of the present invention in combination with at least one other anti-inflammatory or immunosuppressive agent. Examples of therapeutic agents that may be used in combination therapy, are described in more detail below in the section on the use of antibodies according to the invention.

Under used herein "pharmaceutically acceptable carrier" is understood as any and all solvents, dispersion media, covering substances, antibacterial and antifungal agents, isotonic and slowing down the absorption of the agents and the like that are physiologically compatible. Typically, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration of the active compound, i.e. the antibody, it antigennegative plot, immunoconjugate or bespecifically molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

In some embodiments, the implementation of the antibodies of the present invention may be in neutral form (in the including zwitterionic form) or in the form of positively or negatively charged compounds. In some cases, the antibodies may be in combination with a counter-ion, forming a pharmaceutically acceptable salt. Thus, the pharmaceutical compounds of the invention can include one or more pharmaceutically acceptable salts.

By "pharmaceutically acceptable salt" understand salt that retains the desired biological activity of the parent compound (e.g., antibodies) and has no undesired Toxicological effects (see, for example, article Berge, S.M.,et al. (1977)J. Pharm. Sci.66:1-19). For example, the term "pharmaceutically acceptable salt" refers to a complex that contains one or more antibodies and one or more counter-ions, where counter-ions come from pharmaceutically acceptable inorganic and organic acids and bases.

Examples of such salts include acid additive salts and basic additive salt. To acid additive salts include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, Hydrobromic, uudistoodetena, phosphorous and the like, as well as from nontoxic organic acids, such as aliphatic mono - and dicarboxylic acids, phenyl-substituted alcamovia acid, hydroxyalkanoate acids, aromatic acids, aliphatic and aromatic sulfonic acid is you and the like. It basically additive salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenziletilendiaminom, N-methylglucamine, chloroprocaine, choline, diethanolamine, Ethylenediamine, procaine and the like.

Moreover, pharmaceutically acceptable inorganic bases include metal ions. Ions of metals include, but are not limited to, the corresponding salts of alkali metals, salts of alkaline earth metals and other physiologically acceptable metal ions. Salts derived from inorganic bases include aluminum salts, ammonium, calcium, cobalt, Nickel, molybdenum, vanadium, manganese, chromium, selenium, tin, copper, ferric iron, ferrous iron, lithium, magnesium, manganese salt of trivalent, divalent manganese, potassium, rubidium, sodium and zinc in its normal valence.

Pharmaceutically acceptable acid additive salt antibodies of the present invention can be obtained from the following acids, including, without limitation, formic, acetic, acetamidobenzoyl, adipic, ascorbic, boric, propionic, benzoic, camphoric, carboxylic, ciclamino, dehydrocholic acid, malonic acid, ethylenediaminetetraacetic, atillery, fendizoic, metaphosphoric, and the RNA, glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric, ascorbic, glucuronic, maleic, folic acid, fumaric, propionic, pyruvic, aspartic, glutamic, benzoic, hydrochloric, Hydrobromic, yodiewonderdog, lysine, solomonow, triperoxonane, pambou, propionic, orthoaminophenol, mailovou, Orotava, oxalic acid, salewoman, oleic, stearic, salicylic acid, aminosalicylic, silicon, para-hydroxybenzoic, nicotine, phenylacetic, almond, ambalavao, sulfonic, methansulfonate, phosphoric, fosfonovoi, econsultancy, etanislao, ammonium, benzosulfimide, Pantothenic, naphthalenesulfonate, toluensulfonate, 2-hydroxyethanesulfonic, sulfanilic, sulphuric, nitric, nitrous, onomatology ether sulfuric acid, cyclohexanesulfonic, β-hydroxybutyrate, glycine, glycylglycine, glutamic, cockadile, diaminohexane, camphorsulfonate, gluconic, ticinobuy, oxoglutarate, pyridoxal-5-phosphate, chloroperoxidase, undecanol, N-acetyl-L-aspartic, muzinovy and galacturonic acid.

To pharmaceutically acceptable organic bases include trimethylamine, diethylamine, N,N'-dibenziletilendiaminom, chloroprocaine, choline, dibenzylamine, diethanolamine, Ethylenediamine, meglumine (N-is ethylglycine), procaine, cyclic amines and cations of Quaternary ammonium, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol, 2-Diethylaminoethanol, 2-dimethylaminoethanol, amandemen, butylamine, ethanolamine, Ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylparaben, glucamine, glucosamine, histidine, geranamine, imidazole, Isopropylamine, methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine, polianinova resins, procaine, purines, theobromine, triethylamine, Tripropylamine, triethanolamine, tromethamine, methylamine, taurine, Holt, 6-amino-2-methyl-2-heptanol, 2-amino-2-methyl-1,3-propandiol, 2-amino-2-methyl-1-propanol, aliphatic mono - and dicarboxylic acids, phenyl-substituted alcamovia acid, hydroxyalkanoate acids, aromatic acids, aliphatic and aromatic sulfonic acid, strontium, tricin, hydrazine, phenylcyclohexylamine, 2-(N-morpholino)econsultancy acid, bis(2-hydroxyethyl)amino-Tris(hydroxymethyl)methane,N-(2-acetamido)-2-aminoetansulfonovaya acid, 1,4-piperazineethanesulfonic acid, 3-morpholino-2-hydroxypropanesulfonic acid, 1,3-bis[Tris (hydroxymethyl)methylamino]propane, 4-morpholinepropanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-econsultancy acid, 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]econsultancy acid, N,N-bis(2-hydroxyethyl)-2-aminoethanol is about acid, 4-(N-morpholino)butanesulfonic acid, 3-(N,Nbis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, 2-hydroxy-3-[Tris(hydroxymethyl)methylamino]-1-propanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid)dihydrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,N,Nbis (2-hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N-[Tris(hydroxymethyl)methyl]-3-aminopropiophenone acid, N-Tris(hydroxymethyl)methyl-4-aminoethanesulfonic acid, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, 2-(cyclohexylamino)econsultancy acid, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, 3-(cyclohexylamino)-1-propanesulfonic acid,N-(2-acetamido)iminodiacetate acid, 4-(cyclohexylamino)-1-butanesulfonic acid,N-[Tris(hydroxymethyl)methyl]glycine, 2-amino-2-(hydroxymethyl)-1,3-propandiol and trometamol.

The pharmaceutical composition according to the invention may also include pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, systeemiteoria, sodium bisulfate, sodium metabisulfite, a sulfite soda is I, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, bottled hydroxyanisol (BHA), bottled hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol and the like; and (3) halirous agents that bind metal ions, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Examples of suitable aqueous and nonaqueous carriers which may be used in the pharmaceutical compositions according to the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures of vegetable oils, such as olive oil, and a complex organic esters for injection, such as etiloleat. Inherent condition of fluidity can be saved, for example, using covering materials, such as lecithin, maintaining the desired particle size in the case of dispersions and by applying a surface-active substance.

These compositions may also contain fillers such as preservatives, moisturizing agents, emulsifying agents and dispersing agents. Protection from microorganisms can be ensured by using procedures sterilization,aboveand by including a large number of antibacterial and antifungal agents, e.g. the, paraben-free, chlorbutanol, fenolcarbonove acid and the like. The composition may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. In addition, prolonged absorption of the injectable pharmaceutical form may be due to the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

To pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporanea preparation of sterile solutions or dispersions for injection. The use of such media and agents for pharmaceutically active substances is well known in this field. Except when any of the accepted medium or agent is incompatible with the active compound, it is assumed their use in pharmaceutical compositions according to the invention. The composition can also be introduced additional active compounds.

Therapeutic compositions typically must be sterile and stable under conditions of manufacture and storage. The composition may be in the mix in the form of a solution, microemulsion, liposome, or other ordered structure suitable for high concentration of the drug. The carrier can be a solvent or dispersion medium is, containing, for example, water, ethanol, polyhydric alcohol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures. Own fluidity can be maintained, for example, using a coating such as lecithin, maintaining the desired particle size in the case of dispersion and by applying surfactants. In many cases it will be preferable to include in the composition is isotonic agents, for example, sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be due to the inclusion in the composition an agent that delays absorption, for example, salts of monostearate and gelatin.

Sterile injectable solutions can be obtained by introducing the active compound in the required amount in an appropriate solvent with one or a combination of desirable ingredients enumerated above, followed by sterilization microfiltration. Generally, dispersions are obtained by introducing the active compound into a sterile vehicle, which contains the basic dispersion medium and the desired other ingredients listed above. In the case of sterile powders for obtaining sterile injectable solutions, the methods of production include, but are not limited to, air drying, vacuum drying and the freeze (freeze-drying), which yield a powder of the active ingredient plus any additional desired ingredient from their solution, pre-sterilized filter.

The amount of active ingredient that may be combined with the material of the carrier for the production of a single dosage form will vary depending on the individual being treated and the particular route of administration. The amount of active ingredient that may be combined with the material of the carrier for the production of a single dosage form will generally be that amount of a composition which creates a therapeutic effect. In General, one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferred from about 1 percent to about 30 percent of active ingredient in combination with pharmaceutically acceptable carrier.

Regimens adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, there may be only one tablet can be entered multiple fractional dose over a period of time or the dose may be proportionally reduced or increased, as will indicate the severity of terapeutiche is such situation. For ease of administration and uniformity of dosage particularly preferably be parenteral compositions in a standard dosage form. Under the standard dosage form used in the present description, understand physically discrete units suitable as single doses to individuals treated; each unit contains a predetermined quantity of active compound calculated for the production of the desired therapeutic effect in Association with a suitable pharmaceutical carrier. Specification standard dosage forms according to the invention is determined by and directly dependent on (a) the unique characteristics of the active compound and the particular achieved therapeutic effect and (b) constraints in the field of preparation of compounds such as an active compound for the treatment of sensitivity in individuals.

For the introduction of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg and more from 0.01 to 5 mg/kg of body weight of the host. For example, a dose can be 0.3 mg/kg of body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg of body weight, or to be in the range from 1 to 10 mg/kg Typical regimen includes the introduction of once per week, once per two weeks, once every three weeks, every four weeks, is in once a month, once in 3 months or once every three to 6 months. Regimens antibodies against α5β1 or antigennegative plot according to the invention include, for example, 1 mg/kg body weight or 3 mg/kg body weight by intravenous administration, with the antibody receive using one of the following schemes of receiving: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) once 3 mg/kg of body weight, then 1 mg/kg of body weight every three weeks.

In some methods, two or more monoclonal antibodies with different specificnosti linking entered at the same time, in this case the dosage of each input antibodies decreases in the specified ranges. The antibody is usually injected a large number of times. Intervals between single dosages can be, for example, once a week, once a month, every three months or once a year. Intervals can also be irregular, indicated by measuring blood levels of antibody to the target antigen in the patient. In some ways the dose adjusted to achieve the concentration of antibodies in plasma from about 1 to 1000 μg/ml and in some methods from about 25 to 300 mcg/ml.

Alternatively, the antibody can be introduced in the form of formulations of extended release, in this case, a lower rate of injection. Dosage and frequency vary depending on the period is olivedale antibodies in the patient. In General, antibodies of humanity has the longest half-life, followed by humanized antibodies, chimeric antibodies and antibodies, non-human. Dose and frequency of injection can vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use introduces a relatively low dose with relatively long intervals over a long period of time. Some patients continue to receive treatment throughout life. Therapeutic application sometimes requires a relatively high dosage at relatively short intervals to reduce or stop the progression of the disease, and preferably as long as the patient will not experience a partial or complete mitigation of symptoms. Then the patient may receive prophylactic scheme.

Actual dosage levels of active ingredients in the pharmaceutical compositions according to the present description can be varied so as to obtain the amount of active ingredient which is effective to achieve the desired therapeutic response in a particular patient, compositions and routes of administration, without being toxic to the patient. The selected dosage level will depend on a large number pharmacokineti the definition of factors, including the activity of the specific compositions used in the present description or of ester, salt or amide, the method of administration, time of administration, rate of excretion of the specific compound, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions used, age, sex, weight, condition, General health and prior medical history of the patient being treated, and like factors well known in these fields of medicine.

"Therapeutically effective dose" antibodies against α5β1 according to the invention preferably leads to decrease in severity of disease symptoms, an increase in the frequency and duration of periods with no symptoms of the disease or to prevent deterioration or disturbance due to the severity of the disease. For example, in the treatment of α5β1-positive tumors "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60% and most preferred, at least about 80% relative to individuals who did not receive treatment. The ability of compounds to inhibit tumor growth can be estimated is as a model system for animals predictive effectiveness for human cancers. Alternatively, this property of a composition can be measured by the ability of the compound to inhibit, such inhibitionin vitrocan be assessed by assays known to practitioners. A therapeutically effective amount of a therapeutic compound can reduce the size of the tumor, or otherwise alleviate the symptoms of the individual. The person skilled in the art could determine such amount on the basis of factors such as the size of the individual, the severity of the symptoms observed in the individual, and selected a particular composition or method of administration.

The composition according to the present description can be entered by one or more routes of administration using one or more of the large number of methods known in this field. As will be clear to the person skilled in the art, the path and/or the method of administration will vary depending on the desired results. The routes of administration of antibodies or their antigenspecific plots according to the invention include intravenous, intramuscular, nutritionally, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g. by injection or infusion. The phrase "parenteral administration"as used in this document, understand the ways of introduction, the balance of the data from enteral and local administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, vnutriobolochechnoe, vnutricapsulino, intraorbital, intracardiac, nutritionally, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, vnutrispinalnaya, epidural and epigastric injection and infusion.

Alternatively, an antibody or antigennegative plot according to the invention can be introduced by other than parenteral, such as local, epidurally route of administration or route of administration through the mucosa, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be obtained with carriers that will protect the compound against rapid release, such as preparative form of controlled release, including implants, transdermal patches, microencapsulation delivery system. Can be used biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, complex poliorcetes, polylactic acid. Many methods for such preparative forms are patented or generally known to experts in this field. See, for example,i> Sustained and Controlled release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be introduced with medical devices known in the field. For example, a therapeutic composition according to the invention can be introduced through without needle device for hypodermal injections, such as the devices described in U.S. patent 5399163; 5383851; 5312335; 5064413; 4941880; 4790824 or 4596556. Examples of well-known implants and modules according to the present description include: U.S. patent 4487603 that describes implantable microinfusion pump for injection of a medicinal product with a controlled rate; U.S. patent 4486194 that describes a therapeutic device for the administration of drugs through the skin; U.S. patent 4447233 that describes an infusion pump for delivery of a drug with a precise infusion rate; U.S. patent 4447224, which describes an implantable apparatus for infusion with a variable flow for continuous drug delivery; U.S. patent 4439196 that describes the osmotic delivery system of the medicinal product, with multi-departments; and U.S. patent 4475196 that describes the osmotic delivery system of the drug. Professionals in this field are known, and many other such implants,delivery systems, and modules.

In a case of monoclonal human antibodies or their antigennegative plots according to the invention can be mixed to achieve a suitable distribution ofin vivo. For example, the blood-brain barrier (BBB) does not pass many high hydrophilic compounds. For the passage of therapeutic compounds according to the invention through the BBB (if necessary) they can be mixed, for example, in liposomes. In relation to methods of producing liposomes, see, for example, U.S. patents 4522811; 5374548 and 5399331. Liposomes can contain one or more fragments, which are selectively transported into specific cells or organs, thus enhancing the delivery of targeted drugs (see, for example, article V.V. Ranade (1989)J. Clin. Pharmacol.29:685). Typical aims fragments include folate or Biotin (see, for example, U.S. patent 5416016 authors Lowet al.); mannoside (Umezawaet al., (1988)Biochem. Biophys. Res. Commun.153:1038); antibodies (P.G. Bloemanet al. (1995)FEBS Lett.357:140; M. Owaiset al. (1995)Antimicrob. Agents Chemother.39:180); the receptor for surfactant protein A (Briscoeet al. (1995)Am. J. Physiol.1233:134); p120 (Schreieret al. (1994)J. Biol. Chem.269:9090); see also K. Keinanen; M.L. Laukkanen (1994)FEBS Lett.346:123; J.J. Killion; I.J. Fidler (1994)Immunomethods4:273.

The use and methods according to the invention

Antibodies, particularly human antibodies is a, compositions of the antibodies and methods according to the present description have a large number ofin vitroandin vivodiagnostic and therapeutic applications, including diagnosis and treatment of α5β1-mediated disorders. For example, these molecules can be introduced into cells in culturein vitroorex vivoor person, for example,in vivofor the treatment, prevention and diagnosis of a large number of diseases. Under used herein, the term "individual" understand humans and animals. Animals include all vertebrates, e.g., mammals and animals other than mammals, such as primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles. To preferred individuals include people with a disorder mediated by the activity of α5β1. The methods are particularly suitable for the treatment of people suffering from a disorder associated with abnormal expression of α5β1. With the introduction of antibodies against α5β1 along with another agent, they may be entered in any order or simultaneously.

Taking into account the specific binding of the antibodies according to the invention with α5β1 antibodies of the invention can be used for specific determination of the expression of α5β1 on the surface of cells and, moreover, can be used to clean α5β1 by immunoaffinity cleanup.

Moreover, taking what I consider the expression of α5β1 on a large number of tumor cells (see, for example, example 6, 7) and its involvement in angiogenesis, human antibodies, compositions of the antibodies and methods according to the present description can be used for the treatment of an individual with abnormal cell growth, for example, suffering from a disorder characterized by the presence of tumor cells expressing α5β1, including, for example, mesothelioma, cancer of the hepatobiliary system (liver and biliary tract), primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, cancer of the gastrointestinal tract (stomach, colorectal and 12 duodenal ulcer), breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal glue the face-to-face carcinoma, carcinoma of the renal pelvis, neoplasms of the Central nervous system (CNS), primary CNS lymphoma, nahodkinskuju lymphoma, spinal cord tumours, glioma, brain stem, pituitary adenoma, adrenocortical cancer, gall bladder, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above malignant tumors.

In one case, the antibodies (e.g. monoclonal antibodies person, multispecificity and bespecifically molecules and compositions) or antihistamie plots according to the invention can be used for detecting levels of α5β1 or levels of cells which contain α5β1 on their membrane surface, and these levels can then be linked to certain disease symptoms. Alternatively, antibodies can be used for inhibiting or blocking α5β1 function that, in turn, may be associated with the prevention or mitigation of certain disease symptoms, thereby taking α5β1 as a mediator of the disease. This can be achieved by bringing the sample and the control sample in contact with the antibody against α5β1 in conditions that promote the formation of a complex between the antibody and α5β1. Any complexes formed between the antibody and α5β1, detect and compare the indicate in the sample and the control.

In another case, antibodies (e.g., human antibodies, multispecific and bespecifically molecules and compositions) or antihistamie plots according to the invention can be initially tested against binding activity associated with therapeutic or diagnostic usein vitro. For example, the composition of the invention can be tested using the analysis of flow cytometry as described in the examples below.

Antibodies (e.g., human antibodies, multispecific and bespecifically molecules, immunoconjugates and composition) or antihistamie plots according to the invention is additionally used in the treatment and diagnosis of diseases associated with α5β1. For example, monoclonal human antibodies multispecificity or bespecifically molecules and immunoconjugates can be used to identify thein vivoorin vitroone or more of the following biological activities: inhibition of growth and/or destruction of cells expressing α5β1; mediating phagocytosis or ADCC of a cell expressing α5β1, in the presence of effector cells or blocking the binding of α5β1 ligand with α5β1.

In the specific case of antibodies (e.g., human antibodies, multispecific and bespecifically molecules and compositions) or antigenes the surrounding areas of use in vivofor the treatment, prevention or diagnosis of a large number of diseases associated with α5β1. Examples of diseases associated with α5β1 include, among others, abnormal cell growth, such as cancer. In one of the embodiments of the abnormal cell growth is a cancer, including but not limited to, mesothelioma, cancer of the hepatobiliary system (liver and biliary tract), primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, cancer of the gastrointestinal tract (stomach, colorectal and 12 duodenal ulcer), breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell to which rcinoma, carcinoma of the renal pelvis, neoplasms of the Central nervous system (CNS), primary CNS lymphoma, nahodkinskuju lymphoma, spinal cord tumours, glioma, brain stem, pituitary adenoma, adrenocortical cancer, gall bladder, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above malignant tumors.

Suitable routes of administration of the compositions of antibodies (e.g. monoclonal antibodies person, multispecificity and bespecifically molecules and immunoconjugates) or antigenspecific plots according to the inventionin vivoandin vitrowell known in this field can be selected by the person skilled in the art. For example, the antibody composition can be introduced by injection (e.g., intravenous or subcutaneous). Suitable doses of the used molecules will depend on the age and weight of the individual and the concentration and/or composition of antibodies.

The previously described human antibodies against α5β1 or antihistamie plots according to the invention can be introduced together with one or more other therapeutic agents, for example, a cytotoxic agent, radiotoxicity agent or immunosuppressive agent. The antibody may be associated with the agent (immunocomplex) or which may be introduced separately from the agent. In the latter case, a separate introduction) antibody can be introduced before, after or simultaneously with the agent, or may be introduced in conjunction with other known methods of treatment, for example, anti-cancer treatments such as radiation. Such therapeutic agents include, among other antineoplastics agents, such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide hydroxyurea, which themselves are only effective when levels of toxic or subtoxic for the patient. Cisplatin can be administered intravenously at 100 mg/dose once every four weeks, and adriamycin injected 60-75 mg/ml dose once in 21 days. The combined antibodies against α5β1 or antigenspecific fragments of the present invention with chemotherapeutic agents provides two anti-cancer agent, which is influenced through various mechanisms, resulting in a cytotoxic effect to human tumor cells. The combined introduction can solve the problems caused by the development of drug resistance or the change in the antigenicity of tumor cells that would make them immune to the antibody.

Target-specific effector cells, e.g., effector cells associated with the compositions (e.g., antibodies which mi man multispecificity and bespecifically molecules),according to the invention can also be used as therapeutic agents. Effector cells to target may be a human leukocytes, such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other cells bearing receptors of IgG or IgA. If necessary, effector cells can be obtained from the individual receiving treatment. Target-specific effector cells can be introduced in the form of a cell suspension in a physiologically acceptable solution. The number of input cells may be of the order of 108up to 109but that will change depending on therapeutic purpose. In General, the quantity must be sufficient to achieve localization at the target cell, for example, a tumor cell expressing α5β1, and to effect the destruction of cells by, for example, phagocytosis. Route of administration may also vary.

Treatment with target-specific effector cells can be carried out along with other techniques of removal nesenevich cells. For example, antitumor therapy using the compositions (e.g., human antibodies, multispecific and bespecifically molecules) according to the invention and/or effector cells, reinforced these comp what dispositions, can be used along with chemotherapy. In addition, the combined immunotherapy can be used for two different populations of cytotoxic effector cells in the rejection of tumor cells. For example, antibodies against α5β1-related receptor anti-Fc-gamma RI or anti-CD3 may be used together with agents that specifically bind to the receptor of IgG or IgA.

Bespecifically and multispecificity molecules according to the invention can also be used to modulate FcγR or levels of FcγR on effector cells, such as capping and elimination of receptors on the cell surface. For this purpose, can also be used a mixture of anti-Fc-receptor.

In the presence of complement can also be used in the composition (e.g., human antibodies, humanized or chimeric antibodies, multispecific and bespecifically molecules and immunoconjugates) according to the invention, which have the binding sites of complement, such as plots from IgG1, -2, or -3 or IgM which bind complement. In one case, the processing of theex vivoa population of cells containing target cells with a binding agent according to the invention and suitable effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells, dormancy is itih binding agent according to the invention, can be improved by binding proteins of the complement. In another case, the target cells coated with the compositions (e.g., human antibodies, multispecific and bespecifically molecules) according to the invention can also be lysed using the complement. In one case, the composition according to the invention do not activate complement.

Compositions (e.g., human antibodies, humanized or chimeric antibodies, multispecific and bespecifically molecules and immunoconjugates) according to the invention can also be introduced along with complement. Accordingly, the invention encompasses compositions containing human antibodies multispecificity or bespecifically molecules and serum or complement. These compositions have the advantage, because the complement is in close proximity to human antibodies, multispecific or bespecifically molecules. Alternatively, human antibodies, multispecific or bespecifically molecules according to the invention and the complement or serum can be entered separately.

The present invention also encompasses kits containing the compositions of the antibodies of the invention (e.g., human antibodies, bespecifically or multispecificity molecules or immunoconjugates) and instructions for use. The kit may optionally with erati one or more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or radiotoxicity agent, or one or more additional human antibodies or their antigenspecific plots according to the invention (for example, a human antibody having an extension activity, which binds to the epitope on the antigen α5β1, distinct from the epitope recognized by the first human antibody).

Accordingly, patients receiving the antibody composition according to the invention, may be added before, simultaneously with or after the introduction of the human antibodies of the invention) another therapeutic agent such as a cytotoxic or radiotoxicity agent that enhances or augments therapeutic effect of the antibody of the person.

In other cases, the individual can get an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fcγ or Fcγ-receptors, through, for example, treatment of an individual with the help of the cytokine. The cytokines for administration during treatment multispecific molecule include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon γ (IFNγ) and tumor necrosis factor (TNF).

Compositions (e.g., human antibodies, multispecific and bespecifically molecules) on the image the structure can also be used to target cells, expressing FcγR or α5β1, for example for labeling such cells. For such applications, the binding agent can be linked to a molecule that can be detected. Thus, the invention relates to methods of localizationex vivoorin vitrocells expressing Fc receptors, such as Fcγ or α5β1. Detected label can represent, for example, a radioisotope, fluorescent compound, enzyme or cofactor of the enzyme.

In the specific case of the invention relates to methods of detecting the presence of antigen α5β1 in the sample, or measuring the amount of antigen α5β1, comprising bringing into contact of the sample and the control sample with a monoclonal antibody to human anatomy or antigennegative portion which specifically binds to α5β1, under conditions that allow to form a complex between the antibody or a part of it and α5β1. The formation of a complex is then detected, and the formation of different complexes between the compared sample and a control sample indicates the presence of antigen α5β1 in the sample.

In other cases, the invention relates to methods for treating α5β1-mediated disorder in an individual, for example, abnormal cell growth, such as cancer, by introducing the individual antibodies person or their antigenspecific plots described above. Such antibodies and the derivatives are used for inhibition of α5β1-induced activities, associated with certain disorders, such as angiogenesis, proliferation and differentiation. Contact antibodies to α5β1 (for example, by introducing antibodies to the individual) the ability of α5β1 to induce such activity is inhibited and, thus, the treatment of associated disorders is effective. Composition antibodies can be injected alone or together with another therapeutic agent such as a cytotoxic or radiotoxicity agent, which acts on par or synergistic composition antibodies for the treatment or prevention of α5β1-mediated diseases.

In one case, immunoconjugate according to the invention can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxins, immunosuppressants, etc.) to cells which have the receptor on the cell surface α5β1, by linking such compounds to the antibody. Thus, the invention also relates to methods of localizationex vivoorin vivocells expressing α5β1 (for example, using a detectable label such as a radioisotope, fluorescent compound, enzyme or cofactor of the enzyme). Alternatively, immunoconjugate can be used to kill cells which have the receptor on the cell surface α5β1 by natalian what I cytotoxins or radiotoxins on α5β1.

The present description is additionally illustrated by the following examples which should not be construed as limiting. The content of all shapes and all references, patents and published patent applications cited throughout this description, the right provided in this document as a reference in full.

EXAMPLES

Example 1

Getting hybridoma producing antibody against α5β1

Visual antibodies according to the invention was obtained, was collected and analyzed as follows:

Immunization and create hybridoma:

To obtain hybridoma used the following immunogen: purified recombinant protein integrin α5-Fc man; integrin α5-His (production company R&D Systems, order); NIH3T3 cells, transfetsirovannyh for the expression of α5 person; cell line Jurkat cell line U-937 (ATCC Cat no CRL-1593) and cell line K-562 (ATCC Cat no CCL-243), which in natural conditions Express the integrin α5β1 person.

Purified recombinant protein integrin α5-Fc man is a chimeric construct of the extracellular domain of integrin α5 person (amino acids 42-995), fused with the Fc domain of the rat, which was cloned in the vector pSecTag2 and expressed using the system 293-FreeStyle (production company Invitrogen). The NIH3T3 cells, transfetsirovannyh for the expression of α5 person, etc who were dotirovala according to the following. cDNA full-length integrin α5 person (the production company Invitrogen, Cat. No. FL1002, clone ID:3629647) cloned from clone MGC full-length integrin α5 person (Invitrogen) and was subcloned into the retroviral expressing vector (pBabe). Viral particles were obtained and used to infect NIH3T3 cells (ATCC Cat. CRL-1658). For positive selection of stable cell clones were added puromycin. For selection of stable cell clones with the highest expression level was carried out by analysis of the expression of the protein human α5 Western blot and FACS.

Fully human monoclonal antibodies against integrin α5β1 person received using strain transgenic for human Ig mice NSO/NSO and strain transamazonic/transgenic genes on human mice KM (production company Medarex, Inc.). All these strains Express fully human antibodies that are indistinguishable from antibodies isolated from people. In these strains of mice endogenous gene Kappa light chain mouse was destroyed homozygous manner, as described in Chen et al. (1993) EMBO J. 12:811-820, and endogenous gene of the heavy chain of the mouse was destroyed homozygous manner as described in example 1 of PCT publication WO 01/09187. Each of these strains of mice carries the transgene Kappa light chain human XO described in article Fishwild et al. (1996) Nature Biotechnology 14:845-851. Strain NSO carries the transgene, the heavy chain of human NSO, described is in the U.S. patents 5545806, 5625825 and 5545807. Strain NSO carries the transgene, the heavy chain of human NSO described in example 2 of PCT publication WO 01/09187. Strain NSO/NSO carries both the heavy chain transgene NSO and NSO. Strain KM is minichromosome person described in article Ishida et al., (2002), Cloning and Stem Cells, 4:91-102.

To produce fully human monoclonal antibodies against α5β1 HuMab mice strains NSO/NSO and KM were immunized with the recombinant protein integrin α5-Fc man or integrin α5-His, NIH3T3 cells, transfitsirovannykh for the expression of α5 man and the cell line Jurkat cell line U-937 and cell line K-562, which in natural conditions, Express α5β1 person. The General scheme of immunization of HuMab mice is described in articles Lonberg, N. et al.(1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and PCT publication WO 98/24884. If the first infusion of antigen were taken of the mouse at the age of 6-16 weeks. For intraperitoneal (IP) and subcutaneous (Sc) immunization of mice HuMab used purified recombinant preparation of antigen integrin α5-Fc man or integrin α5-his (15-20 μg), drug transfected NIH3T3 cells or Jurkat cells, cells U-937 cells and K-562 (1×107cells).

Transgenic mice were immunized with antigen in Freund Ribi intraperitoneally and subcutaneously at intervals of 1-4 weeks (up to 16 immunizations). Immune response was observed in the blood taken by R is troubeling of bloodletting. Serum was skanirovali by FACS analysis (described below), and mice with sufficient titers of immunoglobulin against α5β1 used for fusions. Mice stimulated by the intravenous injection of antigen for 3 and 2 days before slaughter and extraction of the spleen and/or lymph nodes. Typically, for each antigen was carried out by 10-20 m. All were immunized 60 mice NSO/NSO and KM. Each antigen were immunized several dozen mice.

Selection of HuMab mice producing antibodies against α5β1:

To select HuMab mice producing antibodies that are associated with α5β, the serum of immunized mice were skanirovali by flow cytometry (FACS) in relation to binding to cell line expressing full-integrin α5β1 person and do not associate with a control cell line not expressing α5β1. Briefly, NIH3T3 cells expressing α5, incubated with serum from immunized mice diluted 1:20. Cells were washed, and specific binding of antibodies was detected using Ab against human IgG labeled with FITC. Analysis flow cytometry was performed on the device is a flow cytometer FACS (production company Becton Dickinson, San Jose, CA). Mice who had developed the highest titers of antibodies against α5β1, used for fusions. The merger was carried out as described below, and g is Britanie supernatant were tested using FACS in respect of activism against α5β1.

Development of hybridomas producing monoclonal antibodies against α5β1:

Splenocytes and/or lymphocytes from lymph nodes of the mouse isolated from the HuMab mice were merged using electroline (E-merge, technology Cyto PulseTM, Cyto PulseTMSciences, Inc., Glen Burnie, MD), with the myeloma cell line of mouse Sp2/0 (ATCC, CRL-1581, Manassas, VA)using protocols recommended by the manufacturer. Briefly, suspensions of single cells lymphocytes of the spleen and/or lymph nodes of immunized mice were merged with the equivalent number of " non-secretory myeloma cells of mouse Sp2/0 using E-merge. The cells were placed in flat-bottomed tablets for micrometrology in an amount of about 2×104the splenocytes/well and incubated for 10 to 14 days in selective medium containing 10% fetal bovine serum, 10% air-conditioned cells, P388D1 (ATCC, CRL TIB-63) Wednesday, from 3 to 5% (IGEN) in DMEM (production company Mediatech, Herndon, VA, Cat. No. CRL 10013) with high glucose, L-glutamine and sodium pyruvate), 5 mm HEPES, 0.055 mm 2-mercaptoethanol, 50 mg/ml gentamicin and 1-fold NAT (production company Sigma, Cat. NO. CRL P-7185). After 1-2 weeks the cells were cultured in the medium in which the nut was replaced with HT. After about 10-14 days after placing the cells supernatant from individual wells were skanirovali first in the content of antibodies to ha is mA Kappa-chains of human rights. Supernatant, which was noted as positive in respect of antibodies to gamma - Kappa-chains of a human, then was skanirovali by FACS (described above) in respect of antibodies against α5β1.

Monoclonal antibody IgG:

Hybridoma, secreting antibodies transferred in 24-hole tablets, again skanirovali and upon confirmation of positivity in relation to IgG-monoclonal antibodies against α5β1 was subclinically at least twice by limiting dilution. Stable subclones were then culturedin vitroto obtain small amounts of antibody in tissue culture medium with additional features. The procedure described above, used for the production of several monoclonal antibodies against α5β1, including antibodies, designated as "V", "S", "1D9" and "2D2", which are described in this document.

Example 2

Structural characterization of monoclonal antibodies person W

Sequence of cDNA encoding the variable regions of the heavy and light chains of the monoclonal antibodies W received from hybridoma V using standard techniques of PCR, and sequenced using standard techniques of DNA sequencing.

Nucleotide and amino acid sequences of variable regions thee who Eloy chain antibodies V shown in figa and 1B and in SEQ ID NO:11 and 7, respectively. Nucleotide and amino acid sequences of variable region of the light chain antibodies V shown in figs and 1D and SEQ ID NO:12 and 8 respectively.

The original molecule V belonged to the IgG4 subclass. The IgG subclass switch to IgG1 to ensure binding to FcγR and effector functions such as ADCC. To further increase binding to FcγR receptors and the activity of effector functions in the constant region IgG1 was injected three mutations (S247D, A338L and I340E) via site-specific mutagenesis. Antibody V with the subclass IgG1 and content of these three mutations is designated as "22B5/DLE" or "22B5 IgG1 DLE". Two mutations variable domain of the heavy chain was returned to the germ line to minimize immunogenicity (I30S and N33S). In the variable sequence of the light chain mutations were found. Heavy chain 22B5/DLE presented as SEQ ID NO:9, whereas the light chain 22B5/DLE presented as SEQ ID NO:10.

Comparison of the sequences of the heavy chain immunoglobulin V with the known sequences of the heavy chains of immunoglobulin germline human showed that the heavy chain of the antibody V used segment VHfrom gene VH4-39 germline human D segment of the gene germline 3-10 man and a JH segment from gene JH 6b germline person. Sequence alignment of the VHis ntitle V sequence V H4-39 the germ line are presented in figure 2. Additional sequence analysis of the VHantibodies V using the Kabat system determining region CDR, led to the image regions CDR1, CDR2 and CDR3 of the heavy chain, presented on figv and SEQ ID NO:1, 2, and 3, respectively.

Comparison of the sequences of the light chain of the immunoglobulin V with known sequences of immunoglobulin light chain germline human showed that in the light chain of the antibody V used segment VLfrom gene VK L6 germline of the person and the segment from JK JK gene 4 germline person. Sequence alignment of the VLantibodies V sequence VK L6 germline presented in figure 2. Additional sequence analysis of the VLantibodies V using the Kabat system determining region CDR, led to the image regions CDR1, CDR2 and CDR3 of light chain, presented on fig.1D and in sequences of SEQ ID NO:4, 5, and 6, respectively.

Example 3

Characterization of binding specificity and kinetic analysis of the binding of monoclonal antibodies person W

In this example, the affinity of binding and kinetic analysis of binding of human antibodies against α5β1 V with mutations IgG1 DLE (22B5/DLE) was assessed by Biacore analysis. The binding specificity was assessed by the th flow cytometry (FACS).

The affinity of binding and kinetic analysis of binding

Kinetic analysis of binding and par avidity antibodies W/DLE to the extracellular domain of integrin α5β1 was determined using Biacore analysis (Biacore AB, Uppsala, Sweden). To implement kinetic analyses BIAcore antibody W/DLE was immobilized on a biosensor chip, and a large number of concentrations of the extracellular domain of recombinant human α5β1 was passed through the surface when 25,0°C (see figure 3). Data binding was summed up in the General model for the binding of one-to-one model with drifting baseline. W/DLE reversibly binds to α5β1. The binding constant (KDfor the extracellular domain of recombinant human α5β1 ranged from 2.7 to 4.1 nm. KDturned out to be higher when measured in the absence of divalent cations, consistent with the cation-dependent integrin activation. Kinetic binding parameters were within the range of from 3.4×10-5up to 4.5×10-5M-1with-1for speed direct reaction and from 1.2×10-3up to 1.4×10-3with-1for the rate of reverse reaction (see figure 3 and table 1).

To measure par avidity of the extracellular domain of recombinant human α5β1 was immobilized on a biosensor chip, and a large number of concentrations W/DLE was passed through the surface when 25,0°C. the Magnitude of the nominal value of the Noi the avidity was 0.05 PM in 20 mm HEPES, a pH of 7.4; 150 mm NaCl; of 0.005% P20 with 4.0 mm MgCl2.

Table 1
The resulting data binding and kinetic analysis based on research BIAcore
Affinity W/DLE to α5β1 (Biacore)
KD(CaCl2)2,7 nm
KD(MgCl2)of 4.1 nm
Speed direct reaction (kon) (CaCl2)4,5×10-5M-1s-1

Speed direct reaction (kon) (MgCl2)of 3.4×10-5M-1s-1
Speed direct reaction (kon) (CaCl2)of 1.2×10-3s-1
Speed direct reaction (kon) (MgCl2)of 1.4×10-3s-1
The avidity W/DLE to α5β1 (KD, Biacore)0,05 PM
Under CaCl2understand a buffer containing 10 mm HEPES, pH of 7.4; 150 mm NaCl; of 0.005% P20 with 0,1575 inches CaCl2/sub> ; under the MgCl2understand a buffer containing 10 mm HEPES, pH of 7.4; 150 mm NaCl; of 0.005% P20 with 4.0 mm MgCl2.

Under CaCl2understand a buffer containing 10 mm HEPES, pH of 7.4; 150 mm NaCl; of 0.005% P20 with 4.0 mm CaCl2; under the MgCl2understand a buffer containing 10 mm HEPES, pH of 7.4; 150 mm NaCl; of 0.005% P20 with 4.0 mm MgCl2.

Using FACS affinity to cells expressing α5β1

Antibody W/DLE tested for its affinity for binding to integrin cell surface α5, using FACS analysis, using cells expressing endogenous integrin α5β1 human (HUVEC). Briefly, cells were isolated using trypsin-EDTA and washed with chilled PBS. After making aliquot in 96-well tablets, cells were blocked with serum and incubated with different concentrations of specific mAb for 1 hour at 4°C. Then cells were washed and incubated with a secondary antibody against the κ-chain man, conjugated with fluorophore R-PE, and analyzed using flow cytometer FACSCalibur. For each sample collected 10000 events without applying any datirovaniya. To determine the KDexpected geometric mean of the histogram of each sample and expressed as a function of the concentration of mAb. KDwas calculated after summing up under equilibrium model with two States. Antibody W/DLE associated with endogenous α5β1 with K of 2.15 nm (n=4) in HUVEC cells (see figure 4).

Example 4

Affinity for Fcγ-receptors determined using Biacore analysis

The ability of antibodies W/DLE, which contains three mutations IgG1 DLE (described previously in example 2), to enhance binding to FcγR receptors were evaluated in the experiment Biacore binding. The affinity of binding to FcγR antibodies W/DLE was compared to the affinity of binding of IgG1 wild-type (wt). It was tested on three classes of FcγR receptors: 1) high-affinity receptor FcγRI; 2) two polymorphic variants of low-affinity receptor FcγRIIa/N and FcγRIIa/131R; and 3) two polymorphic variants of the receptor with an average affinity FcγRIIIa/158F and FcγRIIIa/158V. The results (presented in figure 5 and summarized in table 2) show approximately 6-fold increase for the high-affinity receptor FcγRI. Observed 122 - and 70-fold increased binding to receptors with moderate affinity FcγRIIIa/158F and 158V respectively. Comparison of the affinity of the antibody binding sites W/DLE and IgG1 wild-type was also evaluated against the Fcγ-receptors of the mouse. The maximum increase binding was observed for mFcγRIV, then for mFcγRI and mFcγRIII (see figure 5).

Table 2
Binding to FcγR receptors in the Biacore analysis (KDthe ratio of gain relative to wt IgG1
FcγR KD(the ratio of the gain relative to wt IgG1)
FcγRI manof 5.8 nm (6-fold)
FcγRIIa/131H man1000 nm (1,6 times)
FcγRIIa/131R man650 nm (2,3-fold)
FcγRIII/158F man27 nm (122 times)
FcγRIII/158V manof 9.4 nm (70-fold)
FcγRI mouse96 nm (14-fold)
FcγRIII mouse1700 nm (5-fold)
FcγRIV mouseof 9.1 nm (260-fold)

Example 5

Cellular functional activity of antibodies W/DLE

Blockade of cell adhesion

Was tested the ability of antibodies W/DLE to disrupt cell adhesion mediated by integrin α5β1. Analyses of cell adhesion was performed by pre-incubation of HUVEC cells with the antibody (V/DLE, V IgG1, variant subclass IgG2 and IgG4 or negative control mAb (VNA IgG1)and then placed in the tablets coated with fibronectin (FN) or collagen. Cells HUVEC (15000) was mixed with the antibody in the buffer and is gasii (Hepes-buffered saline, containing glucose and bovine serum albumin) for 20 minutes at room temperature. Cells were added to wells of 96-hole tablet, coated with fibronectin or collagen, and left for adhesion to the plate for one hour at 37°C/5% CO2. Naughtyallie cells were removed by washing each well three times. Measured quantity desirously cells remaining in each well. Adgeziruetsya cells were literally by adding a buffer containing dye CyQUANT GR, and fluorescence was measured at Ex/Em: 485/535 nm with a spot reader. As can be seen in Fig.6, mAb, functionally blocking the integrin α5β1, selectively inhibit the adhesion of HUVEC cells to fibronectin, but not collagen (negative control). As shown in Fig.6, the value of the IC50for W/DLE and variants of its subclass (IgG1 wild-type IgG1 DLE, IgG2 and IgG4) were similar in this one-hour analysis. These data indicate that binding of antibodies to α5 and/or α5β1 inhibits the binding of an integrin with fibronectin. These data additionally indicate that the antibody binds to α5β1, if it is expressed on the cell surface. That is, the epitope recognized by the antibody is available, if the chain α5 and β1 is associated, and the epitope is available for binding when the integrin is expressed on the cell surface.

Example 6

ADCC-effector function in vitro

Activity of the effector functions was assessed by analyses of ADCC in the presence of mononuclear cells in human peripheral blood (RVMS) against α5β1-positive target cells using standard methods. The levels of expression of integrin α5 for a number of cell lines was measured using analysis Western blot in accordance with the following. Cells were literally in lytic buffer RIPA (the production company Upstate)containing protease inhibitors (production company Roche) and phosphatase (production company Calbiochem). Was carried out by electrophoresis of lysates (3 µg of total protein each) in the gel SDS-PAGE and Western blot turns with antibodies against integrin α5. Immunoreactive bands were identified and analyzed by infrared system image the Odyssey LI-COR Bioscience. An example of a number expression is shown in Fig.7.

To measure ADCC effector cells (RVMS) were isolated using centrifugation in a gradient of ficoll, samples from the blood Bank of San Diego according to standard methods. Ten thousand target cells (HUVEC or tumor lines) pre-incubated with the antibody in the growth medium at room temperature for 20 minutes, then added one million cells RVMS person (E:T=100:1), and the mixture is incubated at 37°C/5% CO2within 4 hours. ADCC-mediated cell lysis was measured using the detection wisweb the Denia lactate dehydrogenase (LDH) (Roche) or set for bioanalysis ToxiLight (Cambrex) according to standard protocols.

Antibody W/DLE associated with swelling or integrin α5β1, expressed by the endothelium and stimulate ADCC (up to 80% lysis of target cells) with EC500,04 nm compared to 0.23 nm for wt 22B5 IgG1. These data indicate that mutations DLE increased ADCC activity compared to the IgG1 type. In a four-hour analysis or for V IgG2, nor for V IgG4 ADCC activity was not detected. As a negative control mAb used KLH IgG1. Examples of ADCC in HUVEC cells and typical tumor lines (U87MG) shown in Fig (reflects both methods of detection).

As shown by the data presented in Fig.9, cell cytotoxicity induced W/DLE and wt 22D5 IgG1 correlates with the level of expression of α5β1 cell line. In cells with the highest expression of α5β1 (panel A, U87MG) observed the highest level of cell cytotoxicity, whereas the cell lines with an average expression (Fig.9, Hs578T and 3T3, 5) observed intermediate cytotoxicity. It is noted that the antibody V/DLE increased ADCC effects in M24met cells, which expressed low levels of α5β1 (examples in the bottom panel of figure 9). These data suggest that ADCC is mediated, at least in part, by the binding of antibodies to α5β1 expressed by the cell.

Example 7

Antimetastatic efficacy in vivo

In addition to his proliferation activities, the moment the promotion and survival of endothelial and tumor cells, integrin α5β1 is also involved in tumour metastasis by stimulating the extravasation of tumor cells and migration to distant organs. The following study is intended to demonstrate antimetastatic efficacy W/DLE in preclinical models.

Inhibition of experimental metastasis A-Luc easy

A-Luc is a cell line non-small cell human lung cancer (NSCLC), transtitional gene luciferase. When intravenous implantation of cells A-Luc preferably localized in the lung that can be measured by bioluminescent image (BLI). In this study, cells A-Luc was injected (3×106on the animal) via the tail vein in mice SCID BALB/c mice that received a prior dose of the appropriate antibodies for two days prior to injection. After implantation, the animals received a dose once a week up to 8 weeks. The bioluminescence of each animal was measured once per week up to 20 weeks.

The antibodies W/DLE showed the highest efficacy against tumor metastasis with TGI (inhibition of tumor growth) 97% 8 week (the final week of the dose)that is statistically significant (p<0.01) compared with the control group. The dose of antibody V IgG1, which are assumed not to be mediates ADCC is this model also led to significant TGI, amounting to 89%, 8 week (figa). Values of R comparison between V/DLE and V IgG2 5, 6, 7 and 8 weeks was 0.07; 0,03; of 0.08 and 0.17, respectively. All treatment was stopped after 8 weeks, while daily monitoring of undesirable physiological signs and weekly measurements using BLI animals continued.

Tumors in the group treated W/DLE remained cupressiforme through 13 weeks, whereas in the group treated W IgG2, began to grow rapidly after about 2 weeks after discontinuation of the medicinal product (pigv). The end point of the study believed achieved when reaching BLI tumor 1×108fonorow/second, at this time, the mice were scored. The graph of the Kaplan-Meier (figs) shows that the antibody V/DLE was significantly prolonged the mean survival time of animals up to 20 weeks compared with 14 weeks for the animals treated W IgG2, and 10 weeks for the control group.

These data indicate that in this model, the antitumor effect of antibodies W not mediated exclusively ADCC activity. That is V-IgG2 inhibited growth/tumor invasion, although unknown, mediates whether IgG2 human effector functions, including cellular cytotoxicity and indirect antibody anti-tumor effects. In addition, the data showed that grew the tumor, the remaining supression after the introduction V-IgG1-DLE, stopped. These data indicate that blocking the binding of α5β1 with FN mediates antitumor effect (V-IgG2), which is additionally enhanced ADCC activity (V-IgG1/DLE).

Example 8

Structural characterization of monoclonal antibodies person S, 1D9 and 2D2

Sequence of cDNA encoding the variable regions of the heavy and light chains of monoclonal antibodies S, 1D9 and 2D2, was obtained from hybrid S, 1D9 and 2D2 (respectively), using standard techniques of PCR, and sequenced using standard techniques of DNA sequencing.

Nucleotide and amino acid sequences of variable regions of heavy chain antibodies IS shown in fige and 1F and the sequences SEQ ID NO:21 and 19, respectively. Nucleotide and amino acid sequences of variable region of the light chain antibodies 24C7 shown in fig.1G and 1H and in sequences of SEQ ID NO:22 and 20, respectively.

Nucleotide and amino acid sequences of variable regions of the heavy chain of the antibody 1D9 shown in Fig and 1J and in sequences of SEQ ID NO:31 and 29, respectively. Nucleotide and amino acid sequences of variable region of the light chain of the antibody 1D9 shown in FIGC and 1L and the sequences SEQ ID NO:32 and 30, respectively.

Nucleate the Naya and amino acid sequences of variable regions of heavy chain antibodies 2D2 shown in figm and 1N and in sequences of SEQ ID NO:41 and 39, respectively. Nucleotide and amino acid sequences of variable region of the light chain of the antibody 1D9 shown in Figo and 1P and in sequences of SEQ ID NO:42 and 40, respectively.

The original molecule S, 1D9 and 2D2 belonged to the IgG1 subclass. In respect of the subclass IgG1, to further improve binding to FcγR receptors and the activity of effector functions in the constant region IgG1 was injected three mutations (S247D, A338L and I340E) via site-specific mutagenesis. Thus, the antibody IS related to the subclass IgG1 and contains these three mutations, referred to as S/DLE" or "S IgG1 DLE". Likewise, similar antibodies to these three mutations denoted in this document as "1D9/DLE" or "1D9 IgG1 DLE" and "2D2/DLE" or "2D2 IgG1 DLE". Similarly, antibodies with two mutations S247D and I340E denoted in this document as the mutants "DE", for example, "24C7/DE" or "24C7 IgG1 DE". Similar names of any of the antibodies described herein, and with any one, two or three of the above mutations indicate in this document in the same way. For example, under "1D9/DE" understand antibody 1D9 described in this document relating to the IgG1 subclass, with the following mutations in the Fc-region - S247D and I340E. Similarly, "2D2/L" would apply to the 2D2 antibody, described herein, with isotype IgG1 containing the mutation A338L and so on.

the alignment sequence of the heavy chain immunoglobulin S with the known sequences of the heavy chains of immunoglobulin germline man showed in the heavy chain S used segment VHfrom gene VH3-30 .3 germline human D segment of the gene 7-27 germline man and a JH segment from gene JH 6b germline person. Sequence analysis of the VHantibodies S using the Kabat system of CDR define region, led to the image regions CDR1, CDR2 and CDR3 of the heavy chain, presented on fig.1F and in sequences of SEQ ID NO:13, 14 and 15, respectively.

Comparison of the sequences of the light chain of the immunoglobulin 24C7 with known sequences of immunoglobulin light chain germline human showed that in the light chain 24C7 used segment VLfrom gene VK L6 germline of the person and the segment from JK JK gene 2 germline person. Sequence analysis of the VLantibodies 24C7 using the Kabat system of CDR define region, led to the image regions CDR1, CDR2 and CDR3 of light chain, presented on fign and in sequences of SEQ ID NO:16, 17 and 18, respectively.

Comparison of the sequences of the heavy chain of an antibody 1D9 with the known sequences of the heavy chains of immunoglobulin germline human showed that the heavy chain of the 1D9 used segment VHfrom gene VH3-30 .3 the germ line of a man and a JH segment from gene JH 6b germline person. Segment D could not be what opstellen with the genome of the germ line because of the common mutations. Sequence analysis of the VHantibodies1D9, using the Kabat system of CDR define region, led to the image regions CDR1, CDR2 and CDR3 of the heavy chain, presented on fig.1J and in sequences of SEQ ID NO:23, 24 and 25, respectively.

Comparison of the sequences of the light chain of an antibody 1D9 with known sequences of immunoglobulin light chain germline human showed that in the light chain of the 1D9 used segment VLfrom gene VK L6 germline of the person and the segment from JK JK gene 1 germline person. Sequence analysis of the VLantibodies1D9, using the Kabat system of CDR define region, led to the image regions CDR1, CDR2 and CDR3 of light chain, presented on fig.1L and in sequences of SEQ ID NO:26, 27 and 28, respectively.

Comparison of the sequences of the heavy chain immunoglobulin 2D2 with the known sequences of the heavy chains of immunoglobulin germline human showed that the heavy chain 2D2 used segment VHfrom gene VH3-30 .3 germline human D segment of the gene 7-27 germline man and a JH segment from gene JH 6b germline person. Sequence analysis of the VHantibodies2D2, using the Kabat system of CDR define region, led to the image regions CDR1, CDR2 and CDR3 of the heavy chain represented by the WMD on fig.1N and in sequences of SEQ ID NO:33, 34 and 35, respectively.

Comparison of the sequences of the light chain of the immunoglobulin 2D2 with known sequences of immunoglobulin light chain germline human showed that in the light chain 2D2 used segment VLfrom gene VK L6 germline of the person and the segment from JK JK gene 3 germline person. Sequence analysis of the VLantibodies2D2, using the Kabat system of CDR define region, led to the image regions CDR1, CDR2 and CDR3 of light chain, presented on figr and in sequences of SEQ ID nos:36, 37 and 38, respectively. In the following table 3 presents the use of genes in monoclonal antibodies B, 2D2, S and 1D9.

Example 9

Characterization of binding specificity and kinetic analysis of binding of monoclonal antibodies person V, S, 1D9 and 2D2

In this example, the binding specificity of human antibodies against α5β1 V, S, 1D9 and 2D2 subclass IgG1 in the presence and in the absence of mutations IgG1 DLE (i.e. "22B5 G1 DLE", "22B5 G1", "1D9 G1 DLE", "1D9 G1" and so on) were evaluated by flow cytometry using the sorting of fluorescently-activated cells (FACS).

Determination of affinity for cells expressing α5β1, using FACS

Antibodies W/DLE, S/DLE, 1D9/DLE and 2D2/DLE tested for their affinity of binding to the integrin cell is poverhnosti α5, using FACS analysis using cells expressing endogenous integrin α5β1 human (HUVEC). Briefly, cells were isolated using trypsin-EDTA and washed with chilled PBS. After making aliquot in 96-well tablets, cells were blocked with serum and incubated with different concentrations of specific mAb for 1 hour at 4°C. Then cells were washed and incubated with a secondary antibody against the κ-chain man, conjugated with fluorophore R-PE, and analyzed using flow cytometer FACSCalibur. For each sample collected 10000 events without the use of any datirovaniya. To determine the KDexpected geometric mean of the histogram of each sample and expressed as a function of the concentration of mAb. KDwas calculated after summing up under equilibrium model with two States. The results are presented on figa and 11B.

Example 10

In vitroADCC-effector function of monoclonal antibodies person V, S, 1D9 and 2D2

Activity of the effector functions were evaluated by analyses of antibody-dependent cell-mediated cytotoxicity (ADCC), described previously in example 6. Size EU50regarding stimulate ADCC (up to 80% lysis of target cells) in a typical tumor lines (U87MG) presented on Fig for antibody 1D9, 1D9/DLE, 2D2/DLE, 22B5/DLE and 24C7/DLE.

The use of the 11

In vivo ADCC-dependent antitumor efficacy in a syngeneic model of metastatic melanoma

To demonstrate ADCC-specific response monoclonal human antibody 1D9/DLE (which binds to α5β1 human and mouse, but not functionally neutralize α5β1) was tested on the model of inhibition of tumor growth (TGI) in syngeneic mice with intact immune effector cells. Specifically, the ADCC-mediated antitumor activity was evaluated on the model of syngeneic mice, in which cells of mouse melanoma B16F10 expressed α5β1. Line of mouse melanoma B16F10 expressing integrin α5 is highly metastatic and intravenously initially colonize the lung. Tumor cells were injected intravenously in immune-competent C57BL/6 mice. The B16F10 cells (2×105for animal) were injected via tail vein in syngeneic mice host C57BL/6, which previously 1 day before the injection was given antibody (10 mg/kg, n=10 per group). Animals received subcutaneously antibody once a week for only 3 weeks. On day 21, animals were scored and resellable light.

As shown by the results on Fig, antibody 1D9/DLE has produced significant antitumor efficacy ofin vivo, indicating that for the production of TGI enough ADCC response. To display specific ADCC effect in the dressed animals were processed using 1D9 IgG2 (no data about the mediating ADCC) and 1D9 IgG1 DLE (isotype IgG1 with increased Fc due to mutations DLE). Greater antitumor efficacy was observed with 1D9 IgG1 DLE than 1D9 IgG2. As couples isotypes have identical amino acid sequences in antigennegative domain andin vitroactivity data indicate that increased efficiency 1D9 IgG1 DLE was responsible enhanced ADCC as a result of mutations DLE.

Information about Deposit

Applicants deposited variable regions of the heavy and light chains of the antibodies identified in this document as V, American Type Culture Collection (ATCC) Manassas, VA 20110-2209 U.S.A. VH-region of the antibody V deposited on July 16, 2008 and assigned Deposit number ATSS MOUTH-9377. VL-region antibody V deposited on July 16, 2008 and assigned Deposit number ATSS MOUTH-9377. These deposits are made in accordance with the provisions of the Budapest Treaty on the international recognition of the Deposit of microorganisms for purposes of patent procedure and the regulations under that Treaty (the Budapest Treaty). These deposits must be maintained without restriction in the Depository ADS within a period of 30 years, or 5 years after the most recent request, or for the term of the patent, regardless of its duration, and must be replaced if the Deposit becomes non-viable during that period. Availability of the deposited material should not be construed as permission to perform l is any of the aspects of the present invention, in violation of the rights data under the authority of any government in accordance with its laws patenting. It is assumed that the above description should be enough for the person skilled in the art could carry out all aspects of the present invention. The present invention should not be limited in the amount of deposited material, deposited as an implementation option is understood as one of the illustrations of some aspects of the invention and any constructs that are functionally equivalent are covered by this invention. Deposition of material in this document is not an admission that the herein description is not enough to implement any aspect of the invention, including its best way, and it should not be construed as limiting the scope of the claims, the specific illustrations that it represents. In fact, experts in this field from the foregoing description it should be clear large number of modifications of the invention in addition to presented and described in this document, and they are covered by the attached claims.

1. The selected monoclonal antibody to integrin α5β1 person that contains:
(a) variable regions CDR1 of the heavy chain, containing SEQ ID NO:1;
(b) CDR2 of variabel the Oh region of the heavy chain, containing SEQ ID NO:2;
(c) the CDR3 of the variable region of the heavy chain, containing SEQ ID NO:3;
(d) variable regions CDR1 light chain containing SEQ ID NO:4;
(e) a CDR2 variable region of the light chain containing SEQ ID NO:5; and
(f) a CDR3 of the variable region of the light chain containing SEQ ID NO:6.

2. The antibody according to claim 1, containing:
(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:7; and
(b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:8.

3. The antibody according to claim 1 or 2, where the aforementioned antibody is a full-IgGl-human antibody, and where at least one amino acid in the Fc-region of the specified antibodies metirovan, and additionally, where the specified antibodies observed ADCC greater than that of identical antibodies, which indicated at least one amino acid not metirovan.

4. The antibody according to claim 3, in which at least one mutation occurs at amino acid position serine 247, alanine 338 or isoleucine 340.

5. The antibody according to claim 3, where at least one mutation selected from the group consisting of mutations: serine 247 on aspartic acid (S247D), alanine 338 on leucine (A338L) and isoleucine 340 to glutamic acid (I340E).

6. The antibody according to claim 5, where the antibody contains mutations S247D, A338L and I340E.

7. The antibody according to claim 1, where the specified antibody contains a heavy chain, asteasu of the amino acid sequence, shown in SEQ ID N0:9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:10.

8. Composition for inhibiting the growth of tumor cells expressing the integrin α5β1 person, containing an effective amount of the antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. The selected nucleic acid molecule encoding the antibody according to any one of claims 1 to 7.

10. Expressing the vector containing the nucleic acid molecule of claim 9.

11. A host cell for expression of the antibody according to any one of claims 1 to 7, containing expressing the vector of claim 10.

12. A method of obtaining antibodies against α5β1, including the expression of antibodies in the cell host according to claim 11.

13. Method of inhibiting the growth of tumor cells expressing the integrin α5β1 person, including the conversion of these cells into contact with an antibody according to any one of claims 1 to 7 or the composition of claim 8 in an amount effective for inhibiting the growth of tumor cells.



 

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7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: invention refers to the field of biotechnology and food industry. Presented is a barley plant that yields grain and is homozygotic in at least two loci for the genetic variations having been bred, representing: a) allele wherein most of or all the genes coding B-hordein in Locus Hor2 are removed, and b) mutant allele in the barley Locus Lys3 so that the grain contains neither B-, nor C- hordeins, the said genetic variations present in Lines Riso 56 and Riso 1508 barley accordingly; absence of B-hordeins is to be revealed by absence of amplified DNA using primers: 5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3', 3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3', while absence of C-hordeins is to be revealed by absence of the 70 kDa strip during study of the grain alcohol-soluble extract by means of SDS-PAGE. Additionally presented are: barley grain cropped from the said plant; B- and C-hordein-free products produced from the said grain such as flour, malt and beer. Additionally described are methods for production of food products barley (flour, whole-grain flour, starch, malt) and beverages using grain cropped from the barley plant having the above characteristics. Proposed is a method for identification of barley grain suitable for production of a malt-based food product and/or beverage suitable for consumption by a person suffering from gluten-sensitive enteropathy which method includes: a) production of one or more materials: i) sample of a plant capable to yield the said grain, ii) grain, iii) malt produced from the grain, and/or iv) extract of the said grain; b) analysis of Stage a) material for presence of at least one hordein and/or at least one hordein-coding gene with selection of grain having the gene pattern of the above plant.

EFFECT: invention allows to manufacture B- and C-hordein-free malt-based food products or beverages.

27 cl, 14 dwg, 10 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology. There are presented versions of the recombinant bacterial strain Escherichia coli that are succinic acid producers and contain a gene coding pyruvate carboxylase. The bacterial strain Escherichia coli SGM1.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE inactivated genes. The bacterial strain Escherichia coli SGM1.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE and icIR inactivated genes. The bacterial strain Escherichia coli SGM2.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE inactivated genes and enhanced expression of aceE, aceF and lpdA genes. The bacterial strain Escherichia coli SGM2.1 [pPYC] possesses ackA, pta, poxB, ldhA, adhE, icIR inactivated genes and enhanced expression of aceE, aceF and lpdA genes. The bacterial strain Escherichia coli SGM3.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE, pflB inactivated genes and enhanced expression of aceE, aceF and lpdA genes. The bacterial strain Escherichia coli SGM3.1 [pPYC] possesses ackA, pta, poxB, ldhA, adhE, pflB, iclR inactivated genes and enhanced expression of aceE, aceF and lpdA genes. What is also presented is a method for preparing succinic acid with using the above strains.

EFFECT: group of inventions provides higher succinic acid yield.

17 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention concerns biotechnology and nanotechnology. The method includes transforming archaeal cells with a recombinant plasmid, growing cells, selecting flagella and modifying the surface of the flagella. The plasmid structure contains recombinant genes for synthesis of flagellins A1 and A2 which form flagella, wherein the sequence of flagellin A1 or flagellin A2 or sequences of flagellin A and flagellin A2 contain at least one peptide insert for selective binding of metal ions or nanoparticles. The point of the peptide insert in flagellin A1 is defined in the region between first and second glycosylation sites located between position 86 and position 96 of SEQ ID NO:2, and the point of the peptide insert in flagellin A2 is defined in the region between first and second glycosylation sites located between position 82 and position 92 of SEQ ID NO:3, where the length of the peptide insert is 5 to 60 amino acids. The method includes selecting archaeal flagella containing peptide inserts for non-covalent bonding with metal ions, performing fragmentation of flagella into fragments and modifying the surface of flagella by binding peptide inserts with metal ions and oxidising metals, washing, drying and packing the obtained nano-structured material.

EFFECT: method enables to obtain a coating for forming active surfaces on flexible and solid substrates or capsules using archaeal flagella, which enable non-covalent bonding of a wide range of substances such as metal ions, metal nanoparticles, semiconductors and other ligands.

6 cl, 11 dwg, 1 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.

EFFECT: invention enables providing higher methane-producing cell permeability.

18 cl, 35 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a plant, having high resistance to an AHAS-inhibiting herbicide, which includes at least one Shiloh-8 IMI nucleic acid, parts thereof, a plant cell and seeds. Described is a nucleic acid which encodes a polypeptide which increases herbicide resistance of a plant. Disclosed are an expression cassette and a plant transformation vector which include said nucleic acid. Described are methods of controlling weeds growing near a plant having high resistance to an AHAS-inhibiting herbicide. Disclosed is a method of producing a plant having high resistance to an AHAS-inhibiting herbicide, as well as a method of increasing AHAS activity in a plant. Described is a method of selecting a cell transformed by a vector containing IMI nucleic acid. Also disclosed is a method of increasing resistance to an AHAS-inhibiting herbicide and a weed control method which includes treatment with an AHAS-inhibiting herbicide.

EFFECT: invention enables to obtain a plant which is resistant to an AHAS-inhibiting herbicide, which provides effective control of weeds growing near said plant.

57 cl, 3 dwg, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to methods for preparing next generation drug preparations and biologically active additives in bioreactors on the basis of transgenic producing mammals. The method for creating transgenic animals producing a protein with stable and high expression in milk, involves producing transgenic mammals with using a vector containing a reporter gene coding a target protein that is a goat beta-casein gene promoter, a bovine growth factor terminator and effective two-fold transcription terminators. The terminators surround an expression cartridge and possess an ability to break genome transcripts effectively in a mammalian genome by the effective protection of transgene expression in the mammalian genome against further repression. The effective two-fold terminators represent any mammalian genome site fulfilling the following conditions: 3'-sites of the two simultaneously expressing and opposite genes containing a site of the second-to-last exon, the last intervening sequence, the last exon and a polyadenylation signal, a space of two polyadenylation signals at different DNA strands is no more than 100 base pairs.

EFFECT: method can be used for creating the transgenic animals with high and stable target protein production in milk for medical and research purposes.

4 dwg, 4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to the field of biotechnology. Synthetic 5'UTR regions are applied to enhance the transgene expression, with the 5'UTR regions being located between a promoter and a sequence, presenting an interest in an expression vector. The claimed invention also claims vectors, which contain the 5'UTR regions, and a method of enhancing the transgene expression in their application.

EFFECT: claimed invention provides the synthetic 5'UTR regions, which contain the first polynucleotide fragment in the form of the second intron of gene of calcium ATphase of the sarcoplasmic/endoplasmic reticulum and the second polynucleotide fragment, represented by a part of the 5' untranslated region (5'UTR) of casein gene.

25 cl, 17 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biochemistry, in particular to clostridial neurotoxins with a modified persistence. Claimed is a polypeptide, containing HC-domain, the first and, at least, one additional LC-domain with amino acid sequences, at least, 90% identical to the respective sequences of a neurotoxic component of botulotoxin of a serotype A, B, C1, D, E, F or G. Also claimed are nucleic acid, an expression vector and a host cell, intended for the expression of the said polypeptide. Also claimed are a method of obtaining and application of the said polypeptide, including as a component of a pharmaceutical composition, for treatment of a condition, associated with hyperactive cholinergic innervations of a muscle or a exocrine gland, and for cosmetic procedures, associated with wrinkles.

EFFECT: invention makes it possible to controllably vary a period of activity of clostridial neurotoxins.

12 cl, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a recombinant strain of Escherichia coli bacteria - a producer of biologically active flagellin. The described strain is obtained by transformation of an E. coli BL21[DE3] cell culture with recombinant plasmid DNA pET151FliC, which is obtained based on a pET151FliC vector in which was embedded a fliC gene which codes biologically active flagellin, having a nucleotide sequence represented in Seq ID No 3. The strain is deposited in the Russian National Collection of Industrial Microorganisms (RCIM) of the Research Institute for Genetics and Selection of Industrial Microorganisms under No B-11369.

EFFECT: present solution has higher production capacity with respect to recombinant flagellin, which is an effective adjuvant.

1 dwg, 2 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: carrier is proposed for targeted delivery of nucleic acids to cells expressing the receptor CXCR4, which consists of a sequence-ligand to the receptor CXCR4 with the amino acid sequence KPVSLSYRSPSRFFESH, the linker part of two molecules of ε-aminohexanoic acid linking the sequence-ligand to the sequence for compaction of nucleic acids, the sequence providing compaction of nucleic acids and the complex output from endosomes CHRRRRRRHC.

EFFECT: invention can be used for targeted delivery of genetic structures into cells with the receptor CXCR4 on the surfaces, such as malignant tumour and stem cells, in order to correct genetic defects, influence on processes of implementation of the genetic information and prevention of diseases.

3 cl, 7 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

Pcsk9 antagonists // 2528735

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented: an isolated antibody or its variant specifically recognising PCSK9, and a based pharmaceutical composition for lowering LDL-cholesterol. There are described: using for lowering blood cholesterol and/or LDL; reducing an incidence rate and/or correcting abnormal cholesterol and/or LDL; using for preparing a drug for lowering blood cholesterol and/or LDL; reducing the incidence rate and/or correcting abnormal cholesterol and/or LDL. There are disclosed versions of cell lines producing PCSK9 antibody or its antigen-binding portion and deposited in the American typical culture collection (ATCC) under Nos. PTA-8986, ATCC PTA-8984, ATCC PTA-8983, respectively. What is described is a coding nucleic acid and a host cell for producing the based antibody.

EFFECT: invention provides PCSK9 agonist antibodies that can find application in medicine for lowering cholesterol.

18 cl, 24 dwg, 9 tbl, 9 ex

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