Non-mouse anti-m-csf-antibody (versions), preparation and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to anti-M-CSF-specific antibodies based on RX1 or originating from RX1, and which more than 785% compete with monoclonal antibodies RX1, MC1 and/or MC3 for bonding with M-CSF (macrophagal colony-stimulating factor). The non-mouse antibody is two-stranded, contains a certain amino acid sequence (given in the formula of invention and list of sequences) and retains high affinity towards M-CSF. The invention discloses an isolated nucleic acid which codes the said antibody, an expression vector, a host cell and a method of producing the anti-M-CSF-antibody using a host cell or hybridome, particularly ATCC PTA-6263 or ATCC PTA-6264 hybridome. The invention describes a pharmaceutical composition containing said antibodies, sets containing pharmaceutical compositions and methods of preventing and treating osteoporosis in a person suffering from an osteolytic disease.

EFFECT: disclosed antibodies can inhibit osteoclast differentiation, which facilitates their use as highly effective preparations for treating osteolysis, cancer with metastases and osteoporosis associated with cancer metastases.

131 cl, 44 dwg, 12 tbl, 16 ex

 

In the present application claims the priority of provisional application U.S. No. 60/535181, filed January 7, 2004, and provisional application U.S. No. 60/576417, filed June 2, 2004, each of which in its entirety is introduced into the present description by reference.

The scope to which the invention relates.

The present invention relates to methods for prevention and treatment of osteolysis, cancer metastasis and osteoporosis associated with cancer metastasis, through the introduction of M-CSF-specific antibody to the individual.

Prior art

Cancer metastases are the main cause of postoperative or posttherapeutic recurrence of cancer in patients. Despite intensive attempts to develop a method of treatment of cancer, cancer metastasis, mostly, are not amenable to treatment. Bone is one of the most common areas in which spreads cancers of various types (for example, breast cancer, lung, prostate and thyroid). The appearance of metastases, causing osteolysis of the bone, is the cause of the development of serious diseases, manifested by a constant pain, high risk of fractures, compression of the nerves and hypercalcemia. Despite the severity of these clinical symptoms, there are very few ways for the treatment of osteoporosis associated the metastasis of cancer.

Osteoclasts mediate readsorption bones. Osteoclasts are multinucleated cells that differentiate from hematopoietic cells. It is generally assumed that the osteoclasts are formed by the merger of mononuclear precursors, derived from hematopoietic stem cells in the bone marrow, but not as a result of incomplete cell division (Chambers, Bone and Mineral Research, 6:1-25, 1989; Göthling et al., Clin. Orthop. Relat R. 120:201-228, 1976; Kahn et al., Nature 258:325-327, 1975, Suda et al., Endocr. Rev. 13:66-80, 1992; Walker, Science 180:875, 1973; Walker, Science 190:785-787, 1975; Walker, Science 190:784-785, 1975). These cells originate from the same stem cells, and cell lines differentiation of monocytes-macrophages (Ash et al., Nature 283:669-670, 1980, Kerby et al., J. Bone Miner.Res. 7:353-62, 1992). For differentiation of precursors into Mature osteoclasts multinuclear osteoclasts requires the presence of various factors, including hormonal and local stimuli (Athanasou et al., Bone Miner. 3:317-333, 1988; Feldman et al., Endocrinology 107:1137-1143, 1980; Walker, Science 190:784-785, 1975; Zheng et al., Histochem. J.23:180-188, 1991), and it was shown that viable bone tissue and bone cells play a crucial role in the development of osteoclasts (hagenaars on et al., Bone Miner 6:179-189, 1989). For differentiation of osteoclasts also requires the presence of osteoblasts or stromal cells of the bone marrow. One of the factors produced by cells, contributing to the formation of osteoblasts, is acromegaly colony-stimulating factor, M-CSF (Wictor-Jedrzejczak et al., Proc. Natl. Acad. Sci. USA 87:4828-4823, 1990; Yoshida et al., Nature 345:442-444, 1990). Another signal (Suda et al. Endocr. Rev. 13:66-80, 1992) is an activator of the receptor for ligand NF-kB (RANKL, also known as TRANCE, ODF and OPGL), through which osteoblasts/stromal cells stimulate the formation and resorption of osteoclasts, namely receptor RANK (TRANCER), localized on the osteoclasts and the precursors of osteoclasts (Lacey et al., Cell 93:165-176, 1998; Tsuda et al., Biochem Biophys Res. Co.234:137-142, 1997; Wong et al., J. Exp. Med. 186:2075-2080, 1997; Wong et al., J. Biol. Chem. 272:25190-25194, 1997; Yasuda et al., Endocrinology 139:1329-1337, 1998; Yasuda et al., Proc. Natl. Acad. Sci. US 95:3597-3602, 1998). Osteoblasts also secrete a protein that is a strong inhibitor of the formation of osteoclasts and is called osteoprotegerin (OPG, also known as OCIF), which acts as a receptor-“trap” for RANKL, i.e. inhibits positive signal between osteoclasts and osteoblasts by RANK and RANKL.

Osteoclasts are responsible for the dissolution of mineral and organic bone matrix (Blair et al., J. Cell Biol. 1164-1172, 1986). Osteoclasts are cells terminal differentiation with unique polarized morphology with specialized membrane areas and several membrane and cytoplasmic markers such as tartrate-resistant acid phosphatase (TRAP)(Anderson et al. 1979), (carbonic acid)-anhydrase I (Väänänen et al., Histochemistry 78:481-485, 1983), calcitonin receptor (Warshafsky et al., Bone 6:179-185, 1985) and the vitronectin receptor (Davies et al., J. Cell. Biol. 109:1817-1826, 1989). Multinuclear osteoclasts usually contain less than 10 cores, but they can contain up to 100 cores, the diameter of which ranges from 10 to 100 microns (Göthling et al., Clin. Orthop. Relat. R. 120:201-228, 1976). This makes it relatively easy to identify such cells under an optical microscope. These cells, in their active state are highly vacuolation, and contain many mitochondria, which indicates their high metabolic rate (Mundy, in Primer on the metabolic bone diseases and disorders of mineral metabolism, pages 18-22, 1990). Because osteoclasts play a major role in osteolysis associated with bone metastases, it is necessary to identify new tools and develop methods of stimulation and function of osteoclasts.

Thus, the need to identify new tools and develop methods of prevention or treatment of osteolysis or metastasis of cancer, including metastases, causing osteolysis of the bone remains extremely relevant.

Description of the invention

Materials and methods according to the invention satisfy the above-mentioned and other requirements relating to this area. In one of its variants the present invention relates to a murine monoclonal antibody, including NGF the national fragment, which specifically binds to the same epitope of M-CSF as any one of the mouse monoclonal antibody RX1, MC1 or MS having the amino acid sequence shown in figures 4, 14 and 15, respectively. In his related embodiment, the present invention relates to the aforementioned antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, including antibody Human Engineered™ (human engineered antibody); gumanitarnogo antibody; a human antibody; chimeric antibodies; fragments of antibodies: Fab, F(ab')2, Fv, ScFv or SCA; dyatel; linear antibodies or mutein any of the above mentioned antibodies, where the specified antibody preferably retains the binding affinity of equal to at least 10-7, 10-8or 10-9or higher. The present invention also is not a murine monoclonal antibody, including its functional fragment, which more than 75% compete with monoclonal antibody RX1, MC1 and/or MS having the amino acid sequence represented in figure 4, for binding to M-CSF.

In another embodiment, the present invention relates to a murine monoclonal antibody, including its functional fragment, where not specified mouse monoclonal antibody or its functional FR is gment binds to the epitope of M-CSF, which includes at least 4, 5, 6, 7 or 8 contiguous amino acids 98-105 of the sequence in figure 12.

In another embodiment, the present invention relates to a murine monoclonal antibody, including its functional fragment, where not specified mouse monoclonal antibody or functional fragment binds with an epitope of M-CSF that includes at least 4, 5, 6, 7 or 8 contiguous amino acid residues in positions 65-73 or 138-144 sequence represented in figure 12 (corresponding to the epitope of M-CSF, recognizable by antibodies N or S).

In yet another embodiment, the present invention relates to the aforementioned antibody or its fragment that binds to an epitope of M-CSF that includes amino acids 98-105, shown on figure 12. In his related embodiment, the present invention relates to the aforementioned antibody containing the CDR3 region shown in figure 4A. In another embodiment, the present invention relates to an antibody that contains at least, 1, 2, 3, 4, 5 or 6 CDRs of murine antibody RX1 presented on figure 4A. This antibody containing at least 1, 2, 3, 4 or 5 of the 6 CDRs of murine antibody RX1, may also contain at least any of 1, 2, 3, 4 or 5 of the 6 CDR regions of the antibodies (5H4 presented on figure 16A-Century Altern is effective, the antibody that contains at least 1, 2, 3, 4, or 5 CDR regions of mouse antibodies RX1, may also contain at least any of 1, 2, 3, 4 or 5 of the 6 CDRs of the antibodies MS presented on figure 16A-C. In another alternative embodiment, the aforementioned antibody may also contain any of at least 1, 2, 3, 4 or 5 of the 6 CDRs of the antibodies MS presented on figure 16A-Century In its related embodiment, the present invention relates the antibody that contains at least 1, 2, 3, 4, or 5 CDR regions of mouse antibodies RX1 and which may contain at least 1, 2, 3, 4 or 5 regions CDR consensus sequences presented on figure 16A-C. In another related embodiment of the invention, one or more residues in the consensus sequence of the CDR regions in the above-mentioned antibody is replaced with an appropriate balance of any of the CDRs of murine antibody RX1, (5H4, MS or MS. When this desirable affinity binding can be maintained even if one or several amino acids in the antibody are mutated, for example, conservative substitutions in the CDR, and/or conservative or nonconservative substitutions in the residues of low and moderate risk.

In another embodiment, the present invention relates to variants of the above-mentioned antibodies containing the amino acid pic is egovernance variable regions of the heavy chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence shown in figures 4A, 13, 14 or 15. In a related embodiment of the invention, the specified antibody includes the amino acid sequence of the variable region of the light chain that is at least on 60, 65, 70, 75, 80, 85, 90, 91, 92 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence shown in figures 4A, 13, 14 or 15.

In yet another variant of the invention, the specified antibody contains a constant region and one or more variable frame regions of the heavy and light chain sequences of a human antibody. In a related embodiment of the invention, the specified antibody contains a modified or unmodified constant region of human immunoglobulin IgG1, IgG2, IgG3 or IgG4. In a preferred embodiment of the invention, the constant region is a human IgG1 or IgG4, which may be optional, is modified in order to enhance or diminish some of the properties of this antibody. In the case of IgG1, modifications made to a constant region, and in particular in the hinge area or CH2-region, can increase or decrease effector function, including ADCC and/or CDC activity. In other embodiments of the invention, the constant region of IgG2 modified in C the order of the lower education level of a complex of the antibody-antigen”. In the case of IgG4, modifications in the constant region, and in particular in the hinge region, can lead to lower levels of education poloitical.

In yet another variant of the invention, the aforementioned antibody is derived, or obtained on the basis of, or contains part of the human consensus sequence, the sequence of human germline, consensus sequences of the human germline, or any one of the sequences of a human antibody, represented in databases Kabat, NCB1 Ig Blast, http://www.ndci.nlm.nih.gov/igblast/showGermline.cgi in the Kabat database http://www.bioinf.org.uk/abs/seqtest.htlm on the FTP site for the version Kabat Release 5.0 (1992) ftp:/ftp.ndci.nih.gov/repository/kabat/Rel5.0 in the data ImMunoGeneTics (Montpellier France), https://imgt.cnusc.fr:8104/, V-Base http://www.mrc-cpe.cam.ac.uk/LIST.php?menu=901, Zurich University http://www.unizh.ch/~antibody/Sequences/index.html, therapeutic Antibody Human Homology Project (TAHHP) http://www.path.cam.ac.uk/~mrc7/humanisation/TAHHP.html, Protein Sequences and Structure Analysis of Antibody Domains http://how to/AnalyseAntibody/, Humanisation by design, http://people.cryst.bbk.ac.uk/~ubcg07s/, Antibody Resources http://www.antibodyresource.com/education.html, Antibody Engineering (by Wu TT), Humana Press.

In a preferred aspect of the present invention, the aforementioned antibody is an antibody of Human Engineered™. For example, the sequence of the antibody is Human Engineered™ is any one of the sequences indicated in figures 23-24. Also examines other antibodies Human Engineered™ Il is their options.

For example, in one of its variants the present invention relates to the aforementioned antibody derived from the antibody RX1, where the variable region of the heavy chain of the antibody includes the amino acid sequence of

where X represents any amino acid. In his related embodiment, the present invention relates to an antibody in which the variable region of the heavy chain contains the amino acid sequence of

where X represents any amino acid.

In another embodiment, the present invention relates to the aforementioned antibody in which the variable region of the heavy chain contains the amino acid sequence of

where X represents any amino acid. In his related embodiment, the present invention relates to an antibody in which the variable region of the heavy chain contains the amino acid sequence of

where X represents any amino acid. In another embodiment, the present invention relates to an antibody in which the variable region of the heavy chain contains the amino acid sequence of

In another embodiment, the present invention relates to an the body, in which the variable region of the heavy chain contains the amino acid sequence of

In another embodiment, the present invention relates to the aforementioned antibody in which the variable region of the light chain contains the amino acid sequence of

where X represents any amino acid. In his related embodiment, the present invention relates to an antibody in which the variable region of the light chain contains the amino acid sequence of

where X represents any amino acid. In yet another embodiment, the present invention relates to an antibody in which the variable region of the light chain contains the amino acid sequence of

where X represents any amino acid.

In another embodiment, the present invention relates to the aforementioned antibody in which the variable region of the light chain contains the amino acid sequence of

where X represents any amino acid. In his related embodiment, the present invention relates to an antibody in which the variable region of the light chain contains the amino acid sequence of

where Predstavljaet any amino acid. In another embodiment, the present invention relates to an antibody in which the variable region of the light chain contains the amino acid sequence of

In another embodiment, the present invention relates to the aforementioned antibody in which the variable region of the light chain contains the amino acid sequence ofIn another embodiment, the present invention relates to an antibody in which the variable region of the light chain contains the amino acid sequence of

In another embodiment, the present invention relates to the aforementioned antibody, in which at least one X represents the corresponding amino acid in the amino acid sequence represented in figure 4A. In his related embodiment, the present invention relates to an antibody in which at least one X is a conservative substitution (table 1) corresponding amino acids in the amino acid sequence represented in figure 4A. In another related embodiment, the present invention relates to an antibody in which at least one X is not a conservative substitution (table 1) corresponding amino acids in the am is nomikoto sequence, presented in figure 4A. In yet another embodiment, the present invention relates to an antibody in which at least one X represents the corresponding amino acid sequence of a human antibody.

The above-mentioned antibody is Human Engineered™ is derived, or obtained on the basis of, or contains part of the consensus sequences of human antibody sequences of the human germline, consensus sequences of the human germline, or any one of the sequences of human antibodies in the database Kabat, NCB1 Ig Blast, http://www.ndci.nlm.nih.nih.gov/igblast/showGermline.cgi in the Kabat database http://www.bioinf.org.uk/abs/seqtest.htlm on the FTP site for the version Kabat Release 5.0/ (1992) ftp:/ftp.ncbi.nih.gov/repository/kabat/Rel5.0/, database ImMunoGeneTics (Montpellier France), https://imgt.cnusc.fr:8104/, V-Base http://www.mrc-cpe.cam.ac.uk/LIST.php?menu=901, Zurich University http://www.unizh.ch/~antibody/Sequences/index.htlm, therapeutic Antibody Human Homology Project (TAHHP) http://www.path.cam.ac.uk/~mrc7/humanisation/TAHHP.html, Protein Sequence and Structure Analysis of Antibody Domains http://how to/AnalyseAntibodies/, Humanisation by design, http://people.cryst.bbk.ac.uk/~ubcg07s/, Antibody Resources http://www.antibodyresource.com/educational.html, Antibody Engineering (by Wu TT), Humana Press.

In another embodiment, the present invention relates to the aforementioned antibody is Human Engineered™, the sequence of which represents one of the sequences indicated in figures 23-24 or 29-30. In other variants of the f invention, the aforementioned antibody includes the amino acid sequence of the variable region of the heavy chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to one of the amino acid sequence presented in figure 19C. In another embodiment of the invention, the specified antibody includes the amino acid sequence of the variable region of the light chain that is at least on 60, 65, 70, 75, 80, 85, 90, 91, 92 93, 94, 95, 96, 97, 98 or 99% identical to one of the amino acid sequences of the light chain shown in figures 20B-22B.

In yet another variant of the invention the above-mentioned antibody, such as (5H4, MS or MS having the sequence shown on one of the figures 24C-24TH, is an antibody Human Engineered™, obtained by the methods described in U.S. patent No. 5766886, Studnicka et al., in example 4A of the present invention, and these sequences are numbered according to the numbering Kabata, as shown in figures 24C-24TH, with the identification of residues substitutions low, moderate or high risk. In his related embodiment, the present invention relates to the aforementioned antibody, where all traces of low risk in heavy or light chain or both chains were modified, if necessary, so that these residues were identical to the remnants of the original sequence of a human immunoglobulin. Similarly, in another embodiment, the present invention relates vysheupomyanutomu antibody, where all the remains low + moderate risk in heavy or light chain or both chains were modified, if necessary, so that these residues were identical to the remnants of the original sequence of a human immunoglobulin. Heavy chain, where all traces of low risk are modified, can be combined with a light chain, where everything remains low and moderate risk are modified, and Vice versa. Similarly, the light chain and heavy chain of the above-mentioned antibodies Human Engineered™ can be combined with a light or heavy chain gumanitarnogo antibodies or chimeric antibodies.

In another embodiment of the invention, the specified antibody includes the amino acid sequence of the variable region of the heavy chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to one of the amino acid sequences of the heavy chain of the above-described antibodies are Human Engineered™, obtained in accordance with the method Studnicka et al. In yet another variant of the invention, the specified antibody includes the amino acid sequence of the variable region of the light chain that is at least on 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to one of the amino acid sequences of the light chain of the above-described antibodies are Human Engineered™, obtained in accordance with the method of the m Studnicka et al.

In another embodiment, the present invention relates to an antibody containing heavy chain described above, and light chain described above.

In yet another variant of the invention, the aforementioned antibody has an affinity Kd of at least 10-7. In a related embodiment of the invention, the said antibody has an affinity Kd of at least 10-9.

In another embodiment, the present invention relates to the aforementioned antibody, which is a polyclonal antibody; a monoclonal antibody, including antibody Human Engineered™; humanitariannet antibody; a human antibody; a chimeric antibody; fragments of antibodies: Fab, F(ab')2, Fv, ScFv or SCA; ditelo; linear antibody or mutein any of these antibodies. In a related embodiment of the invention, the monoclonal antibody is a selected antibody.

In yet another embodiment, the present invention relates to a selected nucleic acid containing a nucleotide sequence encoding a light chain of the above-mentioned antibodies. In a related embodiment of the invention the specified selected nucleic acid contains a nucleotide sequence of the heavy chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the nucleotide sequence of the heavy chain, the pre is presented in figures 4A, 13, 14 or 15. In another related embodiment of the invention the specified selected nucleic acid contains a nucleotide sequence of light chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the nucleotide sequence of the light chain shown in figures 4A, 13, 14 or 15.

In another embodiment, the present invention relates to a vector containing the above-mentioned selected nucleic acid. In a related embodiment, the present invention relates to the aforementioned vector, which specified the selected nucleic acid is functionally linked to a regulatory sequence. In another embodiment, the present invention relates to the cell host containing the above-mentioned vector.

In this application discusses several ways of implementing the present invention. For example, in the present invention discusses a method of producing the above-mentioned antibody, comprising culturing the above-mentioned host cell, so that expressed in this cell selected nucleic acid was produced this antibody. In a related embodiment, the present invention also relates to a method involving the extraction of antibodies from the culture of host cells. In a related embodiment, the present invention is e refers to the selected antibody produced by the aforementioned method.

The present invention also relates to hybridoma, which secretes the above-mentioned antibody according to the invention. In addition, the present invention relates to the aforementioned antibody anywherefrom with the toxin.

In another embodiment, the present invention relates to pharmaceutical compositions containing one of the aforementioned antibodies and a pharmaceutically acceptable carrier, excipient or diluent. In a related embodiment, the invention specified in the pharmaceutical composition further comprises a second therapeutic agent. In another related embodiment, the present invention relates to pharmaceutical compositions, where the specified second therapeutic agent is a cancer chemotherapeutic agent. In yet another related embodiment, the present invention relates to pharmaceutical compositions in which the specified second therapeutic agent is biphosphonate. In another embodiment of the invention the specified second therapeutic agent is another antibody.

The present invention describes antibodies, which have a number of desirable properties, is effective for the treatment of diseases and disorders. In one of its variants the present invention relates to Liu the WMD of the above antibodies, which binds to M-CSF and can be used to prevent the patient has a condition that causes or stimulates the development of osteolysis, and which contributes to a significant weakening of the severity of osteoporosis associated with the specified disease. Similarly, the present invention relates to any of the above antibody, which binds to M-CSF and which can be used to treat a patient suffering from a disease that causes or stimulates the development of osteolysis, where the specified antibody contributes to a significant weakening of the severity of osteoporosis associated with the specified disease.

The present invention addresses some of the diseases and disorders that can be treated using the antibodies in accordance with the invention. In one of its variants the present invention relates to the aforementioned antibody that can be used for the treatment of diseases selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrinopathies (including hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce status (including rickets/disorder, scurvy, malnutrition), the HRO is systematic diseases (including malabsorption syndromes, chronic renal failure (including renal osteodystrophy), chronic liver disease (including hepatic osteodystrophy)), diseases associated with medicines (including glucocorticoids (induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (imperfect osteogenesis, homocystinuria), cancer, osteoporosis, osteoporosis, inflammation associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

In his related embodiment, the present invention relates to the aforementioned antibody that binds to M-CSF and which can be used for prevention or treatment of cancer with bone metastases, where the specified metastatic cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma is ecnica, endometrial cancer, cancer of the vagina and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; skin cancer, including malignant melanoma or squamous cell carcinoma.

In another embodiment, the present invention relates to a method of screening for M-CSF-specific antibody, comprising a stage of contacting environment metastatic tumor cells, osteoclasts and antibodies candidate; detecting the formation of osteoclasts, proliferation and/or differentiation of osteoclasts and identification of the specified antibodies candidate as M-CSF-specific antibody, if it was found to be lower education level, proliferation and/or differentiation of osteoclasts. Similarly, the present invention relates to the aforementioned method, in which the specified environment metastatic tumor cells include tumor cells.

In another embodiment, the present invention relates to the aforementioned method where the specified stage contacting (a) flowsin vivo; specified detection stage (b) includes the detection of the size and/or number of bone metastases, and antibody-candidate identify as M-CSF-specific antibody, if it was found a reduction in the size and/or number of bone metastases. In his related embodiment, the present invention otnositsa the above-mentioned method, additionally, providing the stage of determining the presence of binding antibodies candidate with M-CSF. Similarly, in another embodiment, the present invention relates to the aforementioned method, which additionally provides for the stage of determining whether inhibition of an interaction between the indicated antibodies candidate with M-CSF and its receptor M-CSFR.

In another embodiment, the present invention relates to a method for the identification of M-CSF-specific antibody, which may contribute to the prevention or treatment of cancer metastases to bone, where the specified method comprises the stages of: (a) detection of binding antibodies candidate epitope of M-CSF, comprising at least 4 contiguous amino acid balance 98-105 indicated on figure 12; and (b) analysis of the ability of the specified antibodies candidate for the prevention or treatment of metastatic cancer in bonesin vitroorin vivo.

In another embodiment, the present invention relates to a method for the identification of M-CSF-specific antibody, which may contribute to the prevention or treatment of cancer metastases in bone, where the specified method comprises the stages of: (a) detection of binding antibodies candidate epitope of M-CSF, comprising at least 4 contiguous amino acid balance 65-73 or 138-144 indicated on figure 12 (corresponding to the epitope of M-CSF, recognizing the th antibody (5H4 or MS); and (b) analysis of the ability of the specified antibodies candidate for the prevention or treatment of metastatic cancer in bonesin vitroorin vivo.

In yet another embodiment, the present invention relates to a method of modifying CDR antibody, which binds to the epitope of M-CSF that includes amino acids 98-105 presented on figure 12, where the method includes: modification of amino acids in the CDR amino acid sequence represented in figure 4A, and selection of antibody that binds to M-CSF with the affinity of the AC component of at least 10-7. In another embodiment, the present invention relates to a method, system modifications up to 60% amino acid sequence of the heavy chain represented in figure 4A, where the method provides for the modification of any residues X1-H in amino acid sequence:and testing of antibodies containing a modified amino acid sequence, binding to the epitope of M-CSF that includes amino acids 98-105, shown on figure 12.

In his related embodiment, the present invention relates to a method, system modifications up to 60% amino acid sequence of the light chain represented in figure 4A, where the method provides for the modification of any residues X1-HW amino acid sequence: and testing of antibodies containing a modified amino acid sequence, binding to the epitope of M-CSF that includes amino acids 98-105, shown on figure 12.

In yet another embodiment, the present invention relates to a method of modifying CDR antibody, which binds to the epitope of M-CSF that includes amino acids 65-73 or 138-144 shown on figure 12 (corresponding to the epitope of M-CSF, recognizable by the antibody (5H4 or MS), where the method includes the modification of amino acids in the amino acid sequence of the CDR field presented on one of the figures 13, 14 and 15, and selection of antibody that binds to M-CSF with the affinity of the AC component of at least 10-7. In another embodiment, the present invention relates to a method of system modifications to 60% amino acid sequence of the heavy chain, presented on one of the figures 13, 14 and 15, where the method includes a modification of the above-mentioned sequences, obtained by the methods described in U.S. patent No. 5766886, Studnicka et al., in example 4A of this application, and are numbered according to the numbering Cabatu, as shown in figures 24C-24TH; and testing of antibodies containing a modified amino acid sequence, binding to the epitope of M-CSF that includes amino acids 65-73 or 138-144, shown on Fig is re 12 (corresponding to the epitope of M-CSF, recognized by the antibody (5H4 or MS). In a related embodiment of the invention all traces of low risk are modified. Similarly, in another embodiment of the invention, all the remains low and moderate risk are modified. In yet another variant of the invention, all the remains low and moderate risk, except Proline, are modified.

In another embodiment, the present invention relates to a method of expression of the antibody with CDR field, constructed in the above way. In another embodiment, the present invention relates to a pharmaceutical composition comprising the antibody binding to M-CSF, where the specified antibody receive the above method.

In yet another embodiment, the present invention relates to a method for prevention or attenuation of osteoporosis, comprising the administration to an individual suffering from a disease that causes or stimulates the development of osteolysis, a therapeutically effective amount of any of the above antibodies, which prevents or reduces osteoporos associated with the specified disease. In his related embodiment, the present invention relates to a method of treatment of an individual suffering from a disease that causes or stimulates the development of osteolysis, where the p is educative introduction to the specified individual a therapeutically effective amount of any of the above antibodies, decreasing the severity of osteoporosis associated with the specified disease.

In his related embodiment, the present invention relates to the aforementioned method, in which the disease is selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrinopathies (including hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce status (including rickets/disorder, scurvy, malnutrition), chronic diseases (including malabsorption syndromes, chronic renal failure (including renal osteodystrophy), chronic liver disease (hepatic osteodystrophy)), diseases associated with medicines (including glucocorticoids (including that induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (including imperfect osteogenesis, homocystinuria), cancer, osteoporosis, osteoporosis, inflammation associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

In yet another embodiment, the present invention relates to a method for prevention or treatment of cancer metastases in bone, including the introduction of an individual the mind, suffering from metastatic cancer, a therapeutically effective amount of any of the above antibodies. In his related embodiment, the present invention relates to a method in which the specified metastatic cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; skin cancer, including malignant melanoma or squamous cell carcinoma.

In yet another embodiment, the present invention relates to a method for prevention of osteoporosis and tumor growth, where this method provides an introduction to the individual in need, a therapeutically effective amount of any of the above antibodies. In a related embodiment of the invention the method also provides for the maintenance of the second therapeutic agent. In another related embodiment, the present invention relates to a method in which the second therapeutic agent is a cancer chemotherapeutic agent or biphosphonate. In yet another related embodiment, the present invention relates to a method in which the specified biphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate. In yet another related embodiment, the present invention relates to the aforementioned methods, in which the specified therapeutic agent is a cytotoxic chemotherapeutic agent. In another embodiment, the present invention relates to the aforementioned method, wherein said individual has not undergone the treatment biphosphonates.

In yet another related embodiment, the present invention relates to the aforementioned method, in which the indicated antibody is effective to reduce the dose of the second therapeutic agent required to achieve a therapeutic effect. In another embodiment of the invention specified therapeutic tool is another, non-M-CSF, colony-stimulating factors, such as colony-stimulating factor (G-CSF or antibody against RANKL, or soluble RANKL receptor.

In another embodiment, the present invention relates to the aforementioned methods for the am, in which the specified individual is a mammal. In a related embodiment, the invention specified mammal is man.

In another embodiment, the present invention relates to the aforementioned methods, in which this antibody inhibits the interaction between M-CSF with its receptor (M-CSFR). In another related embodiment of the invention, the specified antibody inhibits proliferation and/or differentiation of osteoclasts induced by tumor cells. In yet another embodiment, the present invention relates to the aforementioned methods, in which this antibody is administered at a dose of approximately 2 mg/kg to 30 mg/kg, 0.1 mg/kg to 30 mg/kg or 0.1 mg/kg to 10 mg/kg of body weight.

In another embodiment of the invention describes the use of antibodies for the manufacture of a medicinal product in a quantity effective to prevent or ameliorate osteoporosis in a patient with symptoms of osteolysis, and for the manufacture of a medicinal product for the treatment of a patient suffering from disease causing or stimulating the development of osteolysis. In addition, the above antibody can be used for the treatment of diseases selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrine is patii (including, hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce state (including rickets/disorder, scurvy, malnutrition), chronic diseases (including malabsorption syndromes, chronic renal failure (including renal osteodystrophy), chronic liver disease (including hepatic osteodystrophy)), diseases associated with medicines (including glucocorticoids (induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (including, imperfect osteogenesis, homocystinuria), cancer, osteoporosis, osteoporosis, inflammation of bone associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

In another embodiment of the invention describes the use of antibodies for the manufacture of a medicine for the prevention or treatment of cancer metastases to bone in a patient suffering from metastatic cancer. In a related embodiment, the invention specified metastatic cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; gastric cancer-isicelo tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; or skin cancer, including malignant melanoma or squamous cell carcinoma.

In yet another embodiment discusses the use of the antibodies according to the invention for the manufacture of a medicinal product for the treatment of a patient suffering from cancer.

In any of the above treatment options specified drug is combined with treatment with the second therapeutic agent. In a related embodiment of the invention the specified second therapeutic agent is a cancer chemotherapeutic agent. In related embodiments of the invention the specified second therapeutic agent is another, non-M-CSF, colony stimulating factor, or an antibody against RANKL, or soluble RANKL receptor, or biphosphonate. In a related embodiment of the invention the specified biphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate.

In another VA who ianthe invention is considered any of the above-mentioned applications, where the patient is not treated by biphosphonates, and/or where the patient has undergone prior treatment with a second therapeutic agent. In a related embodiment of the invention the specified second therapeutic agent is a cancer chemotherapeutic agent, other non-M-CSF, colony stimulating factor, or an antibody against RANKL, or soluble RANKL receptor, or biphosphonate. In yet another related embodiment of the invention the specified biphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate. In yet another related embodiment of the invention, the specified patient not treated with biphosphonates.

In another embodiment of the invention discusses the use of a synergistic combination of antibodies according to the invention for obtaining a medicinal product for the treatment of a patient with symptoms of osteolysis, where the use of this medicinal product coordinated with treatment with the second therapeutic agent. In a related embodiment of the invention the specified second therapeutic agent is a cancer chemotherapeutic agent, other non-M-CSF, colony stimulating factor, or an antibody against RANKL, or soluble RANKL receptor, or biphosphonate. In a related embodiment, the invention specified bipost which the gateway is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate. In yet another related embodiment of the invention, the specified patient not treated with biphosphonates.

There are variants of any of the above mentioned applications, where in the specified drug antibody is present in a quantity effective to reduce the dose of the second therapeutic agent required to achieve a therapeutic effect. In any of the above options related to osteoporosis associated with cancer, the number of antibodies in the medicinal product is preferably effective for inhibiting proliferation and/or differentiation of osteoclasts induced by tumor cells.

In any of the above medicines number of antibodies may be a dose of from about 2 μg/kg to 30 mg/kg of body weight. In a related embodiment of the invention, the number of antibodies in medicine is the dose from about 0.1 mg/kg to 30 mg/kg of body weight. In yet another variant of the invention, the number of antibodies in medicine is the dose from about 0.1 mg/kg to 10 mg/kg of body weight.

The present invention also addresses the sets. In one of its variants the present invention relates to a kit containing a therapeutically effective quantity is the number of antibodies according to the invention, Packed in a container such as a jar or bottle, and, in addition, contains a label attached to the specified container or enclosed in the container where the specified label includes a description of the contents of this container, indications and/or instructions for using the contents of this container for the prevention or attenuation of osteoporosis.

In another embodiment, the present invention relates to a kit containing a therapeutically effective amount of the antibody according to the invention, packaged in a container such as a jar or bottle, and, in addition, contains a label attached to the specified container or enclosed in a container, where the specified label includes a description of the contents of this container, indications and/or instructions for using the contents of this container for the treatment of a patient suffering from disease causing or stimulating osteolysis.

In his related embodiment, the present invention relates to a kit used for the treatment of diseases selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrinopathies (including hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce state is based (including rickets/disorder, scurvy, malnutrition), chronic diseases (including malabsorption syndromes, chronic renal failure (including renal osteodystrophy), chronic liver disease (including hepatic osteodystrophy)), diseases associated with medicines (including glucocorticoids (induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (including imperfect osteogenesis, homocystinuria), cancer, osteoporosis, osteoporosis, inflammation associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

In another embodiment, the present invention relates to a kit containing a therapeutically effective amount of the antibody according to the invention, packaged in a container such as a jar or bottle, and, in addition, contains a label attached to the specified container or enclosed in the container where the specified label includes a description of the contents of this container, indications and/or instructions for using the contents of this container for the prevention or treatment of cancer metastases in bone. In a related embodiment, the invention specified metastatic cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, cancer of the thyroid is the gland, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; or skin cancer, including malignant melanoma or squamous cell carcinoma.

In yet another embodiment, the present invention relates to a kit containing a therapeutically effective amount of the antibody according to the invention, packaged in a container such as a jar or bottle, and, in addition, contains a label attached to the specified container or enclosed in the container where the specified label includes a description of the contents of this container, indications and/or instructions for using the contents of this container for cancer treatment.

In another embodiment of the invention the specified collection also includes a second therapeutic agent. In a related embodiment of the invention the specified second therapeutic agent are the two who is anti-cancer chemotherapeutic agent, other, non-M-CSF, colony stimulating factor, or an antibody against RANKL, or soluble RANKL receptor, or biphosphonate. In a related embodiment of the invention the specified biphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate. In yet another variant of the invention the specified set includes instructions for its use to treat a patient who is not treated by biphosphonates.

In another embodiment, the present invention relates to the aforementioned kit, containing the dose of the antibody effective to reduce the dose of the second therapeutic agent required to achieve a therapeutic effect. In another embodiment of the invention the specified set contains a synergistic dose of antibody. In yet another variant of the invention the specified set contains the dose of the antibody effective to inhibit proliferation and/or differentiation of osteoclasts induced by tumor cells.

In yet another variant of the invention above set contains a dose of the antibody component of from about 2 μg/kg to 30 mg/kg of body weight. In another embodiment of the invention the specified set contains the dose of the antibody component of from about 0.1 mg/kg to 30 mg/kg of body weight. In yet another variant of the invention the specified set contains the dose of the antibody component of from about 0.1 mg/to the 10 mg/kg of body weight.

In another embodiment, the present invention relates to packaging, vessel or container, including medicinal product containing one or more of the above antibodies, and the user manual, which States that this medicine should be used in combination with surgery or radiation therapy. In another embodiment, the present invention relates to a method for prevention or treatment of cancer metastases in bone, including the introduction phase of any of the above antibodies to the individual and the individual surgery or radiation therapy. In another embodiment, the present invention relates to a method for targeted treatment of tumor cells expressing on their surface membrane-bound M-CSF, where the specified method comprises the stage of introduction of any of the above antibodies, where the specified antibody conjugated with a radionuclide or other toxin. In another embodiment, the present invention relates to a method of treatment of an individual suffering from cancer, where the method involves the introduction of a specified individual a therapeutically effective amount of any of the above antibodies.

In yet another embodiment, the present invention relates to a method for prevention of osteoporosis, vkluchaya is the introduction to the individual, suffering from disease causing or stimulating osteolysis, any of the above antibodies in the amount effective to neutralize M-CSF produced by cells of a particular individual, where the specified amount exceeds the amount effective to neutralize M-CSF produced by cancer cells. In a related embodiment, the present invention relates to a method of treatment of an individual suffering from a disease causing or stimulating osteolysis, any of the above antibodies in the amount effective to neutralize M-CSF produced by cells of a particular individual, where the specified amount exceeds the amount effective to neutralize M-CSF produced by cancer cells.

In one embodiment of the invention the present invention relates to pharmaceutical compositions containing the antibody RX1, (5H4, MC1 and/or MS, or not murine antibody derived from the antibody RX1, (5H4, MC1 and/or MS, or an antibody that competes with antibody RX1, (5H4, MC1 and/or MS, and anti-cancer therapeutic agent. In another embodiment, the present invention relates to packaging, vessel or container, including medicinal product containing the antibody RX1, (5H4, MC1 and/or MS, or non-murine antibody derived from the antibody RX1, (5H4, MC1 and/or MS, or an antibody that competes with antibody RX1, (5H4, MC1 and/and and MS, and the user manual, which States that this medicine should be used in combination with surgery or radiation therapy.

In yet another embodiment, the present invention relates to a method of treatment of an individual suffering from cancer, in which cancer cells to secrete M-CSF, where the specified method comprises the stage of introduction of any of the above antibodies.

Brief description of the graphical material

The figure 1 presents the topological graph, which shows the disulfide bond in a truncated dimeric M-CSF.

The figure 2 presents stereotigra With-alpha-skeleton of M-CSF, where marked every tenth residue and where cristallografia axis of symmetry is indicated by the dashed line.

The figure 3 illustrates the comparison of osteoclast-inducing activity of purified M-CSF and conditioned medium (CM) from cells MDA-231 and MCF7.

Figure 4A presents the amino acid sequence of M-CSF-specific murine antibody RX1 (SEQ ID NO: 2 and 4) (encoded by cDNA insert of the plasmid deposited with the American type culture collection, Manassas, VA, USA, and in ATSC number of Deposit of the MOUTH-6113) and the corresponding nucleic acid sequence (SEQ ID NO: 1 and 3). Region CDR numbered and marked in bold.

In figures 4B and 4C presents the amino acids of the s sequence light (SEQ ID NO: 5) and heavy (SEQ ID NO: 6) circuits M-CSF-specific murine antibody RX1, accordingly, with the remains of high risk (in bold), moderate risk (underlined) and low risk, identified by the method Studnicka et al., WO 93/11794.

In figure 5A it is shown that anti-M-CSF antibody RX1 and A are species-specific. Figure 5B shows the M-CSF-neutralizing activity of the antibodies MS and MS.

In figure 6 it is shown that the antibody RX1 effectively inhibits osteolysis in a model of human xenograft at a concentration of 5 mg/kg

The figure 7 shows that the number of metastases is reduced with the introduction of antibodies RX1 “Nude” mice bearing cancer cells of human breast cancer MDA-MB-231 at a concentration of 5 mg/kg

In figure 8A and 8B it is shown that M-CSF-specific antibody binds to the cell line breast cancer MDA-MB-231 and cancer cell line multiple myeloma ARH77.

In figure 9 it is shown that M-CSF is prevalent on the surface of some cancer cells.

The figure 10 shows the amino acid sequence of M-CSFα (SEQ ID NO: 7).

The figure 11 presents the amino acid sequence M-CSFβ (SEQ ID NO: 8).

The figure 12 presents the amino acid sequence M-CSFγ (SEQ ID NO: 9). Various polymorphisms in DNA sequences can lead to modifications of amino acids. For example, widespread polymorphism leads to the replacement of Pro Ala in which ogenyi 104.

In figures 13, 14 and 15 shows the amino acid sequence of M-CSF-specific mouse antibodies (5H4 (SEQ ID NO: 10 and 11), MS (SEQ ID NO: 12 and 13) (produced by hybridomas deposited in ATSC number of Deposit of the MOUTH-6263) and MS (SEQ ID NO: 14 and 15) (produced by hybridomas deposited in ATSC number of Deposit of the MOUTH-6264), respectively.

In figures 16A and illustrates the alignment of amino acid sequences of the CDR regions of the heavy and light chains of human M-CSF-specific murine antibody RX1, (5H4, MS and MS (SEQ ID NO: 16-38).

The figure 17 shows the neutralizing activity of intact antibodies RX1 or its Fab fragment compared to the neutralizing activity of intact antibodies (5H4 or its Fab fragment.

The figure 18 shows the structure of M-CSF with epitopes (light) for antibodies RX1, (5H4 and MS (SEQ ID NO: 120, 122, and 123).

Figure 19A shows (a) risk areas (H = high risk M = moderate risk L = low risk) for the heavy chain of murine RX1; (b) amino acid sequence of the heavy chain of the antibody RX1 (SEQ ID NO: 6); (C) the amino acid sequence of the most related human consensus sequence, i.e. the consensus sequence Vh2 Kabat, aligned with the RX1 sequence (SEQ ID NO: 39), and (d) substitutions that were made for the production of two representative sequences of the antibody is Human Engineered™ (SEQ ID NO: 41 and 43). Figure 19C shows a representative amino acid sequences of the two heavy chains of the antibody is Human Engineered™ (SEQ ID NO: 41 and 43), called “low risk” and “low + moderate risk”, as well as the corresponding nucleic acid sequence (SEQ ID NO: 40, and 42).

Figure 20A shows (a) risk areas (H = high risk M = moderate risk L = low risk) for the light chain of the murine RX1; (b) amino acid sequence of the light chain of the antibody RX1 (SEQ ID NO: 5); (C) the amino acid sequence of the most related human consensus sequence, i.e. the consensus sequence Vk3 Kabat, aligned with the RX1 sequence (SEQ ID NO: 49), and (d) substitutions that were made for the production of two representative sequences of the antibodies are Human Engineered™ (SEQ ID NO: 45 and 47). Figure 20B shows a representative amino acid sequence of two light chains of the antibody is Human Engineered™ (SEQ ID NO: 45 and 47), called “low risk” and “low + moderate risk”, as well as the corresponding nucleic acid sequence (SEQ ID NO: 44 and 46).

Figure 21A shows (a) risk areas (H = high risk M = moderate risk L = low risk) for the light chain of the murine RX1; (b) amino acid sequence of the light chain of the antibody RX1 (SEQ ID NO: 5); (C) amino acid p is the sequence of the most related human consensus sequence, that is the consensus sequence Vk3 Kabat, aligned with the RX1 sequence (SEQ ID NO: 49), and (d) an alternate representative amino acid sequence in which residues at positions 54-56 are unmodified (i.e represent the remains of the murine sequence) (SEQ ID NO: 48). In the figure 21B presents a representative amino acid sequence of two alternative light chain of the antibody is Human Engineered™ (SEQ ID NO: 48 and 136), and the corresponding nucleic acid sequence (SEQ ID NO: 137 and 135).

Figure 22A shows (a) risk areas (H = high risk M = moderate risk L = low risk) for the light chain of the murine RX1; (b) amino acid sequence of the light chain of the antibody RX1 (SEQ ID NO: 5); (C) the amino acid sequence of the most related consensus sequences of the human germline, Vk6 subgroups 2-1-(1) A14 aligned with the RX1 sequence (SEQ ID NO: 50), and (d) substitutions that were made for the production of two representative sequences of the antibodies are Human Engineered™ (SEQ ID NO: 51 and 53). Figure 22B shows a representative amino acid sequence of two light chains of the antibody is Human Engineered™ (SEQ ID NO: 51 and 53), called “low risk” and “low + moderate risk”, and the corresponding sequence of nuclei the OIC acid (SEQ ID NO: 52).

In figures 23A and 23C shows an alignment of the amino acid sequence of the light chain of the murine RX1 (SEQ ID NO: 54) with various human consensus sequences and the consensus sequences of the human germline carried out in accordance with the numbering system on Kabuto (numbering of amino acids is indicated in the row labeled “POS”) (SEQ ID NO: 55-82).

In figures 24A and 24 shows an alignment of the amino acid sequence of the heavy chain of murine RX1 (SEQ ID NO: 83) with various human consensus sequences and the consensus sequences of the human germline carried out in accordance with the numbering system on Kabuto (numbering of amino acids is indicated in the row labeled “POS”) (SEQ ID NO: 84-112). In figures 24C-24TH illustrated accordance amino acid residues of the antibody (5H4, MS and MS the numbering system Kabata (SEQ ID NO: 10 and 11; SEQ ID NO: 12 and 13; SEQ ID NO: 14 and 15, respectively).

Figure 25 illustrates the comparative neutralization of recombinant human M-CSF recombinant murine antibody RX1 marked rmRX1, and three variants of the antibody is Human Engineered™, RX1-1 (which were made to replace low risk), each of which has a different constant region (IgG1, IgG2 or IgG4), marked heRX1-1.G1, heRX1-1.G2 and heRX1-1.G4.

In the figure 26 polluter is by comparative neutralization of human serum recombinant murine antibody RX1, marked rmRX1, and several different versions heRX1-1 (which were made to replace low risk), each of which has a different constant region (IgG1, IgG2 or IgG4), denoted by RX2, RX1-1-IgG2, RX1-1-IgG1, RX1-1-IgG1, RX1-1-IgG4, RX1-a-IgG4.

Figure 27 illustrates the comparative neutralization environment MDA231 (cell lines breast cancer) recombinant murine antibody RX1 marked rmRX1, and several different versions heRX1-1 (which were made to replace low risk), each of which has a different constant region (IgG1, IgG2 or IgG4), denoted by RX2, RX1-1-IgG2, RX1-1-IgG1, RX1-1-IgG1, RX1-1-IgG4, RX1-a-IgG4.

Figure 28 illustrates the effect on osteoclastogenesis (measured by TRAP-activity) recombinant murine antibody RX1 marked rmRX1, and two different variants heRX1-1.G1, each of which has a different constant region (IgG1 or IgG2), marked heRX1-1.IgG1 and heRX1-1.IgG2.

Figure 29A shows the amino acid sequence (SEQ ID NO: 114) and nucleotide sequence (SEQ ID NO: 113) for heRX1-1.IgG1 with amino acid modifications at low risk. Figure 29B shows the amino acid sequence (SEQ ID NO: 116) and nucleotide sequence (SEQ ID NO: 115) for heRX1-1.IgG1 with amino acid modifications low + moderate risk.

The figure 30 shows the amino acid sequence (SEQ ID N: 119) and nucleotide sequence (cDNA (SEQ ID NO: 118) and genomic DNA (SEQ ID NO: 117)) for heRX1-1.IgG4 with amino acid modifications at low risk.

Detailed description of the invention

The ability to metastasize is a characteristic feature of cancer. Metastasis is called the spread of cancer cells to other parts of the body or condition caused by such spread of cancer cells. Metastasis is a complex multistage process that involves changes in the genetic material of cells, uncontrolled proliferation of the modified cells with the formation of the primary tumor, the development of a new circulatory system for supplying a specified primary tumor, invasion of the specified circulatory system cells of the primary tumor, the spread of small aggregates of cells of the primary tumor to other parts of the body and the growth of secondary tumors at these sites.

Bone is one of the most common sites of metastasis of cancerous tumors of the breast, lung, prostate and thyroid gland of man, as well as other cancers, and at the autopsy it was found that almost 60% of patients with cancer was observed bone metastases. Metastasis causing osteolysis of the bone, includes a unique stage osteoclastic bone resorption, which is not observed in metastases in other organs. The bone loss associated with cancer metastasis, Opera is : osteoclasts (multinuclear giant cells, with the ability to resorption of mineralized tissue), which, obviously, are activated by tumor products.

It was found that colony-stimulating factor (CSF-1), also known as macrophage colony-stimulating factor (M-CSF), plays a crucial role in the formation of osteoclasts. In addition, it was shown that M-CSF, in cooperation with other soluble factors that modulate the function of Mature osteoclasts, their migration and viability, as well as intercellular interactions mediated by osteoblasts and fibroblasts (Fixe & Praloran, Cytokine 10:3-7, 1998; Martin et al., Critical Rev. in Eukariotic Gene Expression 8:107-23 (1998)).

Full-size mRNA of human M-CSF encodes the precursor protein consisting of 554 amino acids. In the result of alternative mRNA splicing and differential post-translational proteolytic processing, M-CSF can either secretariats into the bloodstream in the form of a glycoprotein or proteoglycan containing chondroitin sulfate, or be expressed in the form of a transmembrane glycoprotein on the surface of M-CSF-producing cells. Three-dimensional structure expressed in bacteria 150 amino-terminal amino acids of human M-CSF, i.e. the minimum sequence required for full biological activity ofin vitroindicates that this protein is cologically dimer, each monomer consists of four alpha-helical bundles and antiparallel beta-folds (Pandit et al., Science 258:1358-62 (1992)). In the result of alternative splicing of mRNA produced three different types of M-CSF. These three polypeptide precursors are: M-CSFα, consisting of 256 amino acids, M-CSFβ consisting of 554 amino acids, and M-CSFγ consisting of 438 amino acids. M-CSFβ is a secreted protein that is not found in membrane-bound form. M-CSFα is expressed in the form of an integral membrane protein, which is slowly released by proteolytic cleavage. M-CSFα cleaved at amino acids 191-197 sequence presented on figure 10. Membrane-bound form of M-CSF can interact with receptors on neighboring cells, and therefore it can mediate specific intercellular contacts. The term “M-CSF” may also include amino acids 36-436 presented on figure 12.

Various forms of M-CSF act by binding with its receptor M-CSFR on the target cells. M-CSFR is a transmembrane molecule with five extracellular, immunoglobulin-like domains, a transmembrane domain and intracellular faint Src-related tyrosinekinase domain. M-CSFR is encoded by the proto-oncogeneC-fms. The binding of M-CSF with extracellular house is Mr. M-CSFR leads to dimerization of the receptor and thereby activates the cytoplasmic kinase domain and subsequent autophosphorylation and phosphorylation of other cellular proteins (J.A. Hamilton J. Leukoc. Biol., 62(2):145-55 (1997); Hamilton J, A. Measurement Today., 18(7):313-7(1997).

Phosphorylated cellular proteins induce a cascade of biochemical events leading to such cellular responses, as mitosis, secretion of cytokines, active movement in the membrane and regulation of transcription of its own receptor (Fixe & Praloran, Cytokine 10:32-37 (1998)).

M-CSF is expressed in stromal cells, osteoblasts and other cells. It is also expressed in the tumor cells of the breast, uterus and ovary. The level of expression in these tumors correlates with a high degree of malignancy of the tumor and poor prognosis (Kacinski Ann. Med. 27:79-85 (1995); Smith et al., Clin. Cancer. Res. 1:313-25 (1995)). In carcinomas of the mammary gland expression of M-CSF prevalent in invasive tumor cells, but not in intraductal (pre-invasive) cancer cells (Scholl et al., J. Natl. Cancer. Inst. 86:120-6 (1994)). In addition, it was shown that M-CSF stimulates the progression of breast tumors with malignant tumors (Lin et al., J. Exp. Med. 93:727-39 (2001). With regard to breast cancer and ovarian cancer, the production of M-CSF, obviously, leads to the recruitment of macrophages into the tumor.

As shown in this application, M-CSF-specific antibody, such as antibody RX1, (5H4, MS or MS, will neutralize the induction of osteoclasts metastatic cancer cells and/or reduces the number of metastases in bone in his belly who's model of cancer. For example, the present invention relates to compositions and to methods of treatment or prevention of cancer, cancer metastasis and osteoporosis associated with cancer metastasis.

Preferred mouse anti-M-CSF antibody RX1 has been modified so that it is less immunogenic to humans, method Studnicka et al. on the basis of antibodies to Human Engineered™. In a preferred embodiment of the invention 8-12 surface amino acid residues of the variable region of the heavy chain and 16-19 surface residues in the region of the light chain were replaced with residues in human terms, as defined, in all probability, not have a negative effect on binding to the antigen or to the laying of the protein, but at the same time, reduce its immunogenicity to humans. Were designed synthetic genes containing modified variable regions of the heavy and/or light chain, and these genes were attached to the constant regions of human heavy chains γ and/or light chain Kappa. Any constant region, a human heavy and light chains can be used in combination with the variable regions of the antibodies are Human Engineered™. Genes of the human heavy and light chains were introduced into mammalian cells, after which they were obtained and characterized recombinant immunoglobulin products. Other represen is exploring anti-M-CSF antibodies, such as (5H4, MS and MS, also antibodies are Human Engineered™.

The term “antibody, derived from RX1” includes any of the following antibodies:

1) amino acid variant murine antibody RX1, having the amino acid sequence represented in figure 4, including variants containing amino acid sequence of the variable region of the heavy chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence represented in figure 4, and/or containing the amino acid sequence of the variable region of the light chain that is at least on 60, 65, 70, 75, 80, 85, 90, 91, 92 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence represented in figure 4, subject to the same amino acids to determine homology;

2) M-CSF-binding polypeptides (except murine antibody RX1)that contains one or more hypervariable regions (complementarity-determining regions, CDRs) of murine antibody RX1, having the amino acid sequence represented in figure 4, preferably consisting of at least the CDR3 of the heavy chain RX1, and more preferably contains two or more or three or more or four or more or five or more, or all six CDRs;

3) antibodies to Human Engineered™with AMI who kislotnye sequence of the heavy and light chains, shown in figures 19B-22B, or their variants having amino acid sequences of the heavy and light chains, where the specified amino acid sequence at least 60% identical to the amino acid sequence of the heavy or light chain of the original antibody is Human Engineered™shown in figures 19B-22B, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, for example, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%;

4) M-CSF-binding polypeptides (except murine antibody RX1), containing the remains of the high risk of one or more CDRs of antibodies to Human Engineered™shown in figures 19B-22B, and preferably containing the remains of the high risk of two or more or three or more or four or more or five or more, or all six CDRs;

5) Antibodies Human Engineered™ or their variants, preserving the amino acid residues at high risk, presented in figure 4B and contains one or more modifications balances low or moderate risk, presented in figure 4B;

for example, containing one or more modifications in the low risk residues and conservative substitutions of residues of moderate risk, presented the on figure 4B, or,

for example, preserving the amino acid residues of moderate and high risk, presented in figure 4B, and contains one or more modifications in the residuals at low risk,

where these modifications include insertions, deletions or substitutions, and may include conservative substitutions, or can lead to the production of recombinant antibodies, which has a sequence that is most similar to the sequence of human light or heavy chain with a sequence of light chain or the heavy chain of the human germ line, with a consensus sequence of human light chain or heavy chain, or a consensus sequence light chain or heavy chain human germline; where these antibodies retain the ability to bind to M-CSF.

These antibodies preferably bind to M-CSF with an affinity, which constitutes at least 10-7, 10-8or 10-9or higher, and preferably neutralize osteoclastogenic-inducing activity of M-CSF.

Similarly, the term “antibody, derived from MS” includes any of the following antibodies:

1) amino acid variant of mouse antibodies MS having the amino acid sequence represented in figure 15, including variants containing amino acid placenta is the sequence of variable region of the heavy chain, which, at least 60, 65, 70, 75, 80, 85, 90, 91, 92 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence represented in figure 15, and/or containing the amino acid sequence of the variable region of the light chain that is at least on 60, 65, 70, 75, 80, 85, 90, 91, 92 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence, presented on figure 15, subject to the same amino acids to determine homology;

2) M-CSF-binding polypeptides (optionally including or excluding murine antibody MS)containing one or more hypervariable regions (CDR) of murine antibodies MS having the amino acid sequence represented in figure 15, preferably consisting of at least the CDR3 of the heavy chain MS, and more preferably contains two or more or three or more or four or more or five or more, or all six CDRs;

3) antibodies to Human Engineered™, produced by modifying the mouse sequence by the methods described in U.S. patent No. 5766886, Studnicka et al., in example 4 of this application, and are numbered according to the numbering Cabatu, as shown in figures 24C-24TH, to identify residues of low, moderate and high risk; where these antibodies include at least one of the following heavy chain and, at the ore, one of the following light chain, namely: (a) a heavy chain in which, if necessary, all the remains low risk were modified so that these residues were identical to the corresponding residues of the original sequence of a human immunoglobulin, or (b) a heavy chain in which, if necessary, all the remains low and moderate risk were modified so that these residues were similar residues of this sequence is the source of human immunoglobulin, (C) light chain, in which, if necessary, all the remains low risk were modified so as to these residues were identical to the corresponding residues of the original sequence of a human immunoglobulin, or (d) a light chain in which, if necessary, all the remains low and moderate risk were modified so that these residues were identical to the corresponding residues of the original sequence of a human immunoglobulin,

4) variants of antibodies, referred to in the preceding paragraph (3), containing the amino acid sequence of the heavy and light chains that are at least 60% identical to the amino acid sequence of the heavy or light chain of the original antibody is Human Engineered™, more preferably at least 80%, more preferably at least 85%, even Bo is her preferred at least 90%, and most preferably at least 95%, for example, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%;

5) M-CSF-binding polypeptides (optionally including or excluding murine antibody MS), containing the remains of the high risk of one or more CDRs of the mouse antibody MS presented on figure 15, and preferably containing the remains of the high risk of two or more or three or more or four or more or five or more, or all six CDRs;

6) antibodies Human Engineered™ or their variants, preserving high risk amino acid residues of murine antibodies MS and contains one or more modifications balances low or moderate risk;

for example, containing one or more modifications in the low risk residues and conservative substitutions of residues of moderate risk or,

for example, preserving the amino acid residues of moderate and high risk and containing one or more modifications in the residuals at low risk,

where these modifications include insertions, deletions or substitutions, and may include conservative substitutions, or can lead to the production of recombinant antibodies, which has a sequence that is most similar to the sequence of human light or heavy chain with a sequence of light is a Bishop or the heavy chain of the human germ line, a consensus sequence of human light chain or heavy chain, or a consensus sequence light chain or heavy chain human germline; where these antibodies retain the ability to bind to M-CSF.

These antibodies preferably bind to M-CSF with an affinity corresponding to at least 10-7, 10-8or 10-9or higher, and preferably neutralize osteoclastogenic-inducing activity of M-CSF.

The terms “antibody derived from (5H4” or “antibody, derived from MS” have values similar to the values of the terms defined above.

As described in detail in this application, the antibodies derived from antibodies RX1, (5H4, MS or MS, including antibodies, Human Engineered™ or their variants may represent antibodies of different isotypes, such as IgG, IgA, IgM or IgE. Antibodies of the IgG class can include different constant region, for example, IgG2 antibody can be modified so that it contains the constant region is IgG1 or IgG4. In its preferred embodiments, the present invention relates to antibodies Human Engineered™ or its variants containing modified or unmodified constant region is IgG1 or IgG4. In the case of IgG1, modifications made to a constant region, and in particular in the hinge area or in Snobiety, can increase or decrease effector function, including ADCC or CDC activity. In other embodiments of the invention, the constant region of IgG2 modified in order to reduce the education level of a complex of the antibody-antigen”. In the case of IgG4, modifications in the constant region, and in particular in the hinge region, can lead to the suppression of the formation of poloitical. In its specific embodiment, the present invention relates to the production of mutations in the hinge IgG4 sequence Cys-Pro-Ser-Cys obtaining hinge sequence Cys-Pro-Pro-Cys characteristic of IgG1.

In this application it is shown that the antibodies are Human Engineered™, containing a constant region IgG1 or IgG4, have improved properties compared with antibodies to the Human Engineered™, containing a constant region of IgG2. The choice of the Fc region of IgG1 or IgG4 allows to increase the binding affinity of M-CSF-neutralizing activity and activity directed against osteoclasts. In addition, the Fc region of IgG1 or IgG4 allows to obtain complexes of antigen-antibody, which are most similar to the complexes formed by the parent murine antibody.

Thus, it is obvious that the mobility of the hinge region significantly affects the binding of an antibody to dimeric antigen M-CSF, and neutralizing activity of antibodies. In the present invention, in General it is assumed, that obtaining antibody containing a heavy chain comprising a modified or unmodified constant region is IgG1 or IgG4, and in particular the hinge region and the CH2 domain, or preferably, at least the hinge region, will increase the binding affinity of the antibodies and/or slow down the dissociation of the dimeric antibodies from the antigens.

The term “antibody is competing with the RX1 includes:

1) no mouse monoclonal antibody or monoclonal antibody, which is not a rodent antibody, which binds to the same epitope of M-CSF that murine RX1 with a full-sized sequences of light and heavy chains presented on figure 4;

2) mouse monoclonal antibody or monoclonal antibody, which is not a rodent antibody, which binds at least 4 contiguous amino acids 98-105 of the sequence M-CSF presented on figure 12; and

3) mouse monoclonal antibody or monoclonal antibody, non-antibody rodents, which is more than 75%, greater than 80% or more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% competes with murine antibody RX1 with full sequence represented in figure 4, for binding to M-CSF. These antibodies preferably bind to M-CSF with an affinity of at least 10 -7, 10-8or 10-9or higher, and preferably neutralize osteoclastogenic-inducing activity of M-CSF.

The terms “antibody competing with MS” or “antibody that competes with MS” or “antibody that competes with (5H4” similarly defined as antibodies that bind to the same epitope of M-CSF, bound mouse antibodies MS, MS or (5H4, with a full-sized sequences of light and heavy chains are shown in figures 13, 14 or 15, respectively, and which has amino acids 65-73 or 138-144 presented on figure 12 (corresponding to the epitope of M-CSF, recognizable by antibodies (5H4 or MS).

Any chimeric, human or humanitariannet antibody against M-CSF, as described before filing this application, or as described in the application filed before the filing date of this application, is excluded, but not necessarily, from the scope of the present invention.

“Monoclonal antibody, non-antibody rodents” is any antibody defined here in a broad sense that is not full of intact monoclonal antibody rodents generated by hybridomas rodents. Thus, more specifically, antibodies, non-rodent antibodies include, but are not limited to, variants of rodent antibodies, fragments of antibodies rodents, line the s antibodies, chimeric antibodies, humanized antibodies, Human antibodies Engineered™ and human antibodies, including human antibodies produced in transgenic animals or produced by the method of phage view. Similarly, the non-murine antibody include, but are not limited to, variants of mouse antibodies, fragments of mouse antibodies, linear antibodies, chimeric antibodies, humanized antibodies, Human antibodies Engineered™ and human antibodies.

Used herein, the term “tumor” means the growing and proliferous neoplastic cells, regardless of whether they are malignant or benign, and all pre-cancerous and cancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe the physiological condition of mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers are breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, not small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, cancer of the colon, carcinoma endometrium, carcinoma of the salivary glands, kidney cancer, liver cancer, vulvar cancer, thyroid cancer, liver carcinoma and head and neck cancer of different types.

The term “treatment” means the intervention carried out in order to prevent the development of disease or influence the pathological process of the disease. Accordingly, the term “treatment” refers to therapeutic treatment and prophylactic or preventative measures. Individuals who need treatment are individuals who are already suffering from this disease, as well as individuals who need prevention of such diseases. In the treatment of tumor diseases (e.g. cancer) therapeutic agent can directly weaken the pathological processes of tumor cells, or to inform these tumor cells more susceptible to treatment with other therapies, such as radiotherapy and/or chemotherapy. The treatment of patients suffering from clinical, biochemical, radiological or subjective symptoms such as osteolysis, may include the weakening of some or all of these symptoms or reducing the predisposition to this disease. The term “pathology” cancer includes all processes that have a negative impact on the health of the patient. This term includes, but is not ogran is ensured by them, abnormal or uncontrolled cell growth, metastasis, negative impact on the normal functioning of neighboring cells, release of cytokines or other secretions at abnormal levels, suppression or amplification of the inflammatory or immune response, etc. Thus, the positive dynamics of the disease after treatment can be manifested in the decrease in tumor size, decrease in the rate of tumor growth, the destruction of existing tumor cells or metastatic cells, and/or reducing the size or number of metastases.

The term “mammal”, being treated, means any animal belonging to the class of mammals, including humans, domestic animals and farm animals, and animals living in zoos, animals, participating in sports, or Pets, such as dogs, horses, cats, cows, etc. is preferred mammal is a human.

Used herein, the term “metastatic cancer” is a cancer that can spread to other areas of the body, and in particular on the bone. Metastasize to bone cancer can different types, but the most common types of cancer that gives metastases to bone are breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer is prostate cancer. For example, other types of cancer that can metastasize to the bone, are, but are not limited to, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; skin cancer, including malignant melanoma and squamous cell carcinoma. In the present invention, in particular, discusses the prevention and treatment of osteolytic bone lesions induced by tumor.

Used herein, the term “therapeutically effective amount” means a therapeutic or prophylactic amount of an antibody against M-CSF that is suitable for implementing the present invention and producing the desired therapeutic or prophylactic effect or response when its introduction in accordance with the desired treatment regimen.

Used herein, the term “human M-CSF” refers to human polypeptide having, by beings who, the same amino acid sequence as Mature human polypeptide M-CSFα, M-CSFβ or M-CSFγ described in the publication Kawasaki et al., Science 230:291 (1985), Cerretti et al., Molecular Immunology, 25:761 (1988) or Ladner et al., EMBO Journal 6:2693 (1987), each of which is introduced into the present description by reference. This terminology reflects the fact that three of the Mature M-CSF have different amino acid sequences described above, and that the active form of M-CSF is a disulfide dimer, and therefore, if the term “M-CSF” refers to a biologically active form, this implies also its dimeric form. The term “dimer M-CSF” means two polypeptide monomer M-CSF, which are diarizonae and include as homodimer (consisting of two monomers M-CSF of the same type), and heterodimer (composed of two different monomers). The monomers M-CSF can be converted into dimers M-CSFin vitroas described in U.S. patent No. 4929700, which is introduced into the present description by reference.

Anti-M-CSF antibodies

The present invention relates to M-CSF-specific antibody, such as RX1, 5H4, MC1 and/or MS; to pharmaceutical compositions comprising an M-CSF-specific antibody, such as antibody RX1, 5H4, MC1 and/or MS; to methods of producing the above pharmaceutical compositions and to methods of treatment of these patients, the pharmaceutical the ski compositions and compounds. The term “antibody” is used in this application in its broadest sense and encompasses full-sized antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bespecifically antibodies), antibody fragments that can bind antigen (e.g., Fab', F(ab')2, Fv, single-chain antibodies, diatel), and recombinant peptides comprising the above-mentioned antibodies, provided that they possess the desired biological activity.

Used herein, the term “monoclonal antibody” means an antibody obtained from a population of mostly homogeneous antibodies, that is, the individual antibodies comprising such a population, which are identical, if not take into account potential natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, unlike conventional drugs (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies, in addition to their specificity, have the advantage that they are synthesized homogeneous culture, the e containing other immunoglobulins with different specificnosti and properties.

The definition of “monoclonal” indicates the type of antibody, derived mainly from a homogeneous population of antibodies, but it is understood that this antibody does not have to be produced to any particular method. For example, the monoclonal antibodies used in accordance with the present invention, can be obtained hybridoma method first described by Kohler et al., Nature, 256:495 [1975], or they can be obtained by the methods of recombinant DNA (see, for example, U.S. patent No. 4816567). “Monoclonal antibodies” may also be isolated from phage libraries of antibodies by methods described, for example, Clackson et al., Nature, 352:624628[1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991).

Immunoglobulins, depending on the amino acid sequence of the constant domain of their heavy chains, can be assigned to different classes. There are five main classes, IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of the heavy chain corresponding to the different classes of immunoglobulins are called alpha, Delta, Epsilon, gamma and mu, respectively. Structures of subunits and three-dimensional configuration of immunoglobulins of different classes are well known in the art. Immunoglobulins different isotype is to have different effector functions, for example, the isotypes IgG1 and IgG3 have ADCC activity.

“Antibody fragments” contain some of the intact full-length antibody, preferably antigennegative or variable region of the intact antibody. Examples of fragments of antibodies are Fab-, Fab'and F(ab')2and Fv fragments; dyatel; linear antibodies (Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); single-stranded molecule antibodies and multispecific antibodies formed from fragments of antibodies. Hydrolysis of the antibody with papain leads to the production of two identical antigenspecific fragments, called “Fab”fragments, each of which has one antigennegative site, and a residual “Fc”fragment, whose name reflects its ability to easily crystallize. Treatment with pepsin leads to the formation of F(ab')2-fragment, which consists of two “single-chain Fv fragments or scFv-fragments of the antibody containing the VH and VL domains of antibodies, where these domains are present in a single polypeptide chain. Preferably an Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which leads to the formation of the structure necessary for binding to the antigen. Description sFv can be found in the publication Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg & Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

The term “hypervariable region's who” means amino acid residues of the antibody, who is responsible for binding to the antigen. Hypervariable region containing amino acid residues, derived from the complementarity-determining region or CDR [i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain described by Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)], and/or residues, derived from the hypervariable loops [i.e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and residues 26-32 (H2), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain described in the publication Chothia et al., J. Mol. Biol. 196:901-917 (1987)].

“Frame or FR-residues represent residues of the variable region, but are not remnants of the hypervariable region.

The term “diately” means a small antibody fragments with two antihistamine sites, where these fragments contain the variable region of the heavy chain (VH)connected to the variable region of the light chain (VL) in the same polypeptide chain (VH VL). If the linker is too short for pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigenspecific site. Diately described in more detail, the example in EP 404097; WO 93/11161 and work Hollinger et al. Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993).

In some embodiments of the invention it may be desirable to generate multispecific (for example, bespecifically) monoclonal antibody, including a monoclonal antibody, a human antibody, humanitariannet antibody, the antibody is Human Engineered™ or variants of anti-M-CSF antibodies having binding specificity for at least two different epitopes. Representative bespecifically antibodies can bind to two different epitopes of M-CSF. Alternatively, a branch of the anti-M-CSF antibody may be combined with the branch that is associated with stimulating molecule on a leukocyte such as a molecule of T-cell receptor (e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so that the cellular protective mechanisms were focused in cells expressing M-CSF. Bespecifically antibodies can also be used to determine the localization of cytotoxic agents to cells expressing M-CSF. These antibodies have the M-CSF-binding branch and the branch that is associated with a cytotoxic agent (e.g., caporino, antiinterference 60, vinylchloride, a chain of ricin A, methotrexate or hapten labeled with a radioactive isotope). Bespecifically antibodies can be perceived by the s as full-length antibodies or fragments of antibodies (e.g., F(ab')2fragments bespecifically antibodies).

In accordance with another method of producing bespecifically antibodies can be constructed boundary between a pair of antibody molecules to maximize the percentage of heterodimers isolated from recombinant cell culture. This boundary preferably contains at least part of the domainN3 constant region of the antibody. In this way, the side chain of one or more small amino acids in the edge region of the first antibody molecules replace the side chains of larger amino acids (e.g. tyrosine or tryptophan). In the edge region of the second antibody molecules create compensatory “cavities”, having a size that is identical or similar size larger(s) side(s) chain(s), by replacing the side chains of large amino acid side chains of smaller amino acids (e.g. alanine or threonine). This allows to increase the output of heterodimers in relation to other unwanted end-products such as homodimers. Cm. application WO 96/27011 published on 6 September 1996.

Bespecifically antibodies are cross-linking or “heteroconjugate” antibodies. For example, one of the antibodies in this heteroconjugate can be attached to avidin, and the other to Biotin. Heteroconjugate EN is the body can be obtained by any standard method of cross stitching. Suitable cross-linking agents, as well as several methods of cross-stitching is well known in the art and described in U.S. patent No. 4676980.

Methods of producing bespecifically antibodies, fragments of antibodies have also been described in the literature. For example, bespecifically antibodies can be obtained by chemical binding. In the work of Brennan et al., Science, 229:81 (1985) described a procedure in which the intact antibody is subjected to proteolytic cleavage with the formation of F(ab')2-fragments. These fragments regenerate in the presence of the agent, forming diceology complex, such as sodium arsenite to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide bonds. Then, the resulting Fab'-fragments are converted into derivatives of dinitrobenzoate (TNB). After that one of the derivatives of Fab'-TNB again turned into Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB, resulting in a gain bespecifically antibody. Received bespecifically antibodies can be used as agents for the selective immobilization of enzymes. In yet another variant of the invention Fab'-SH fragments, directly isolated fromE. colican be chemically linkedin vitrowith the formation of bespecifically and the antibodies (Shalaby et al., J. Exp. Med. 175:217-225 (1992)).

In the publication Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized molecule F(ab')2especifismo antibodies. Each Fab'fragment was separately secreted fromE. coliand subjected to direct chemical bindingin vitrowith the formation of especifismo antibodies. Thus obtained bespecifically antibody has the ability to bind to cells, sverkhekspressiya the HER2 receptor and normal human T-cells, as well as to stimulate lytic activity of human cytotoxic lymphocytes directed against target cells tumors of human breast cancer.

Were also described various methods of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, bespecifically antibodies were produced using “latinovich lightning” (Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)). Peptides latinboy lightning originating from proteins Fos and Jun were added to the Fab'-parts of two other antibodies by ligating genes. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used for the production of homodimeric antibodies. T is hnology “datel”, described by Hollinger et al., Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993), provides an alternative mechanism for obtaining fragments especifismo antibodies.

These fragments contain the variable region of the heavy chain (VH)that is connected with the variable region of the light chain (VL) through a linker that is too short to create a pairing between the two domains on the same chain. In line with this, VHand VLdomains of one fragment are forced to mate with a complementary VLand VH-domains of another fragment, thereby forming two antigenspecific site. Was also described another strategy to obtain fragments especifismo antibodies using single-chain Fv(sFv)dimers. Cm. Gruber et al., J. Immunol., 152:5368 (1994).

Alternatively, bespecifically antibody may be a “linear antibody”, produced as described in the publication Zapata et al., Protein Eng., 8(10):1057-1062 (1995). In short, these antibodies have a pair of tandem Fd segments (VH-CH1-VH-CH1), which form a pair antigenspecific areas. Linear antibodies can be bespecifically or monospecific.

It also considers antibodies with more than two valencies. So, for example, can be obtained and respecification antibodies (Tutt et al., J. Immunol. 147:60 (1991)).

In some embodiments, is subramania monoclonal antibody, human antibody humanitariannet antibody, the antibody is Human Engineered™ or variant anti-M-CSF antibodies are antibody fragment, such as a fragment of the antibody RX1, (5H4, MS or MS. To obtain fragments of antibodies, various techniques have been developed. Traditionally, these fragments are formed as a result of proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can be produced directly by recombinant cell host. In the work of Better et al., Science 240: 1041-1043 (1998) described the secretion of functional fragments of antibodies bacteria (see, e.g., Better et al., Skerra et al., Science 240: 1041-1043 (1998)). So, for example, Fab'-SH fragments can be directly isolated fromE. coliand chemically bonded with the formation of F(ab')2fragments (Carter et al., Bio/Technology 10:163-167 (1992)). In another embodiment of the invention F(ab')2fragments obtained using “latinboy lightning” GCN4 to stimulate the Assembly of F(ab')2-molecules. In accordance with another approach, an Fv, Fab or F(ab')2fragments can be directly isolated from the culture recombinant host cell. Specialists and other known methods of obtaining fragments of antibodies.

“Isolated” antibody is an antibody that was identified and selected the C components of its natural environment. Contaminant components of its natural environment are substances that can adversely affect diagnostic or therapeutic efficacy of antibodies, and such components may include enzymes, hormones and other protein or no protein solute. In preferred embodiments of the invention, the specified antibody can be purified (1) to the extent of more than 95% by weight of antibody as may be determined by the method of Lowry, and more preferably more than 99% by weight of the antibody, or (2) to the extent sufficient for the introduction of at least 15 residues of N-terminal or internal part of the amino acid sequence, using a sequencing machine, equipped with a centrifuge vessel, or (3) to homogeneity by electrophoresis in LTO-page in reducing or non conditions with staining of Kumasi blue or preferably silver. The term “isolated antibody” includes antibodyin situin recombinant cells, if there is at least one natural component of this antibody. However, usually, the selected antibody can be obtained, at least in one stage of cleaning.

A detailed description of the structure and generation of antibodies can be found in publications D.B. Roth and Craig N.L., Cell 94:411-414 (1998) and in U.S. patent No. 6255458, which in its entirety are introduced in the present is the description by reference. Briefly, the process of generating DNA that encodes the genes for the heavy and light chains of immunoglobulin, occurs mainly in developing b cells. Before rearrangeable and attach different gene segments of the immunoglobulin gene segments regions V, D, J, and constant region (C) are mainly in relative proximity to each other on the same chromosome. In the process of differentiation of b-cells, each of the members of the corresponding family gene segments, V, D and J (or only V and J, in the case of light chain genes) undergoes recombination with the formation of functionally rearranging genes of the heavy and light chains of immunoglobulin. This process of rearrangement of gene segments is obviously consistent. That is, first formed D-J-region of the heavy chain, and then formed V-DJ-region of the heavy chain and V-J-region of the light chain.

Recombination of gene segments of the variable region with the formation of a functional variable regions of the heavy and light chains is mediated by recombination signal sequences (RSS)that flank recombinante competent segments V, D and j feed necessary and sufficient to direct recombination, contain diazo-symmetric heptamer, at-rich nonamer and intron spacer elements region, consisting of 12 or 23 base pairs. These signals are con ervative among different loci and species in which the recombination of D-J or V-J), and functionally interchangeable. Cm. Oettinger et al. (1990), Science, 248, 1517-1523 and work cited therein. This heptamer contains a sequence of CACAGTG or equivalent, followed by spacer elements region nonconservative sequence, and then nonamer having the sequence YAAAAAS or equivalent. These sequences are located in the J-region or area below each gene segment V and D. Immediately before D - and J-segments of the germ line also has two recombinant signal sequence, first nonamer and then heptamer separated nonconservative sequence. Heptagenia and nonamine sequence located at VL-VH- or D-segment, complementary to sequences located in front of JL-, D, or JHsegments with which they recombine. The spacers located between saptamanii and nonmarine sequences have a length of either 12 pairs of nucleotides, or at 22-24 nucleotide pairs.

In addition to the rearrangement of V, D and J segments of other variability is generated in the primary repertoire of the heavy and light chains of immunoglobulin by variable recombination in provisions, which are connected to V - and J-segments of the light chain and in which is connected the D - and J-is egment heavy chain. This variability in the light chain is typically the last codon of the gene V-segment and the first codon J-segment. A similar mismatch in the areas of junction is observed on the chromosome of the heavy chain between the D - and JHsegments and may extend up to 10 nucleotides. In addition, several nucleotides can be inserted between the D - and JHsegments and between the VH- and D-segments that are not encoded in the genomic DNA. The accession of these nucleotides is known as the N-terminal variability.

The cumulative effect of such rurangirwa in the gene segments of variable area and variable recombination that may occur during this accession consists in producing the primary repertoire of antibodies.

“Fv” is the minimum antibody fragment that contains a full-sized antipersonnel website and antigennegative website. This region consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain, rigidly connected with each other by non-covalent bond. This area has such a configuration in which the three CDRs of each variable domain interact with each other so that they form antigennegative site on the surface of the dimer VH-VL. In General, the six CDR inform the antibody specificity is wyzwania with the antigen. However, even a single variable domain (or half of an Fv, containing only three CDRs specific for an antigen) has the ability to recognize the antigen and to contact him, although with lower affinity than the entire binding site.

Fab-fragment also contains the constant domain of the light chain and the first constant domain (SN) the heavy chain. Fab-fragments differ from Fab'-fragments the fact that they have a few additional residues at the carboxy-end SN-domain heavy chain including one or more cysteines, derived from the hinge region of the antibody. Used herein, the designation of Fab'-SH means Fab'in which the cysteine(C) residue(s) constant domain has(have) a free thiol group. F(ab')2-fragments of antibodies, by their nature, are produced as pairs of Fab'-fragments, among which are cysteine hinge region.

The term “neutralizing antibody” means an antibody molecule, is able to eliminate or significantly weaken the effector function of the target antigen to which it binds. Accordingly, a “neutralizing antibody against the target has the ability to eliminate or seriously reduce effector function, such as enzymatic activity, binding to the ligand or the transmission of intracellular signals.

As described in this application, for the achievement of the desired effects, in the compositions and methods of treatment of cancer metastasis and/or osteoporosis associated with cancer metastasis, can be used one or more antibodies, taken separately or in combination with other therapeutic agents. Antibodies according to the invention can be isolated from an animal, which produces this antibody as a result of either direct contact with the antigen, the environment, or immunization with this antigen. Alternatively, antibodies can be produced by the methods of recombinant DNA using one of the systems in the expression of antibodies is well known in the art (see, for example, Harlow &Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)). Such antibodies may include recombinant IgG, chimeric hybrid proteins having a sequence derived from the immunoglobulin or antibody is Human Engineered™, and all these antibodies can be used for the treatment of cancer metastasis and/or osteoporosis associated with cancer metastasis according to the invention. The term “antibody”, in addition to the intact full-sized molecules, also means fragments thereof (such as, for example, scFv-, Fv-, Fd-, Fab-, Fab'and F(ab')2-fragments) or multimer or aggregates of intact molecules and/or fragments that bind to M-CSF or M-CSFR). These antibody fragments bind to the antigen and can be derivatize ofany so, so they have structural characteristics that facilitate their excretion and absorption, for example, through the introduction of galactose residues.

In one embodiment of the present invention a monoclonal antibody against M-CSF can be obtained, mainly, as described in U.S. patent No. 5491065 (1997), Halenbeck et al., which is introduced into the present description by reference. Representative monoclonal antibody against M-CSF antibodies are associated with the apparent conformational epitope associated with recombinant or native dimeric M-CSF, and simultaneously neutralize its biological activity. These antibodies essentially do not react with biologically inactive forms of M-CSF, including Monomeric and chemically derivationally dimeric M-CSF.

In other embodiments, the present invention relates to monoclonal anti-M-CSF antibody Human Engineered™. The term “antibody Human Engineered™” means an antibody derived from non-human antibody, typically from murine monoclonal antibodies. Alternatively, the antibody is Human Engineered™ may occur from chimeric antibody which retains or essentially retains antigennegative properties of the parent non-human antibody, but which, in his introduction to a person, has a lower immunogenicity compared with the specified parent is nitela. Used herein, the term “chimeric antibody” means an antibody containing a sequence derived from two different antibodies (see, for example, U.S. patent No. 4816567), which are typically derived from different species. Most often, chimeric antibodies contain fragments of human and mouse antibodies, as usual, the constant region of human antibody and the variable region of mouse antibody.

The term “complementarity-determining region” or “CDR” means amino acid sequences which together define the binding affinity of and specificity of the natural Fv region of the binding site of native immunoglobulin (see, e.g., Chothia et al., J. Mol. Biol. 196:901-917 (1987); Kabat et al., U.S. Dept. of Health and Human Services NIH Publication No. 91 3242 (1991)). The term “constant region” means the portion of the molecule antibodies, which informs effector functions. In the present invention, the constant region of the mouse antibody is preferably replaced with the constant regions of human antibodies. Constant region under consideration antibodies originate from human immunoglobulins. The constant region of the heavy chain may be selected from the five isotypes: alpha, Delta, Epsilon, gamma or mu.

Antibodies according to the invention are immunospecificity or specifically bind antibodies, if they regard yaytsa to the antigen with K andgreater than or constituting about 106M-1preferably greater than or constituting about 107M-1more preferably greater than or constituting about 108M-1and most preferably greater than or constituting about 109M-1, 1010M-1, 1011M-1or 1012M-1. Anti-M-CSF antibodies can be linked to various natural forms of M-CSF, including forms, expressing in the host tissues/individual, as well as the forms expressed by the tumor. Described here monoclonal antibodies, such as antibodies RX1, (5H4, MS or MS have affinity to M-CSF and are characterized by the equilibrium constant of dissociation (Kd)constituting at least 10-4M, preferably at least about 10-7M - 10-8M, and more preferably at least about 10-8M, 10-10M, 10-11M, 10-12M Such affinity can be easily determined by standard methods such as equilibrium dialysis, the analysis using the BIAcore device 2000 in accordance with the General procedures described by the manufacturer; radioimmunoassay using125I-labeled M-CSF, or other methods known in the art. Data affinity can be analyzed, for example, the methods of the om Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949). Thus, it is obvious that the preferred antibody against M-CSF will have a high degree of specificity to M-CSF and will be in contact with other molecules, mainly with lower affinity. Preferred antibodies bind to M-CSF with an affinity similar to the affinity of binding murine RX1 presented on figure 4, with M-CSF, have low immunogenicity and inhibit metastasis of cancer cells, as determined when the test animal model with metastatic disease. Other representative antibody binds to M-CSF with an affinity similar to the affinity of binding to M-CSF mouse antibodies (5H4, MS or MS shown in figures 13, 14 or 15, respectively.

The antigen used for production of antibodies may represent, for example, intact M-CSF or fragment M-CSF, which preserves the desired epitope and which is optionally attached to another polypeptide that allows you to display the epitope in the native conformation. Alternatively, cells expressing M-CSF on its surface, can be used for producing antibodies. These cells can be transformed so that they are expressed M-CSF, or can be used other natural cells expressing M-CSF. Other forms of M-CSF that is suitable for generating and the antibodies, well-known experts in this field.

Polyclonal antibodies

Polyclonal antibodies are preferably produced in animals after repeated subcutaneous (s.c.) or intraperitoneal (I.P. Pavlova.) injections of the relevant antigen and an adjuvant. Enhanced humoral response can be achieved by conjugation of the relevant antigen to a protein that is immunogenic for species subjected to immunization, for example, hemocyanine lymph snails, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, such as maleimidophenylmethacrylates (conjugated through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic acid anhydride, or other known agents.

Animals subjected to immunization with the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes complete adjuvant's adjuvant and by subcutaneous injection of the solution into several sections. A month later the animals repeatedly subjected to immunization in many areas by subcutaneous injection fraction 1/5-1/10 of the original amount of peptide or conjugate in complete adjuvant who's adjuvant. After 7-14 days after the second injection in animals blood sample and analyze the serum for antibody titer. Then animals again subjected to immunization before reaching the plateau of the title. Repeated immunization of animals preferably carried out by injection of the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. The conjugates can also be obtained in recombinant cell culture as hybrid proteins. In addition, to enhance the immune response can also be used aggregating agents such as alum.

Monoclonal antibodies

Monoclonal antibodies can be produced using hybridoma technology, first described by Kohler et al. Nature, 256:495 (1975), or they can be obtained by the methods of recombinant DNA.

In the method of obtaining a hybrid mouse or other suitable animal host, such as a hamster or macaque, subjected to immunization, as described above, for generating them lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes may be immunitaryin vitro. Then lymphocytes subjected to fusion with myeloma cells using a suitable agent for the merger, such as a polyethylene glycol, a result of which it formed hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

Thus obtained hybridoma cells were seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival not drained parent myeloma cells. For example, if the parental myeloma cells do not contain enzyme gipoksantin-guanine-phosphoribosyltransferase (HGPRT or HPRT), the culture medium to obtain hybridomas typically includes gipoksantin, aminopterin and thymidine environment (NAT), that is, substances that prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are cells that are able to be effective with the merger, to maintain a stable production of high levels of antibodies selected antibody-producing cells, and are sensitive to the environment. For producing human monoclonal antibodies are also used in human myeloma cell lines and heteromyinae cell line mouse-human” (Kozbor, J. Immunol., 133:3001 (1984) & Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)). Examples of murine myeloma lines are lines originating from cells of murine tumors MOR-21 MS-11, available at the Institute Salk Institute Cell Distribution Center, San Diego, California USA, and SP cells-2 or H-Ag8-653, deponere is the R in the American type culture collection, Rockville, Md. USA.

Culture medium for the growth of hybridoma cells examined for the production of monoclonal antibodies against this antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells, determined preferably by thus or by holding thein vitroanalysis of the binding, such as radioimmunoassay (RIA) or solid-phase immunofermentnyi assay (ELISA). The binding affinity of the monoclonal antibody can be, for example, identified through analysis of Scatchard (Munson et al., Anal. Biochem., 107:220 (1980)).

After identification of hybridoma cells that produce antibodies with the desired specificity, affinity and/or activity, the clones may be subcloned by using procedures limiting dilution and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). A suitable culture medium designed to achieve this goal is, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells may be cultivationin vivoas ascitic tumors in animals. Monoclonal antibodies secreted by the subclones, accordingly isolated from the culture medium, ascitic fluid, or serum by standard procedures purification of immunoglobulins, t is such as, for example, chromatography on protein a - sepharose, chromatography on hydroxiapatite, gel electrophoresis, dialysis, or affinity chromatography.

Production of recombinant antibodies

DNA encoding the monoclonal antibodies may be isolated and sequenced from hybridoma cells according to standard procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies). To determine the sequence usually requires the allocation of at least part of the desired gene or cDNA. This usually requires cloning DNA or, preferably mRNA (i.e. cDNA)that encodes a monoclonal antibody. Cloning is carried out by standard methods (see, for example, manual Sambrook et al., (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, which is incorporated into the present description by reference). For example, the cDNA library can be constructed by reverse transcription of polyA + mRNA, and preferably membrane-bound mRNA, and this library can be skanirovana using probes specific for the gene sequences of the polypeptides of human immunoglobulin. However, in the preferred embodiment of the invention, amplification of cDNA (or parts of the full sized the cDNA's), coding representing the interest of the segment of the immunoglobulin gene (for example, a segment of the variable region of the light chain)used polymerase chain reaction (PCR). Amplificatoare sequence can easily be cloned into any suitable vector, such as expression vectors, minigene vectors, or vectors, phage view. It should be noted that the specific method of cloning is not decisive if it can be determined sequence of any part of interest of the immunoglobulin polypeptide. Used herein, the term “isolated molecule nucleic acid” or “isolated nucleic acid sequence” means a nucleic acid molecule which is either (1) identified and separated from at least one impurity molecules of nucleic acid with which it is normally associated in the natural source of the nucleic acid, or (2) cloned, amplified, labeled, or otherwise identified from the background nucleic acids, so that it was possible to determine the sequence of interest of the nucleic acid. The selected nucleic acid molecule has the form or setting in which it does not occur in nature. Therefore, the selected nucleic acid molecules differ from molecules n is Cleanaway acid, present in natural cells. However, the selected nucleic acid molecule is a nucleic acid molecule that is found in cells that normally Express the indicated antibody, where, for example, the nucleic acid molecule is located in the chromosome at the position different from the position in which it exists in natural cells.

One of the sources of RNA used for cloning and sequencing, is hybridoma produced by selection of b-cells from transgenic mice and fusion of these cells with termed cells. The advantage of using a hybrid is that they can be easily skanirovaniya, which may be selected hybridoma producing interest human monoclonal antibody. Alternatively, RNA may be isolated from b-cells (or whole spleen) immunising animal. If your sources are not hybridoma, it may be desirable screening for sequences encoding the antibodies or polypeptides of the immunoglobulin which is capable of specific binding. One method of such screening is the application of techniques of phage view. Phage representation is described, for example, in the publications Dowe et al., WO 91/17271, McCafferty et al., WO 92/01407 and Caton & Koprowski, Proc. Natl. Acad. Sci., USA, 87:6450-6454 (1990), which are introduced in the present description by reference. In one embodiment of the invention, in which the method of phage view, impunitivnoj transgenic mice produce cDNA (for example, a full-sized cDNA spleen) and performed polymerase chain reaction for amplification of cDNA sequences coding for part of the immunoglobulin polypeptide, for example the field of the CDR, and then amplificatoare sequence is inserted into the phage vector. cDNA encoding interest peptides, such as peptides of variable domains with the desired binding properties, identified by standard methods, such as panning”.

Then determine the sequence of the amplified or cloned nucleic acid. Usually determine the sequence encoding a full-sized variable region of an immunoglobulin polypeptide, but sometimes it is enough to sequence only a part of the variable regions, such as CDR-coding part. Usually, sequenced part has a length of at least 30 nucleotides, but most often is sequenced sequence encoding at least about one third or, at least, half of the variable regions.

Sequencing can be carried out n the clones, isolated from a cDNA library, or by PCR, it can be carried out after sublimirovanny amplified sequences or by direct PCR sequencing indicated the amplified segment. Sequencing carried out by standard methods (see, for example, manual Sambrook et al., (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and F. Sanger et al. (1977) Proc. Natl. Acad. Sci., USA, 74:5463-5467, which is incorporated into the present description by reference). By comparing the sequence of the cloned nucleic acid with known gene sequences and cDNA of human immunoglobulin a person skilled in the art can easily determine, depending on sequanorum region, (i) the occurrence of data segments of the polypeptide of hybridoma immunoglobulin germ line (including the isotype of the heavy chain) and (ii) the sequence of the variable regions of the heavy and light chains, including sequences formed by attaching N-terminal region and the process of somatic mutation. One of the sources of information on gene sequences of immunoglobulin is the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.

After selecting this DNA can be incorporated into expression vectors, which are then transferout in cell host, such to the to cells E. coli, monkey COS cells, cells of the human embryo kidney 293 (e.g., cells E), cells of the Chinese hamster ovary (Cho or myeloma cells that produce only protein immunoglobulin, resulting in these recombinant cells masters synthesized monoclonal antibodies. Production of recombinant antibodies is well known to specialists.

Sequence expression regulation are DNA sequences necessary for the expression of functionally attached to the coding sequence in a given organism-host. Regulatory sequences that are suitable for prokaryotes, for example, promoter and, optionally, the operator sequence; and a binding site with the ribosome. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is “functionally connected”if it is in functional relationship with another nucleic acid sequence. For example, DNA encoding proposedvalue or secretory leader sequence is functionally attached to DNA that encodes a polypeptide if it is expressed in the form of a protein precursor which participates in the secretion of the polypeptide; or a promoter or enhanse the R is functionally attached to the coding sequence, if it affects the transcription of the sequence; or a binding site with the ribosome is functionally attached to the coding sequence, if the position of this website facilitates the translation. In General, the term “functionally connected” means that linked DNA sequences are contiguous and, in the case of a secretory leader sequence they are adjacent and are in the same reading frame. However, enhancers do not have to be adjacent to the join sequence. Connection can be made by ligating the appropriate restriction sites. If such sites are present, in accordance with standard practice can be used synthetic oligonucleotide adaptors or linkers.

Used herein, the terms “cell”, “cell line” and “cell culture” are used interchangeably, and they are all the progeny of these cells. For example, the terms “transformants” and “transformed cells” include the primary cells of the individual and deriving from them the culture, regardless of the number of subcultures. It should be noted that all progeny may not be identical in composition to DNA, due to the artificially introduced or spontaneous mutations. These terms also include mutants the e offspring, which has the same function or biological activity, which was established during the initial screening of the transformed cells. A more precise definition of these terms will be clear from the context of the following description.

Alternatively, the amino acid sequence of interest immunoglobulin can be determined by direct protein sequencing. Suitable coding nucleotide sequence can be designed in accordance with table universal codons.

Variants of the amino acid sequence of the desired antibodies can be obtained by introducing appropriate nucleotide modifications in the encoding DNA, or by peptide synthesis. Such variants include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of the antibodies. To obtain the final design can be used any combination of deletions, insertions and substitutions, provided that the final design will have the desired properties. Amino acid modifications can also lead to changes in post-translational processes in the monoclonal antibody, human antibody, humanitariannet the antibody, the antibody is Human Engineered™ or its variants, such as changing the number or positions of the sites glycodelin is found.

The nucleic acid molecules encoding amino acid sequence variants of this antibody, receive various methods known in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of a natural amino acid sequence variants) or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cluster mutagenesis previously received options or unmodified variant antibodies.

The present invention also relates to a selected nucleic acid that encodes the antibody according to the invention and functionally joined to regulatory sequences recognized the host-cell; to vectors and cells of the host containing the nucleic acid; and to methods for recombinant production of antibodies, which may include culturing the host cell for the expression of nucleic acid and, optionally, the selection of the specified antibodies from the culture or culture medium of the host cell.

For producing recombinant antibodies nucleic acid encoding such an antibody is isolated and inserted into the vector can replicate for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal ant the body, can be easily isolated and sequenced by standard methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of this antibody). There are many available vectors. The components of such vectors typically include, but are not limited to, one or more of the components such as: a signal sequence, an origin of replication, one or more selective marker genes, an enhancer element, a promoter and a sequence of termination of transcription.

(1) The signal sequence

The antibody according to the invention can be recombinante produced not only by the direct method, but also in the form of this hybrid polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-Terminus of the Mature protein or polypeptide. The selected signal sequence is preferably a sequence that is recognized and processed (that is cleaved by a signal peptidase) in the cell host. If prokaryotic cells-the owners do not recognize and do not ProcessInput signal sequence of native antibodies, this signal sequence is alnost can be replaced with a signal sequence, selected, for example, from the group consisting of the leader sequences Pictet-LiAZ (e.g., pelB), alkaline phosphatase, penitsillinazy, lpp, or heat-stable endotoxin II. For secretion in yeast of the native signal sequence may be substituted, for example, a leader sequence of yeast invertase, a leader sequence of α-factor (including the leader sequence of the α-factor of Saccharomyces and Kluyveromyces)or leader sequence of acid phosphatase, a leader sequence glucoamylase C. albicans or signal sequence described in WO 90/13646. For expression in mammalian cells are the signal sequence mammals, as well as viral secretory leader sequence, such as the signal sequence of the gD of herpes simplex virus.

The DNA for such areas predecessor are ligated with the antibody-encoding DNA with preservation of the reading frame.

(2) Component - the origin of replication

Expression and cloning vectors contain a nucleic acid sequence, which promotes replication of this vector in one or more selected cells of the host. Generally, in cloning vectors this sequence is a sequence that allows the vector to replicate independently of the t-DNA chromosome of the host and which includes originy replication or offline can replicate the sequence. Such sequences for a variety of bacteria, yeast and viruses are well known. The origin of replication derived from plasmid pBR322 is suitable for most gram-negative bacteria, namely the origin of the 2 µ plasmid is suitable for yeast, and various viral origin can be used for cloning vectors in mammalian cells. Usually, the origin of replication is not necessary for vectors, expressed in mammalian cells (SV40 origin is typically used in cases where it contains the early promoter).

(3) Component - selective marker

Expression and cloning vectors may contain selective gene, also known as selective marker. Typically, selective genes encode proteins that (a) inform the resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, tetracycline, G418, geneticin, histidine or mycophenolate acid, (b) complementary deficit caused by auxotrophies, and (C) ensure the intake of essential nutrients that do not come from complex environments, such as the gene encoding D-alanine-racemaseBacillus.

One example of schema selection is the use of drugs, stopping the growth of the host cell. Cells that were successfully Tran is formed of a heterologous gene, produce protein, indicating resistance to the drug, thereby contributing to the survival of these cells in selective medium. For such dominant selection can be used, for example, drugs such as methotrexate, neomycin, histidine, puromycin, mycofenolate acid and hygromycin.

Other examples of suitable selective markers for mammalian cells are markers that can identify cells that can embed in your genome antibody-encoding nucleic acid, such markers as DHFR, thymidine kinase, metallothionein-I and II, and preferably the genes encoding metallothionein primates, adenosine-deaminase, ornithine-decarboxylase, etc.

For example, cells transformed by selective DHFR gene, first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR suitable cell host is a cell line of Chinese hamster ovary (Cho), deficient in DHFR activity.

Alternatively, cell owners (particularly wild-type hosts that contain endogenous DHFR)transformed or co-transformed with DNA sequences encoding the antibody according to the invention, protein HFR wild-type and the other is a selective marker, such as aminoglycoside-3'-phosphotransferase (ARN), can be selected by culturing cells in a medium containing an agent for sampling conducted using the selective marker such as aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.

Suitable selective gene for use in yeast is the gene oftrp1, present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Gene trp1 is a selective marker for a mutant strain of yeast, which can not grow in the presence of tryptophan, for example, a strain deposited with the ATCC No. 44076 or RER-1. Jones, Genetics, 85:12 (1977). The presence of mutations in the trp1 gene in the genome of the yeast host cell enables efficient detection of transformation by culturing in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20622 or 38626) complementarily known plasmids bearing Leu2 gene.Ura3-deficient yeast strains were complementarily plasmids carrying the gene ofura3.

In addition, the vectors originating from the ring of 1.6 μm-plasmid pKD1 can be used for transformation of yeast Kluyveromyces. Alternative expression system for large-scale production of recombinant calf chymosin was described for K.lactis. Van den Berg, Bio/Technology, 8:135 (1990). the yli also described stable mnogoopytnyi expression vectors for secretion of Mature recombinant human serum albumin industrial strains of Kluyveromyces. Fleer et al., Bio/Technology, 9:968-975 (1991).

(4) Component - promoter

Expression and cloning vectors usually contain a promoter that is recognized by the body-master and functionally attached to the antibody-encoding nucleic acid. Promoters suitable for use with prokaryotic hosts include the arabinose promoter (for example,araB), the promoter ofphoA, promotor system β-lactamase and lactose promoter systems, alkaline phosphatase, and the tryptophan (trp), and hybrid promoters such as the promoter oftac. However, also suitable are other known bacterial promoters. The promoters used in bacterial systems also contain a Shine-dalgarno sequence (S.D.), functionally joined to DNA encoding the antibody according to the invention.

Promoter sequence suitable for eukaryotes, known to specialists. In fact, all the genes of eukaryotes have at-rich region localized around the area of 25-30 nucleotides above the site of transcription initiation. Another such sequence, localized approximately in the area of 70-80 nucleotides above the site of initiation of transcription of many genes, is the area CNCAAT, where N can mean any nucleotide. At 3'-end of most EUCA oticheskih gene is a sequence of AATAAA, which can serve as a signal for attaching A poly-a tail to the 3'-end of the coding sequence. All of these sequences can be properly embedded into eukaryotic expression vectors.

Examples of promoter sequences suitable for use in yeast cells, the owners, are the promoters for 3-phosphoglycerate-kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, glucokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate-isomerase, 3-phosphoglycerate-mutase, piruwatkinaza, trithioacetone, phosphoglucomutase and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage lies in their ability to regulate transcription in certain culture conditions, are the promoter region of the genes for alcohol dehydrogenase 2, sociogram C, acid phosphatase, hydrolytic enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Vectors and promoters suitable for expression in yeast, is also described in EP 73657. Along with yeast promoters also mainly COI is lesuuda yeast enhancers.

Transcription of the antibodies of the vectors in the cells of the host mammal is regulated, for example, by promoters obtained from the genomes of these viruses as leukemia of Abelson, polyomavirus, the smallpox virus in poultry, adenovirus (such as adenovirus 2), the virus bovine papilloma virus sarcoma birds, and most preferably cytomegalovirus, a retrovirus, hepatitis b virus, simian virus 40 (SV40); heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter; and the promoters of heat shock proteins, provided such promoters are compatible with the systems of host cells.

Early and late promoters of SV40 virus are usually obtained in the form of an SV40 restriction fragment which also contains the origin of replication of SV40 virus. Pretani the promoter of the human cytomegalovirus usually obtained in the form of a HindIII restriction fragment that is the System the expression of the DNA in the cells of the host mammal derived from cow papilomavirus used as a vector, is described in U.S. patent No. 4419446. Modification of this system for the expression of cDNA for human β-interferon in mouse cells under the control of the promoter timedancing of herpes simplex virus described in U.S. patent No. 4601878. Cm. also Reyes et al., Nature 297:598-601 (1982). Alternatively, as the promoter may be the used the rous sarcoma virus, having long terminal repeat.

(5) Component - enhancer -

Transcription of DNA encoding the antibody according to the invention in higher eukaryotes is often increased when embedding in vector enhancer sequence. Currently, there are many enhancer sequences derived from mammalian genes (globin genes, elastase, albumin, alpha-fetoprotein, and insulin). However, we typically use the enhancer from a eukaryotic virus cells. An example is the enhancer of SV40 virus, localized in the late region of origin of replication (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of polyomavirus localized in the late region of origin of replication, and enhancers of adenovirus. Cm. publication Yaniv, Nature 297:17-18 (1982), which describes enhancer elements for activation of eukaryotic promoters. The enhancer may be incorporated into the vector in the 5'- or 3'-polozenie with respect to the antibody coding sequence, but preferably, it was in the 5'position from the promoter.

(6) The site of transcription termination

Expression vectors used in eukaryotic cells-the masters (yeast, fungi, insect, plant, animal, human or nucleated cells originating from other multicellular organisms), which may contain the sequence required for termination of transcription and for stabilizing the mRNA. Such sequences are usually located on the side of the 5'-end, and sometimes, 3'-end from the untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in netransliruemoi part of the mRNA that encodes the antibody. One of the suitable components termination of transcription is the region polyadenylation bovine growth hormone. Cm. description expression vectors in WO 94/11026 and in this application. Another component termination of transcription is the transcription terminator light chain of mouse immunoglobulin.

(7) Selection and transformation of host cells

Cells masters, suitable for cloning or expression of the DNA in the vectors described here, cells are prokaryotes, yeast or higher eukaryotes, as described above. Prokaryotes suitable for this purpose are eubacteria, such as gram-negative or gram-positive microorganisms, such asEnterobacteriaceaesuch asEscherichia,for example,E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella,for example,Salmonella typhimurium, Serratia,for example,Serratia marcescansandShigella,andBacillussuch asB.subtilisandB.licheniformis(for example,B.licheniformisR described in patent DD 266710 published the data on 12 April 1989), Pseudomonassuch asP.aeruginosaandStreptomyces. Preferred cloning hostE. coliisE. coli294 ADS 31446), although can be used and other strains, such asE. coliIn,E. coliH ATS 31537), andE. coliW3110 (ATSS 27325). These examples are provided merely to illustrate and not limit the scope of the invention.

In addition to prokaryotes suitable hosts for cloning or expression of the antibody-encoding vectors are eukaryotic microbes, such as hyphomycetes or yeast. From lower eukaryotic microorganisms-hosts are most often usedSaccharomyces cerevisiaeor standard Baker's yeast. However, in this case can be used and other commonly used and available microorganisms other species and strains, such asSchizosaccharomyces pombe; hostsKluyveromycessuch as, for example,K.lactis,K.fragilisADS 12424),K.bulgaricusADS 16045),K.wickeramiiADS 24178),K.waltiiADS 56500),K.drosophilarumADS 36906),K.thermotoleransandK.marxianus; yarrowia(EP 402226);Pichia pastoris(EP 183070);Candida;Trichodermareesia(EP 244234);Neurospora crassa;Schwanniomycessuch asSchwanniomycesoccidentalis; and hyphomycetes, such as, for example, microorganisms-hostsNeurospora,Penicillium, TolypocladiumandAspergillussuch asAspergillus nidulansandAspergillus niger.

Cells hosts suitable for expression of glycosaminoglycan, are multicellular organisms. Examples of invertebrate cells are cells of plants and insects. Were identified various baculovirus strains and variants and corresponding permissive cells are the owners of insects, such asSpodoptera frugiperda(caterpillar shovels),Aedes aegypti(aegypti mosquito),Aedes albopictus(belopotosky Komar),Drosophilamelanogaster(fruit fly) andBombyx mori(silkworms). Currently there are a number of available strains that can be used for transfection, such as option L-1Autographa californicaNPV and strain Bm-5Bombyx moriNPV, and such viruses may be used in the present invention, particularly for transfection of cellsSpodoptera frugiperda.

As host cells can also be used to plant cell cultures of cotton, corn, potatoes, soybeans, Petunia, tomato, tobacco, duckweed and cells of other plants.

However, the biggest interest vertebrate cells, and propagation of vertebrate cells (tissue culture) has become a routine procedure. Examples of commonly used cell lines in host mammalian cells are Chinese hamster ovary, including cells SNACK ADS CCL61), DXB-11, DG-44 cells of the Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); cell line CV1 monkey kidney, trance is ormirovannogo SV40 (COS-7, ATCC CRL 1651); cell line of human embryo kidney (293 cells or 293 cells, subcloned for growth in suspension culture [Graham et al., J. Gen. Virol. 36:59 (1977)]; kidney cells baby hamster (KSS, ATSS CCL 10); mouse Sertoli cells (™4, Mather, Biol. Reprod. 23:243-251 (1980)); the kidney cells of monkeys (CV1 ATCC CCL 70); the kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); carcinoma cells human cervical (HELA, ATCC CCL 2); cells of the kidneys of dogs (MDCK, ATCC CCL 34); liver cells of laboratory rats Buffalo (BRL 3A, ATCC CRL 1442); cells of the human lung (W138, ATCC CCL 75); the cells of the human liver (HepG2, HB 8065); tumor cells of the mouse mammary gland (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annal NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and cell line human hepatoma (HepG2).

Cell host transformed or transferout the above-described expression or cloning vectors for the production of antibodies and cultured in a suitable nutrient media modified, if necessary, for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In addition, new vectors and transfected cell lines with multiple copies of transcription units separated by a selective marker, are particularly suitable and preferred for the expression of antibody against M-CSF.

(8) the Cultivation of CL is the current host

Cell owners used for producing antibodies according to the invention, can be cultured in various media. Media suitable for culturing the host cells are commercially available environment, such as environment Hams F10 (Sigma), minimal maintenance medium ((MEM) (Sigma), RPMI-1640 (Sigma) and modified by way of Dulbecco Wednesday Needle (DMEM), Sigma). In addition, as a culture medium for culturing the host cells can be used in any environment that is described in the publication Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. patent No. 4767704, 4657866, 4927762, 4560655 or 5122469; WO 90103430; WO 87/00195 or in U.S. patent Re. No. 30985. In any of these environments can be added, if necessary, hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride and calcium phosphate and magnesium), buffers (such as HEPES), nucleotides (such as adenosin and thymidine), antibiotics (such as a drug gentamicin™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar doses), and glucose or an equivalent energy source. May also include any other necessary additives in appropriate concentrations, known to specialists in this field. Conditions Kul is cultivated, such as temperature, pH, etc. similar to the conditions used for the expression of the selected host cells, and well known to the average expert in the field.

(9) Purification of antibodies

Using techniques of recombinant DNA antibody can be produced inside cells or in periplasmatic space, or it can be directly secreted into the environment, including the environment from microbial cultures. If in the first stage, the antibody is produced inside the cell, then the cell debris or cell owners or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. In the publication of Better et al., Science 240: 1041-1043 (1998); ICSU Shot Reports 10: 105 (1990); and Proc. Natl. Acad. Sci., USA, 90: 457-461 (1993) described the procedure for the selection of antibodies that are secreted into periplasmatic spaceE. coli(see also Carter et al., Bio/Technology 10:163-167 (1992)).

Composition antibodies derived from microbial cells or mammalian cells can be purified, for example, chromatography on hydroxylapatite, katiana - or anion-exchange chromatography and affinity chromatography, with the preferred method of cleaning is affinity chromatography. The suitability of protein a as an affinity ligand depends on the species and isotype Fc domain of immunoglobulin present in the antibody. Protein a can be used is an for the purification of antibodies, containing the heavy chain γ1, γ2, or γ4 human immunoglobulin (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). For all mouse isotypes and for human γ3 immunoglobulin is recommended protein G (Guss et al., EMBO J. 5:15671575 (1986)). As a matrix, which is bound affinity ligand, the most commonly used imaging systems, but may be used and other matrices. Mechanically stable matrices such as glass with adjustable pore size or poly(Stradivari)benzene, provide faster flow rates and shorter processing time than can be achieved with the use of agarose. If the antibody contains a domain WithN3, the cleaning can be used resin Bakerbond ABX™ (J.T. Baker, Phillipsburg, N.J.). Depending on the secreted antibodies can also be used and other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin-sepharoseTM, chromatography on anyone - or cation-exchange resin (for example, on a column with poliasparaginovaya acid), chromatofocusing, electrophoresis in LTO-PAG and precipitation with ammonium sulfate.

Chimeric and humanized antibodies

As chimeric or humanized antibodies are less immunogenic to humans, h is m parent murine monoclonal antibodies, they can be used for treatment of humans with far less risk of anaphylaxis. Thus, these antibodies may be preferred for the introduction ofin vivoman for therapeutic purposes.

Chimeric monoclonal antibodies in which the variable domains of the Ig mouse monoclonal antibodies attached to the constant domains of human Ig, can be produced by standard methods known in the art (see Morrison, S.L. et al. (1984) Chimeric Human Antibody, Holtcnles; Mouse Antigen Binding Domains with Human Constant Region Domains, Proc. Natl. Acad. Sci. USA 81, 6841-6855 and Boulianne, G.L. et al., Nature 312, 643-646 (1984)). Although it was confirmed that some chimeric monoclonal antibodies are less immunogenic to humans, but it is known that the variable domains of the murine Ig can still cause significant response in humans against mouse antibodies.

Humanized antibodies can be obtained by various methods, including, for example, (1) introduction human hypervariable regions (CDR) in the human frame and a constant region (this method is known as “humanization through “CDR-transplant”), or alternatively, (2) transplantation of a full size not a human variable domains and their “floor” surface similar to the surface of the sequences of human antibodies (this method is known to the to the “masking”). In the present invention, the term “humanized antibody” includes both “humanized”and “masked” antibodies. These methods are described in publications, for example, Jones et al., Nature 321:522-525 (1986); Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Morrison & Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988)); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217 (1994) and Kettleborough C.A. et al., Protein Eng. 4(7):773-83 (1991), which are introduced in the present description by reference.

In particular, after the re-introduction of rodent antibodies manin vivoeither separately or in the form of a conjugate, the recipient will generate an immune response against the antibody rodents, the so-called NAMA-response (human antimurine antibody). If you want to repeat the dose this HAMA response may limit the effectiveness of this pharmaceutical. Immunogenicity antibodies can be weakened through chemical modification of the specified antibodies hydrophilic polymer, such as polyethylene glycol, or by genetic engineering by creating antibodies with binding structure, more similar to human antibodies. So, for example, gene sequences of variable domains of rodent antibodies that bind to CEA, can be substituted for the variable domains of a human myeloma protein with getting re is ominotago chimeric antibodies. These procedures are described in detail in EP 194276, EP 0120694, EP 0125023, EP 0171496, EP 0173494 and WO 86/01533. Alternatively, the gene sequences of CDR rodent antibodies can be isolated or synthesized and replaced with the corresponding regions of sequence homologous to the gene of human antibodies with obtaining human antibodies with the specificity of the original antibody rodents. These procedures are described in EP 023940, WO and WO 90/07861 91/09967. Alternatively, a large number of residues of the surface of the variable domain of the antibody rodents may be substituted residues are usually present in the homologous human antibody with obtaining antibodies rodent that has “masked” external surface and which, therefore, will be recognized by the body as “it”. This approach was demonstrated in the work Padlan et al. (1991) Mol. Immunol. 28, 489.

CDR-transplantation includes the introduction of one or more of the six CDR derived from the variable domains of the heavy and light chains of murine Ig, in the four frame region variable domains of the human Ig and the transplantation is also called “CDR-grafting”. In this method (L. Riechmann et al., Nature 332, 323 (1988)) are conservative framework region (FR1-FR4) as the basis for preservation of the CDR loops, which are mainly in contact with the antigen. However, the residue CDR-transplantation is what results from this transplant humanitariannet antibody has essentially lower the binding affinity of the original murine antibody, since amino acids of frame areas can play an important role in binding to the antigen and since amino acids of the CDR loops can affect the Association of the two variable domains of Ig. To maintain the affinity of gumanitarnogo monoclonal antibody CDR method-transplantation can be improved by selection of the human frame areas most similar to frame the original mouse antibody, and by site-directed mutagenesis of single amino acids in the framework regions or CDRs using computer simulation antigennegative site (see, for example, Co, M. S. et al. (1994) J. Immunol. 152, 2968-2976).

One of the methods of humanizing antibodies includes alignment not a human sequence heavy and light chains of human sequence heavy and light chains, the selection and replacement of the human skeleton human skeleton area on the basis of this alignment; molecular modeling to predict the conformation of the humanized sequence and its comparison with the conformation of the parent antibody. Then repeat back mutation residues in the CDR is blasti, violate the structure of the CDR, as long as the predicted conformation of the humanized model of the sequence is closest to the conformation is not human CDR of the parent non-human antibody. Such humanized antibodies may be further derivatization to facilitate their capture and purification, for example by Ashwell receptors (see, for example, U.S. patent No. 5530101 and 5585089 that are entered into the present description by reference).

Various methods of humanization of a murine monoclonal antibody by rational design described in the literature (see, for example, an application 20020091240, published July 11, 2002, WO 92/11018 and U.S. patent No. 5693762 and 5766866).

Variants of amino acid sequences

The method used for identification of certain residues or regions of the antibody that are preferred localization for mutagenesis is called “alanine scanning mutagenesis” and described Cunningham & Wells, Science 244:1081-1085 (1989). In this method, a residue or group needs identify residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of these amino acids with antigen. Then the localization of amino acids, proabley the x functional sensitivity to such changes, specify by introducing additional or other modifications in the position of a replacement or at some distance from this replacement. For example, the position of introduction of amino acid substitutions may be determined in advance, while the nature of the mutationper sedoes not have to be defined in advance. So, for example, to analyze the performance of a mutation at a given site, Ala scanning or non-specific mutagenesis is carried out in the correct codon or region and the options expressed antibodies sceneroot on the desired activity.

Insertions in the amino acid sequences are inserted at the amino and/or carboxy-ends, forming hybrids, having a length from one residue to polypeptides containing a hundred or more residues, as well as to insert into the sequence, consisting of one or multiple amino acid residues. Examples of terminal insertions is an antibody with an N-terminal nationalsim residue or the antibody (including antibody fragment)that is attached to the epitope tag or epitope to bind to the receptor of salvation. Other insertional variants of the antibody molecules are hybrids, for example, N - or C-ends of the antibodies with the polypeptide, which increases the half-life of antibodies in the serum.

The term “labeled epitope” refers to the antibody attached to the epitope of the th tag. Polypeptide epitope tag has enough residues for the formation of the epitope against which may be generated antibody, but it is short enough not to interfere with the activity of antibodies. Preferably epitope tag should be sufficiently unique to this antibody, essentially, did not engage in cross-react with other epitopes. Suitable polypeptides labels usually have at least 6 amino acid residues, and more about 8-50 amino acid residues (preferably about 9-30 residues). Examples are the polypeptide label hemagglutinin (HA) of influenza and produced antibody against him SA [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the polypeptide tag C-myc and produced antibody against him 8F9, 3C7, 6E10, G4, B7 and E [Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)], glycoprotein D of herpes simplex virus (gD) and produced against him antibody [Paborsky et al., Protein Engineering 3(6):547-553 (1990)]. Another representative label is polyhistidine sequence, typically containing about six his-tag residues and allow to single labeled thus the connection with the agent, forming a chelate complex with Nickel. Other labels, such as label FLAG® (Eastman Kodak, Rochester, NY), well known in the art and described in literature, are included in the scope is altoadige of the invention.

Used herein, the term “receptor-svyazyami epitope salvation” means an epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3or IgG4), which is responsible for increasing the half-life of the IgG molecule in the serum ofin vivo.

Another option is the option of replacing amino acids. These variants have at least one remote amino acid residue in the molecule antibodies and instead built the remainder. It is envisaged that the mutagenesis with substitutions may be made at any hypervariable region or CDR-regions or frame areas. Conservative substitutions are shown in table 1. The most conservative substitutions are indicated in the column under the heading of “Preferred substitutions”. If such replacement does not result in a change in biological activity, can be entered more basic replacements named in table 1 representative substitutions, or replacements, further described below in the description of classes of amino acids, and can be screened products.

Table 1
The original balanceRepresentative
balance
Preferred replacement of residues
Ala (A)val; leu; ile;Val
Arg (R)lys; gln; asnLys
Asn (N)gln; his; asp; lys; glnArg
Asp (D)glu; asnGlu
Cys (C)ser; alaSer
Gln (Q)asn; gluAsn
Glu (E)asp; glnAsp
Gly (G)ala
His (H)asn; gln; lys; arg
Ile (I)leu; val; met; ala; pheleu, norleucine
Leu (L)norleucine; ile; val;
met; ala; phe
Ile
Lys (K)arg; gln; asn;Arg
Met (M)leu; phe; ile Leu
Phe (F)leu; val; ile; ala; tyr
Pro (P)ala
Ser (S)thr
Thr (T)serser
Trp (W)tyr; phetyr
Tyr (Y)trp; phe; thr; serphe
Val (V)ile; leu; met; phe;
ala; norleucine
leu

Significant changes in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their impact on the structure of the polypeptide backbone in the area of the substitution, for example folded and helical conformation, (b) the charge or hydrophobicity of the molecule at the desired site, or (C) the volume of the side chain. On the General properties of their side chains of natural remnants are divided into the following groups:

(1) hydrophobic residues: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic residues: Cys, Ser, Thr;

(3) acid residues: Asp, Glu;

(4) the OS is ESD residues: Asn, Gln, His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic residues: Trp, Tyr, Phe.

Conservative substitutions include substitution of amino acid of one class by another amino acid of the same class. Not conservative substitutions include substitution of amino acid of one class by another amino acid of a different class.

To improve the oxidative stability of the molecule and prevent unwanted cross-linking any cysteine residue not involved in maintaining the proper conformation of monoclonal antibodies, human antibodies, gumanitarnogo antibodies antibody Human Engineered™ or variant of such antibodies can also be replaced, usually by serine. Conversely, the antibody to improve its stability (particularly if the specified antibody is an antibody fragment such as an Fv fragment)may be introduced cysteine(s) relationship(s).

The affinity maturation involves receiving and screening the variants of antibodies that have substitutions in the CDRs of the parent antibody, and the selection of variants with improved biological properties, such as affinity binding compared to the parent antibody. A standard way of generating such variants with substitutions provides for the affinity maturation using method F. the final presentation. Several provisions of the hypervariable region (e.g., 6-7 provisions) is subjected to mutation for the influence of all possible amino acid substitutions at each position. Generated thus variants of the antibodies will be presented as a monovalent molecules on the particles of filamentous phage in the form hybrids with the product of the gene III, Packed in every part of the phage M13. Then the options presented on the phage, sceneroot on their biological activity (e.g. binding affinity of).

To identify residues of the hypervariable region, which play a significant role in binding with the antigen can be performed alanine-scanning mutagenesis. Alternative or additionally, it may be beneficial to analyze a crystal structure of the complex antigen-antibody to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for replacement, carried out in accordance with methods developed here. After generating these options panel of these variants is subjected to screening as described in this application, and antibodies that detect superior properties in one or more relevant assays may be selected for further investigation.

Can be also produced variants of the antibodies to the that have modified the nature of glycosylation compared with the parent antibody, for example, by deletion of one or more carbohydrate groups present in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked glycosylation means attaching carbohydrate group to the side chain of aspartic residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X means any amino acid except Proline, are sequences of recognition for enzymatic joining carbohydrate portion to the side chain of asparagine. The presence of any of these Tripeptide sequences in the polypeptide promotes the creation of a potential site of glycosylation. Thus, the sites of N-linked glycosylation can be added to the antibody by modifying the amino acid sequence in which it will contain one or more specified Tripeptide sequences. O-linked glycosylation means joining one of the sugars, such as N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate mainly to serine or threonine, although they may also be 5-hydroxyproline or 5-guide oxylipin. Sites of O-linked glycosylation can be added to the antibody by insertion or substitution of one or more serine or treoninove residues in the sequence of the original antibody. For example, amino acids RX1 in positions 41-43 in figure 4A (NGS) can be saved. Alternatively, it may be stored only amino acids 41 and 42 (NG).

Basically, amino acid sequence variants of antibodies, Human Engineered™ have an amino acid sequence that is at least 60% identical to the original amino acid sequence of the heavy or light chain of the antibody is Human Engineered™ (for example, as shown in figures 19B-22B), preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. The terms “identity” or “homology”related to this sequence is defined in this application as the percentage of amino acid residues in the sequence candidate, identical residues of the antibody is Human Engineered™, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, without regard to any conservation the x substitutions (as described above in table 1), shall be considered as part of the identity of the sequence. None of these modifications, as N-terminal, C-terminal, or internal extensions, deletions or insertions in the sequence of the antibody should not adversely affect the identity or homology sequences. Thus, the sequence identity can be determined by standard methods commonly used to determine the similarity of amino acids in this position of the two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptide level for optimal matching of their amino acids (or the entire length of one or both sequences, or by the length of a predetermined part of one or both sequences). These programs have default settings, such as the penalty for missing symbol and the penalty for a gap, and an evaluation matrix such as PAM 250 [standard evaluation matrix; see Dayhoff et al. Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978)], which can be used together with a computer program. For example, the percent identity can be calculated as follows: total number of identical matches multiply by 100 and then divided by the sum of the length of the longer sequence in a given area of compliance and the number of gaps introduced into the longer you'll find the activities to align two sequences.

Can also be considered and other modifications of the antibodies. For example, it may be desirable to modify the antibody according to the invention from the point of view of its effector functions in order to increase the effectiveness of this antibody for the treatment of, for example, cancer. For example, the Fc region may be entered(s) to cysteine(C) residue(s)that will contribute to the formation of messagewall disulfide bond in this area. Generated thus homodimeric antibody may have an enhanced ability to internalize and/or increased complement-dependent cytolytic activity and increased antibody-dependent cytotoxicity (ADCC). Cm. Caron et al. J. Exp. Med. 176:1191-1195 (1992) and Shopes, B.J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be obtained using heterobifunctional cross-linking of the linkers described by Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, it may be engineered antibody that has two Fc-region, and therefore it may have enhanced complement-dependent cytolytic activity and increased ADCC. Cm. Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989). In addition, it was shown that the sequence of the CDR may promote the binding of an antibody to MHC class II and run unwanted response by producera the project helper T cells. Conservative substitution allows the preservation of the binding activity of antibodies, but it can lead to the loss of the ability to run unwanted T-cell response. Cm. publication Steplewski et al., Proc. Natl. Acad. Sci., USA 1988; 85(13):4852-6, which is introduced in the present description by reference, and which describes chimeric antibodies, in which the murine variable regions linked to human constant regions of the gamma-1, gamma-2, gamma 3, and gamma-4.

In some embodiments of the invention for increasing the permeability of the tumor may, for example, be desirable to use not intact antibodies and fragments of antibodies. In this case, to increase the time of its half-life in serum may be desirable modification of such a fragment of the antibody, for example, by attaching molecules such as PEG or other water soluble polymers, including polysaccharide polymers, fragments of antibodies. This can be achieved, for example, by incorporating the epitope binding to the receptor of salvation, in the fragment of the antibody (e.g., by mutation of the appropriate region in this fragment of the antibody or by the inclusion of the epitope into a peptide tag that is then attached to the antibody fragment at either end or in the middle of the sequence, for example, by DNA synthesis or peptide synth is over) (see, for example, WO 96/32478).

The epitope to bind to the receptor of salvation, preferably contains an area in which one or more amino acid residues, derived from one or two loops Fc-domain, transferred to a similar position of the fragment of the antibody. Even more preferably, to have moved three or more residue from one or two loops Fc-domain. Even more preferably, the epitope was isolated from the CH2 domain of the Fc region (e.g., IgG) and transferred to the area CN, CH3, or VH indicated antibodies or in more than one region. Alternatively, the epitope was allocated from the CH2 domain of the Fc region and transferred to the region CLor VLor in both of these areas of the fragment of the antibody. Cm. International application WO 97/34631 and WO 96/32478 that describe the Fc variants and their interaction with the receptor of salvation.

Thus, the antibodies according to the invention may contain human Fc part of human consensus Fc-part or a version thereof that retains the ability to interact with Fc-receptor of salvation, including the cases in which cysteine involved in the formation of disulfide bonds, have been modified or deleterow, and/or in which the N-end was added to the Met and/or removed one or more of the 20 N-terminal amino acids, and/or have been deleted region, which interact with the set is a COP, such as the site of binding to C1q and/or was removed ADCC website [see, e.g., Molec. Immunol. 29 (5): 633-9 (1992)].

In previous studies was mapped the binding site of the human and murine IgG on FcR, mainly, to the lower part of the hinge region consisting of residues 233-239 IgG. In other studies were presented additional broader segments, for example Gly316-Lys338 for the human Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for the human Fc receptor III, or a few specific residues outside of the lower part of the hinge region, for example at Asn297 and Glu318 for mouse IgG2b, interacting with murine Fc receptor II. In the description 3,2 E. crystal structure of the Fc fragment of human IgG1 with the human Fc receptor IIIA specified residues Leu234-Ser239, Asp265-Glu269, Asn297-Thr299 and Ala327-Ile332 IgG1 involved in binding to Fc receptor IIIA. Based on the analysis of the crystal structure it has been suggested that the residues located in loops FG CH2 domain of IgG (residues 326-330), when administered in the lower part of the hinge region (Leu234-Gly237), and BC (residues 265-271), may play a role in binding to Fc receptor IIA. Cm. publication Shields et al., J. Biol.Chem., 276(9):6591-6604(2001), which in its entirety is introduced into the present description by reference. Mutation of residues in the binding sites with Fc-receptor can lead the change effector functions, for example to change ADCC or CDC activity or to change the time half-life. As described above, it is possible mutations are insertions, deletions or substitutions of one or several residues, including replacement by alanine, a conservative substitution, a non-conservative substitution or replacement of the relevant amino acid residue other IgG subclass in the same position antibodies (for example, replacement of residue corresponding IgG1 balance IgG2 in the same position).

Shield et al. reported that IgG1 residues involved in binding to all human Fc-receptore are located in the CH2 domain, present in the immediate vicinity of the hinge region, and are divided into the following two categories: 1) residues, which can communicate directly with all FcR, namely Leu234-Pro238, Ala327 and Pro329 (and possibly Asp265); and 2) residues that influence carbohydrate nature or position, namely, Asp265 and Asn297. Other residues of IgG1, which affect the binding to Fc receptor II are the following residues (maximum effect): Arg225, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298 and the following residues (lesser effect): His268, Asn276, His285, Asn286, Lys290, Gln295, Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339, Ala378 and Lys414. A327Q, A327S, P329A, D265A and D270A reduce binding. In addition to the residues identified above for all FcR, other residues of IgG1 that reduce binding to Fc-what eception IIIA 40% or more, are the following balances: Ser239, Ser267 only (Gly), His268, Glu293, Gln295, Tyr296, Arg301, Val303, Lys338 and Asp376. Options that increase the level of binding to FcRIIIA, are TA, K290A, S298A, E333A, and K334A AT. Lys414 gives a 40% reduction in binding to FcRIIA and FcRIIB, Arg416 gives a 30% reduction in binding to FcRIIA and FcRIIIA, Gln419 gives a 30% reduction in binding to FcRIIA and 40% reduction in binding to FcRIIB and Lys360 gives a 23% increase in the level of binding to FcRIIIA. Cm. also Presta et al., Biochem. Soc. Trans. (2001) 30, 487-490.

For example, in U.S. patent No. 6194551, which in its entirety is introduced into the present description by reference, describes variants with altered effector function and contains a mutation in the Fc-region of human IgG in the provisions of amino acids 329, 331 or 322 (numbering Kabata), and some of these variants have reduced C1q-binding activity or CDC activity. So, for example, U.S. patent No. 6737056, which in its entirety is introduced into the present description by reference, describes variants with altered effector function, or altered function of binding to the Fc receptor-gamma, i.e. containing mutations in the Fc-region of human IgG in the provisions of amino acids 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 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, 37, 438 or 439 (numbering Kabata), and some of these options are profiles of the receptor binding associated with reduced ADCC or CDC activity. It was found that, of these mutations, the mutation at amino acid position 238, 265, 269, 270, 327 or 329 lead to lower level of binding to FcRI, and it was found that mutations in positions of amino acids 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 lead to lower level of binding to FcRII, and mutations in the provisions of amino acids 238, 239, 248, 249, 252, 254, 265, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 lead to lower level of binding to FcRIII.

In U.S. patent No. 5624821, which in its entirety is introduced into the present description by reference, it is reported that the C1q-binding activity of mouse antibodies can be modified by introducing mutations in the positions of amino acid residues 318, 320 or 322 of the heavy chain, and also reported that replacing residue 297 (Asn) leads to the loss of lytic activity.

In the publication of the patent application U.S. No. 20040132101, which in its entirety are introduced in the present description by reference, describes variants having mutations at positions of the amino acids 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328 or 332 (numbering Kabata) or in the provisions of amino acids 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 26, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330 or 332 (numbering Kabata), where mutations in the provisions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330 or 332 may result in reduced ADCC activity or to reduce the level of binding to Fc receptor-gamma.

In the publication Chappel et al., Proc. Natl. Acad. Sci., USA, 1991; 88(20):9036-40, which in its entirety is introduced into the present description by reference, reported that sitophila activity of IgG1 is a property inherent in its CH2 domain of the heavy chain. Single point mutations in any of the amino acid residues 234-237 IgG1 lead to a significant reduction or loss of its activity. To restore full binding activity is necessary to replace all IgG1 residues in positions 234-237 (LLGG) to the corresponding residues of IgG2 and IgG4. It was found that the IgG2 antibody containing the full sequence ELLGGP (residues 233-238), was more active than IgG1 wild-type.

In the publication Isaacs et al., J. Immunol. 1998; 161(8):3862-9, which in its entirety is introduced into the present description by reference, reported that mutations in the motif that plays a crucial role in binding to FcR-gamma (where glutamate 233 is replaced by Proline, leucine/phenylalanine 234 is replaced with valine, and leucine 235 is replaced by alanine), prevent the depletion of target cells. Replacement of glutamate 318 by alanine leads to eliminats and effector functions of mouse IgG2b, as well as lower activity of human IgG4.

In the publication Armour et al., Mol. Immunol. 2003; 40(9):585-93, which in its entirety is introduced into the present description by reference, have been identified variants of IgG1, which react with the activating receptor, FcR-gamma IIa, at least 10 times less efficient than IgG1 wild type, but their level of binding inhibitory receptor FcR-gamma IIb was lower only four times. Were made mutations in amino acids 233-236 and/or in the provisions of amino acids 327, 330 and 331. Cm. application WO 99/58572, which in its entirety is introduced into the present description by reference.

In the publication Xu et al., J. Biol. Chem. 1994;269(5):3469-74, which in its entirety is introduced into the present description by reference, it is reported that in IgG1 replacement Pro331 on Ser leads to a marked reduction in C1q-binding activity and the elimination of political activity. In contrast, replacement of Ser331 on Pro IgG4 reports option Pro331-IgG4 partial lytic activity (40%).

In the publication Schuurman et al., Mol. Immunol. 2001; 38(1):1-8, which in its entirety is introduced into the present description by reference, reported that the replacement of one of the hinge cysteines involved in the bond formation between heavy chains, Cys226, serine leads to the formation of a more stable connection between the heavy chains. the Amena sequence Cys-Pro-Ser-Cys hinge region of IgG4 in the sequence Cys-Pro-Pro-Cys hinge region of IgG1 also leads to a significant stabilization of the covalent bonds between the heavy chains.

In the publication Angal et al., Mol. Immunol. 1993; 30(1):105-8, which in its entirety is introduced into the present description by reference, reported that the replacement of serine at amino acid position 241 in IgG4 for Proline (present in the same position in IgG1 and IgG2) leads to the production of homogeneous antibodies, as well as increase the time half-life in serum and to improve its distribution in the tissue, compared to the original chimeric IgG4.

Human antibody and the antibody is Human Engineered™

Human Engineered™

In the work Studnicka [see, for example, Studnicka et al., U.S. patent No. 5766886; Studnicka et al., Protein Engineering 7:805-814 (1994)] has been described a method of obtaining variable domains of the antibody is Human Engineered™ to reduce immunogenicity while retaining binding activity of the antibody molecules. In accordance with this method, each amino acid variable region was established risk such replacement. Amino acid substitutions were divided into three risk categories: (1) replacement of low risk, in which there is the highest probability of reducing immunogenicity with the least chance of violation of binding to the antigen; (2) replacement of moderate risk, which also lead to reduced immunogenicity, but to increase the chance of a violation of binding to the antigen or protein folding; (3) replacement of high risk that are important for binding or DL the save structure of antibodies and create the highest risk of negative effects on the binding of antigen or laying protein. In General, it is believed that because of the three-dimensional structural configuration prolinol modifications in the provisions of prolinol will lead to the replacement of at least moderate risk, even if the change in this position is usually considered replacing low risk.

Variable region light and heavy chains of antibodies rodents, representing Human Engineered™, were obtained by replacing the human amino acid positions, in which, as has been identified, the likelihood of negative effects of such substitutions on the binding of antigen or laying protein is very low, and the probability that such replacement will result in reduced immunogenicity in humans, is quite high. Amino acid residues that are in the “low risk” and who are candidates for modification in accordance with this method, were identified by aligning the amino acid sequences of variable regions of rodents with the sequence of the human variable regions. This can be used any human variable region, including a separate sequence of VH or VL or human consensus sequence of the VH or VL, or individual or a consensus sequence of human germline. Can be modified amino acid residues at every position “low risk” or in the “low risk”. For example, in every position “low risk”, in which amino acid residues aligned to the mouse and human sequences differ from each other, introducing amino acid modification, i.e. amino acid residue sequence of the rodent replace human balance. The alternative, may be made by modifications in amino acid residues at all positions “low risk” and in any position, “moderate risk”. Ideally, to achieve the lowest immunogenicity in all positions of low and moderate risk, amino acid residues rodents replace the remnants of the human sequence.

Were designed synthetic genes containing modified variable regions of the heavy and/or light chains, and these genes were attached to the constant regions of heavy chains γ and/or light chain of human Kappa immunoglobulin. Any of the constant regions of the heavy and light chains of human immunoglobulin can be used in combination with the variable regions of the antibodies are Human Engineered™, including IgA (of any subclass, such as IgA1 or IgA2), IgD, IgE, IgG (any subclass such as IgG1, IgG2, IgG3 or IgG4 or IgM. The genes for the heavy and light chains of human immunoglobulin is injected into the cells of the host, such as mammalian cells, and then receive and Oh racterization recombinant immunoglobulin products.

Human antibodies from transgenic animals

Human antibodies against M-CSF can also be produced in transgenic animals, which is not reproduced endogenous immunoglobulin, and constructed so that they contain the locus of human immunoglobulin. For example, in WO 98/24893 described transgenic animals having a locus of human Ig, where these animals were not producirovanie functional endogenous immunoglobulins due to inactivation of the endogenous loci of the heavy and light chains. In WO 91/741 also described transgenic mammals are the owners of non-primates and capable of producing an immune response to the immunogen, where antibodies are constant and/or variable regions of primates and where gene loci encoding the endogenous immunoglobulin, have been replaced or inactivated. In WO 96/30498 described using the system of the Cre/Lox for the modification of the immunoglobulin locus in a mammal, so as to replace all or part of the constant or variable regions with the formation of the modified molecules are antibodies. In WO 94/02602 described non-human mammal host, having inactivated endogenous Ig loci and functional human Ig loci. In U.S. patent No. 5939598 described methods of creating transgenic mice lacking endogenous heavy d the PI and which is expressed exogenous immunoglobulin locus, containing one or more xenogeneic constant regions.

Using the above-described transgenic animals can be produced immune response to a selected antigenic molecule, and these animals can be taken antibody-producing cells, which can be used for the production of hybridomas secreting human monoclonal antibodies. The immunization protocols, adjuvants, etc. known in the art and used for immunization, for example, transgenic mice, as described in WO 96/33735. This publication describes monoclonal antibodies against different antigenic molecules, including IL-6, IL-8, TNFα, human CD4, L-selectin, gp39 and tetanus toxin. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. In WO 96/33735 described that monoclonal antibodies against IL-8, derived from immune cells of transgenic mice, immunogenic IL-8, block IL-8-induced functions of neutrophils. Human monoclonal antibodies with specificity to the antigen used for immunization of transgenic animals, as described in WO 96/34096, in the application for U.S. patent No. 20030194404 and in the application for U.S. patent No. 20030031667.

Cm. also, e.g., Jakobovits et al., Proc. Natl. Acad. Sci., USA, 90:251 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggerman et al., Year in Measurement, 7:33 (1993) and U.S. patent No. 5591669, 5589369 and 5545807, and application for U.S. patent No. 20020199213. In the application for U.S. patent No. 20030092125 described methods for modifying the immune response of the animal to the desired epitope. Human antibodies can also be producedin vitroactivated b-cells (see U.S. patent No. 5567610 and 5229275).

Human antibodies obtained by phage view

Development of methods of obtaining sets of recombinant genes to human antibody and the representation of the coded fragments of the antibodies on the surface of filamentous bacteriophage give the opportunity to produce human antibodies. Antibodies produced rahovym method, get as antigen-binding fragments, usually, Fv or Fab fragments in bacteria, and these fragments do not have effector functions. Effector functions can be created one of two strategies, for example, fragments can be designed either as full-length antibodies for expression in mammalian cells, either in the form of fragments bespecifically antibodies having a second binding site, capable of stimulating effector function.

Usually, Fd-fragment (VH-CN1) and light chain (VL-CL) antibody clone separately by PCR and subjected to arbitrary p the combination of combinatorial libraries, phage view which can then be selected to bind with a specific antigen. Fab fragments expressed on the surface of phage, that is physically attached to the genes that encode them. For example, a Fab selection by binding to the antigen carried out simultaneously with the selection of Fab-coding sequences, which can then be amplified. Several rounds of binding to the antigen and re-amplification, that is, the procedure called “penninga”, lead to the enrichment of Fab fragment specific for the antigen, after which the fragment isolated.

In 1994, there was described a method for the humanization of antibodies, called “directional selection”. In directional selection, an efficient method of phage view, which allows the humanization of murine monoclonal antibodies (see Jespers L.S. et al., Bio/Technology 12, 899/903 (1994)). For this Fd-fragment of mouse monoclonal antibodies may be provided in combination with a library of light chains of a human antibody and the Fab library-hybrids can then be selected using antigen. Thus, murine Fd-fragment can then be used as a matrix for directional selection. After that, the selected human light chain combined with a library of human Fd fragments. A selection is received from the library results in a full-sized human Fab.

Have been described various procedures for the selection of human antibodies from phage libraries representations (see, e.g., Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991), U.S. patent No. 5565332 and 5573905; Clackson, T. & Wells, J.A. TIBTECH 12, 173-184 (1994)). In particular,in vitroscreening and selection of antibodies from phage libraries representations become in recent times the most effective method (see Burton D.R. & Barbas III, C.F., Adv. Immunol. 57, 191-280 (1994) & Winter, G. et al., Annu. Rev. Immunol. 12, 433-455 (1994), application for U.S. patent No. 20020004215 and WO 92/01047, application for U.S. patent No. 20030190317, published October 9, 2003, U.S. patent No. 6054287 and U.S. patent No. 5877293).

In the publication Watkins “Screening of Phage-Expressed Antibody Libraries by Capture Lift, Methods in Molecular Biology, Antibody Phage Display: Methods and Protocols 178:187-193, and in the application for U.S. patent No. 200120030044772 published on 6 March 2003, described screening methods expressed by the phage libraries of antibodies or other binding molecules by “capturing”, that is, the method involving the immobilization of molecules that communicates with the candidate, on a solid medium.

Products antibodies can be skanirovaniya on the activity and on the possibility of their use in the treatment methods according to the invention using the analyses described in this application in the section entitled “screening Methods”, or using any suitable assays known in the art.

Other covalent modifications

Covalent modifications of the antibody are also included in the scope of the present invention. Such modifications can be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if necessary. The antibody molecule can be made of covalent modification of other types by interacting selected amino acid residues of the antibody with the organic derivatizing agent capable of reacting with selected side chains or the N - or C-terminal residues.

Caseinline remains often subjected to interaction with α-halogenation (and corresponding amines), such as Chloroacetic acid or chloroacetamide, obtaining carboxymethylated or carboxylatomethyl derivatives. Caseinline debris derivateservlet by interacting with BROMOTRIFLUOROMETHANE, alpha-bromo-β-(5-imidazolyl)propionic acid, chloroacetylation, N-alkylamide, 3-nitro-2-pyridyldithio, methyl-2-pyridyldithio, benzoate p-hartati, 2-hartati-4-NITROPHENOL, or chloro-7-nitrobenzo-2-oxa-1,3-diazoles.

Histidine remains derivateservlet by interacting with diethylpyrocarbonate at pH of 5.5 to 7.0 because this agent is relatively specific for histidine side chain. Can also be used para-brompheniramine, and this reaction is preferably carried out in 0.1 M cacodylate sodium at pH 6.0.

Leinil the s and amino-terminal residues are interacting with the anhydrides of succinic acid or other carboxylic acids. The derivatization of these agents leads to a change in charge disinilah residues. Other suitable reagents for derivatization alpha aminecontaining residues are imediately, such as methylphenidate, phosphate method, pyridoxal, Harborside, trinitrobenzenesulfonic acid, O-methylisoleucine, 2,4-pentandiol and glyoxylate, being involved in the catalyzed transaminase reaction.

Argireline residues altered by interaction with one or more standard reagents, for example with phenylglyoxal, 2,3-butanedione, 1,2-cyclohexandione and ninhydrin. For derivatization arginine residues requires that the reaction was performed in alkaline conditions because of the guanidine functional group has a high PKand. In addition, these reagents can interact with the lysine groups, as well as with the Epsilon-amino group of arginine.

Specific modification trailing residues can be carried out by introducing interest spectral labels in tyrosine residues by reaction with aromatic diazonium compounds or tetranitromethane. In most cases, you use the N-acetylimidazole, tetranitromethane, resulting in the formation of O-acetyltyrosine group and 3-nitro-derivatives, respectively the military. Tyrosine residues are jodirovannuju using125I or131I obtaining labeled proteins for use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by interaction with carbodiimide (RN=C=NR', where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-asoni-4,4-dimethylpentyl)carbodiimide. In addition, aspartamine and glutamine remains turned into asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are often subjected to reaction deliciouse education glutamine and aspartyl residues, respectively. These residues are deletirovanie in neutral or basic conditions. Deliciously form of these residues included in the scope of the present invention.

Other modifications are hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups merilnyh or traveling residues, methylation of the alpha-amino groups of the side chains of lysine, arginine and histidine (T.E.Creighton, Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, pp.79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Covalent modify the situation of the other type involves chemical or enzymatic joining of glycosides to the antibody. These procedures have the advantage that they involve the production of antibodies in the cell host, which has the ability to N - or O-linked glycosylation. Depending on the applied method of joining, sugar(sugar) may be attached(s) (a) arginine and histidine, (b) free carboxyl groups, (C) free sulfhydryl groups such as groups of cysteine, (d) free hydroxyl groups such as groups of serine, threonine, or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in the application WO 87/05330, published on September 11, 1987, and in the publication Aplin & Wriston, CRC Crit. Rev. Biochem., pp.259-306 (1981).

Removal of any carbohydrate groups present on the antibody can be carried out by chemical or enzymatic method. For chemical deglycosylation, the antibody must be processed by the connection triftormetilfullerenov acid or equivalent connection. Such processing leads to the elimination of most or all sugars except the linker sugars (N-acetylglucosamine or N-atsetilgalaktozamin), but the antibody remains intact. Chemical deglycosylation described in the publication Hakimuddin et al., Arch. Biochem. Biophys. 259:52 (1987) and Edge et al. Anal. Biochem. 118:131 (1981. Enzymatic cleavage of carbohydrate groups can be achieved under the action of various endo - and ectoparasites described in the publication Thotakura et al., Meth. Enzymol. 138:350 (1987).

Another type of covalent modification of the antibody comprises attaching this antibody to one of the various protein polymers, such as polyethylene glycol, polypropyleneglycol, polyoxyethylene the polyols, polyoxyethylene the sorbitol, polyoxyethylene glucose, polyoxyethylene glycerin, polyoxyalkylene or polysaccharide polymers, such as dextran. Such methods are known in the art and described, for example, in U.S. patents№№4640835, 4496689, 4301144, 4670417, 4791192, 4179337, 4766106, 4179337, 4495285, 4609546 or in EP 315456.

Gene therapy

The delivery of therapeutic antibodies in the appropriate cells can be achieved by gene therapyex vivo,in situorin vitroany suitable method known in the art, including physical DNA transfer (e.g., using liposomes or chemical agents) or the use of viral vectors (e.g. adenovirus, adeno-associated virus, or a retrovirus). For example, ifin vivotherapy nucleic acid encoding the desired antibody and either taken separately or in combination with a vector, liposome, or precipitate, can be directly in the ill individual, in some embodiments of the invention, it can be introduced in the area of the body, in which the desired expression of this antibody. Whenex vivotherapy in an individual take the cells and these cells are introduced nucleic acid, and then the modified cells are again administered to an individual either directly, or, for example, by encapsulation in porous membranes, which are then implanted into the patient. See, for example, U.S. patent No. 4892538 and 5283187. For the introduction of nucleic acids into viable cells there are different methods. These methods vary depending on, carry whether a given nucleic acid in cultured cellsin vitroor in cells of the desired hostin vivo. Techniques suitable for the transfer of nucleic acid into mammalian cellsin vitroare the use of liposomes, electroporation, microinjection, merge cells, mediated by DEAE-dextran transfection and the precipitation of calcium phosphate. Vector, most often used forex vivodelivery of nucleic acid, is a retrovirus.

Other methods of transferring nucleic acidin vivois transfection using viral vectors (such as adenovirus, herpes simplex I or adeno-associated virus) and lipid systems. Nucleic acid and transferowy agent associiruyutsa is, but not necessarily, with the microparticles. Examples transfairusa agents are agents that provide co-precipitation of calcium phosphate or calcium chloride; agents providing mediated by DEAE-dextran transfection; bromide amphiphilic Quaternary ammonium DO™A ((dioleoylglycerol)-trimethylammonium, commercially available under the name Lipofectin, GIBCO-BRL) (Felgner et al. (1987) Proc. Natl. Acad. Sci., USA, 84, 7413-7417; Malone et al. (1989) Proc. Natl. Acad. Sci., USA, 86:6077-6081); lipophilic diesters of glutamic acid from the upper side trimethylammonium groups (Ito et al. (1990) Biochem. Biophys. Acta 1023, 124-132); metabolisable source lipids such as cationic lipids: diastatochromogenes (DOGS, Transfectam, Promega) and dipalmitoylphosphatidylethanolamine (DPPES) (J.P.Behr (1986) Tetrahedron Lett. 27, 5861-5864, J.P.Behr et al. (1989) Proc. Natl. Acad. Sci., USA, 86, 6982-6986); metabolisable Quaternary ammonium salt (DOTB, methyl sulfate N-(1-[2,3-tileorassi]propyl)-N,N,N-trimethylammonium (DOTAP) (Boehringer Mannheim); polyethylenimine (PAYS), valeology esters, ChoTB, ChoSC, DOSC) (Leventis et al. (1990) Biochim. Inter. 22, 235-241); 3-beta-[N-(N',N'-dimethylaminoethyl)carbarnoyl]cholesterol (DC-Chol), dioleoylphosphatidylcholine (DOPE)/3-beta-[N-(N',N'-dimethylaminoethyl)carbarnoyl]cholesterol (DC-Chol) in a mixture in the ratio of 1:1 (Gao et al. (1991) Biochim. Biophys. Acta. 1065, 8-14), spermine, spermidine, lipopolymer (Behr et al., Bioconjugate Chem. 1994, 5:382-389), lipophilic polylysine (LPLL) (Zhou et al. (1991) Biochim. Biophys. Acta. 939, -18), hydroxide [[(1,1,3,3-TETRAMETHYLBUTYL)cresoxy]ethoxy]ethyl]dimethylbenzylamine (hydroxide DEBDA) with an excess of phosphatidylcholine/cholesterol (Ballas et al. (1988) Biochim. Biophys. Acta 939, 8-18), a mixture of bromide of cetyltrimethylammonium (BECOMING)/DOPE (Pinnaduwage et al., (1989) Biochim. Biohys. Acta. 985, 33-37), lipophilic fluids glutamic acid (™AG) with DOPE, CTAB, DEBDA, bromide of deterimine (DDAB) and stearylamine mixed with phosphatidylethanolamine (Rose et al. (1991) Biotechnique 10, 520-525), DDAB/DOPE (TransfectACE, GIBCO BRL) and lipids carrying oligochaetes. Examples of agents amplifiers transfection, which increase the efficiency of transfection include, for example, DEAE-dextran, polybrene, peptide, destroying lysosome (Ohmori N.I. et al., Biochem. Biophys. Res. Commun. Jun. 27, 1997; 235(3):726-9), proteoglycans on the basis chondroitin, sulfated proteoglycans, polyethylenimine, polylysine (Pollard H. et al., J. Biol. Chem. 1998, 273(13):7507-11)that communicates with the integrin peptide CYGGRGDTP, linear dextran-containing nonshared, glycerin, cholesterolemia groups associated with the oligonucleotide at the 3'-end magnolioideae communication (R.L. Letsinger 1989, Proc. Natl. Acad. Sci., USA, 86:(17):6553-6), lysophosphatidic, lysophosphatidylcholine, lysophosphatidylethanolamine and 1-aeolosomatidae.

In some cases, it may be desirable to introduce a nucleic acid through an agent, which carries out a targeted delivery vector containing nucleic acid in the cell mi is Yeni. Such “target” molecules are antibodies that are specific for the membrane protein surface of the target cell, or a ligand for a receptor on the target cell. In the case of liposomes for targeted delivery and/or to facilitate the introduction into the cell can be used for proteins that bind to membrane proteins on the cell surface associated with endocytosis. Examples of such proteins are the capsid proteins and their fragments, target cell type-specific antibodies against proteins which undergo internalization in the bloodstream, and proteins that determine the intracellular localization and increase the half-life inside the cells. In other embodiments of the invention can be used mediated receptor endocytosis. Such methods are described, for example, Wu et al., 1987 or Wagner et al., 1990. Known methods of tagging genes and gene therapy protocols is available in the publication Anderson 1992. Cm. application WO 93/25673 and work cited therein. A more detailed description of the method of gene therapy can be found in the publication Friedmann, Science, 244:1275-1281 (1989); Anderson, Nature, supplement to vol. 392, no 6679, pp.25-30 (1998); Verma, Scientific American:64-84 (1990) and Miller, Nature, 357:455-460 (1992).

Screening methods

Effective treatment depends on identifying an effective agent that does not have any appreciable toxicity. Antibodies can be with minirovali on the binding affinity of known methods. So, for example, can be conducted analyses on mobility in the gel, Western blot analysis, competitive analysis with the use of radioactive labelling, co-fractionation by chromatography, co-precipitation, cross-linking, ELISA and the like described, for example, the manual Current Protocols in Molecular Biology (1999) John Wiley & Sons, N.Y., which in its entirety is introduced into the present description by reference.

For preliminary screening for antibodies that bind to the desired epitope of M-CSF (for example, which block the binding of the antibody RX1, (5H4, MS and/or MS with M-CSF), can be carried out routine analysis on cross-blocking, i.e., the analysis described in the publication Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed. Harlow &David Lane (1988). Can be also carried out routine tests on competitive binding, in which the unknown antibody oharakterizovat by its ability to inhibit the binding of M-CSF M-CSF-specific antibody according to the invention. This can be used intact M-CSF, its fragments or linear epitopes, such as the epitope represented by the amino acids 98-105 of M-CSF in figure 12, or amino acids 65-73 or 138-144 in figure 12 (corresponding to the epitope of M-CSF, recognizable by antibodies (5H4 or MS). Mapping of epitopes described in Champe et al., J. Biol.Chem., 270:1388-1394 (1995).

Also meant odevaetsa, these antibodies should be tested for their impact on osteoclastogenesis, and then introduced animals. Compounds that can be used for prevention or treatment of osteoporosis associated with cancer metastasis, can be skanirovaniya using different analyses. For example, the antagonist-the applicant may first be characterized in cell culture system to determine its ability to neutralize M-CSF induces osteoclastogenesis. Such a system may include joint cultivation of murine osteoblasts cranial vault and spleen cells (Suda et al., Modulation of osteoclast differentiation. Endocr. Rev. 13:6680, 1982; Martin & Udagawa, Trends Endocrinol. Metab. 9:6-12, 1998), joint cultivation of murine stromal cell lines (for example, MT-G2/PA6 and ST2) cells and mouse spleen (Udagawa et al., Endocrinology 125:1805 13, 1989) and the joint cultivation of ST2 cells and bone marrow cells, mononuclear peripheral blood cells or alveolar macrophages (Udagawa et al., Proc. Natl. Acad. Sci., USA, 87:7260 4, 1990; Sasaki et al., Cancer Res. 58:462 7, 1998; Mancino et al., J. Surg. Res. 100:18-24, 2001). In the absence of any antagonist M-CSF multinucleated cells obtained in this co-cultivation, meet the basic criteria of osteoclasts, such as tartrate-resistant ciclofosfamida activity (TRAP, a marker enzyme of osteoclasts), the presence of ka is ecitoninae receptor, p60C-STC and vitronektinove receptor, as well as the ability to form areas of bone resorption and detinova layer. The presence of an effective antagonist M-CSF leads to inhibition of the formation of these multi-core cell.

In addition to the above systems culturing the ability of anti-M-CSF antibodies candidate to inhibit osteoclastogenesis can be estimated in the system, not containing stromal cells or osteoblasts. M-CSF required for osteoclastogenesis, can be produced by culturing with metastatic cancer cells (for example, MDA 231) or in conditioned medium obtained from these cancer cells (Mancino et al., J. Surg. Res. 0:18-24, 2001), or by adding purified M-CSF.

The effectiveness of this antibody against M-CSF in the prevention or treatment of osteoporosis associated with cancer metastasis, can also be estimated in any animal model of bone metastases, known to specialists. Such model systems are systems which provide for direct injection of tumor cells into the bone marrow cavity (Ingall, Proc. Soc. Exp. Biol. Med., 117:819-22, 1964; Falasko, Clin. Orthop. 169:207, 1982), in the abdominal aorta of rats (Powles et al., Br. J. Cancer 28:31621, 1973), lateral tail vein or the left ventricle of mice (Auguello et al., Cancer Res. 48: 687681, 1988). In the absence of an effective antagonist M-CSF Osceola the ical bone metastases, formed after injection of tumor cells, can be detected using radiography (sections osteolytic bone lesions) or by using histological and immunohistochemical analysis (bones and soft tissues). Cm. Sasaki et al., Cancer Res. 55:3551 7, 1995; Yoneda et al., J. Clin. Invest. 99:2509 17, 1997. Clohisy & Ramnaraine, Orthop. Res. 16:660 6, 1998, Yin et al., J. Clin. Invest. 103:197 206, 1999. The presence of effective antibodies against M-CSF may contribute to the prevention of the formation of osteolytic bone metastases or inhibition of metastasis by reducing their number and/or size.

Anti-M-CSF antibodies according to the invention can also be used for prevention or treatment of cancer metastasis. The effectiveness of anti-M-CSF antibody candidate in the prevention or treatment of cancer metastasis can be evaluated using the model of invasion through the basement membrane of human amnion described in Filderman et al., Cancer Res. 52:36616, 1992. In addition, can also be used with any system of an animal model of metastatic cancer of various types. Such systems models are, but not limited to the system described in the publications Wenger et al., Clin. Exp. Metastasis 19:169 73, 2002; Yi et al., Cancer Res. 62:917 23, 2002; Tsutsumi et al., Cancer Lett. 169:77-85, 2001; Tsingotjidou et al., Anticancer Res. 21:971 8, 2001; Wakabayashi et al., Oncology 59:75 80, 2000; Culp & Kogerman, Front Biosci. 3:D67283, 1998; Runge et al., Invest Radiol. 32:212 7; top uchideshi et al., J. Surg. Oncol. 64:122 6, 1997; Ma et l., Invest Ophthalmol Vis. Sci. 37:2293 301, 1996; Kuruppu et al., J. Gastroenterol. Hepatol. 11:26 32, 1996. The presence of effective antibodies against M-CSF may contribute to the prevention of cancer metastasis or inhibition of metastasis by reducing their number and/or size.

Antitumor activity of a specific antibody against M-CSF, or a combination of antibodies against M-CSF can be evaluated byin vivousing a suitable animal model. So, for example, xenogenic models for malignant lymphoma, which enter the cells of the human lymphomas are animals with immunodeficiency, such as “Nude” mouse or a SCID mouse. The effectiveness of the antibodies can be predicted using tests that assess the inhibition of the formation of tumor regression of a tumor or metastasis, etc.

In one variation relating toin vitroanalysis, the present invention relates to a method comprising steps (a) contacting the immobilized M-CSF by antibody-candidate and (b) detecting binding of the antibody candidate with M-CSF. In an alternative embodiment of the invention the antibody candidate is subjected to immobilization and detects the binding of M-CSF. The immobilization is carried out by any method well known in the art, including covalent binding with the carrier, with the spheres or chromatographic resin, as well as not highly covalent is finnoe interaction, such as binding antibody or binding with streptavidin/Biotin, where the aforementioned immobilized connection includes bitenova molecule. Detection of binding can be performed (i) by using a radioactive label on neimmunizirovannah connection, (ii) using the fluorescent label on neimmunizirovannah connection, (iii) using antibodies immunospecific to neimmunizirovannah connection, (iv) using the tag on neimmunizirovannah connection, exciting the fluorescent medium is attached to the immobilized compound, as well as other methods that are well known and used in practice.

Antibodies that modulate (e.g., increase, reduce or inhibit) the activity or expression of M-CSF, can be identified by incubation of the proposed modulator with a cell expressing M-CSF, and determine the impact of this proposed modulator on the activity or the expression of M-CSF. The selectivity of the antibody that modulates the activity of the polypeptide or polynucleotide M-CSF, can be evaluated by comparing its effect on polypeptide or polynucleotide M-CSF with its impact on other related compounds. Selective modulators may be, for example, antibodies and other proteins, peptides or organic molecules, which is specificeski associated with the polypeptides of M-CSF or with nucleic acid, encodes a polypeptide of M-CSF. Modulators of the activity of M-CSF are therapeutically effective for the treatment of diseases and physiological conditions, the development of which is called the normal or aberrant activity of the polypeptide of M-CSF.

The present invention also deals with the analysis by high-throughput (HTS) to identify antibodies that interact with a polypeptide of M-CSF or inhibit its biological activity (i.e. inhibit the enzymatic activity, binding activity and so on). HTS assays allow effective screening large numbers of compounds. To study the interaction between the polypeptides of M-CSF and their binding partners are applied cellular HTS system. HTS-a assays were developed to identify the “best connection” (“hits”) or “substantial connections”with desirable properties, which may be included modifications to improve these desirable properties. Chemical modification of these “hits” or “substantial connection” is often based on an identifiable correlation structure/activity between “hits” and polypeptides of M-CSF.

In another aspect the present invention relates to methods of identifying antibodies that modulate (i.e. reduce) the activity of M-CSF, where pointed to by the second method involves contacting M-CSF with the antibody and determining the ability of this antibody to modify the activity of M-CSF. Activity in the presence of the test antibody is compared with the activity in the absence of the test antibody. If the activity in the sample containing the test antibody is less than the activity in the sample not containing the test antibody, the antibody will inhibit the activity of M-CSF.

For functional expression of recombinant polypeptides there are several heterologous systems, well known to specialists. Such systems are bacteria (Strosberg et al., Trends in Pharmacological Sciences (1992) 13:95-98), yeast (Pausch, Trends in Biotechnology (1997) 15:487-494), insect cells of several species (Vanden Broeck, Int. Rev. Cytology (1996) 164:189-268), cells of amphibians (Jayawickreme et al., Current Opinion in Biotechnology (1997) 8:629-634) and some mammalian cell lines (Cho, HEK293, COS, etc.; see Gerhardt et al., Eur. J. Pharmacology (1997) 334:1-23). These examples are not exclusive of other possible systems of cellular expression, including cell lines derived from nematodes (PCT application WO 98/37177).

In one embodiment of the invention, methods of screening for antibodies that modulates the activity of M-CSF, provide for the contacting of the test antibody with a polypeptide of M-CSF and analysis on the presence of complex specified antibody and M-CSF. In such assays, the ligand is typically labeled. After a suitable incubation, free ligand is separated from the ligands present in bound form, and if estvo free or unbound label is an indicator of the ability of this antibody to contact the polypeptide of M-CSF or M-CSFR.

In another embodiment of the invention uses a highly efficient screening for fragments of antibodies or CDR with appropriate affinity bind to the polypeptide of M-CSF. In short, large numbers of different small peptide test compounds are synthesized on a solid substrate. Then the peptide test antibody is subjected to contact with a polypeptide of M-CSF and washed. Related polypeptides M-CSF detected by methods well known in the art. Purified polypeptides according to the invention can also be applied directly to the plates for use in the above methods of search of drugs. In addition, no neutralizing antibodies can be used to capture the protein and its immobilization on a solid medium.

Combination therapy

To increase the effectiveness of treatment of cancer metastasis and/or osteoporosis associated with cancer metastasis, in the identification of more than one anti-M-CSF antibody that is effective in an animal model, it may also be preferred to use a mixture of two or more of these anti-M-CSF antibodies. Compositions containing one or more anti-M-CSF antibody, can be administered to the human or mammal suffering from a cancer metastasis and/or osteoporosis associated with such cancer metastasis, or euwema predisposition to these diseases. For simultaneous administration of two therapeutic agents is not necessary that such funds were introduced at the same time or the same way, provided that this period coincides with the period of time during which these funds have a therapeutic effect. This will involve simultaneous or sequential introduction, for example the introduction of various days or weeks.

Although therapy anti-M-CSF antibody can be used at all stages of cancer, however, such therapy may be particularly appropriate in case of advanced cancer or cancer metastasis. Combination antibody therapy with chemotherapy or radiation therapy may be preferred for patients who have not had chemotherapy, whereas antibody therapy may be indicated for patients who have undergone one or more cycles of chemotherapy. In addition, antibody therapy can also reduce the dose of concomitant chemotherapy, especially in patients who are not very well tolerate the toxic effects of chemotherapeutic agents.

The method according to the invention provides for the introduction of anti-M-CSF antibody, alone and in combination with other antibodies or with a “cocktail” of other antibodies. Such a “cocktail” of antibodies may have some advantages, PQS is LCU they contain antibodies acting in accordance with various effector mechanisms, or contain a combination of directly acting cytotoxic antibodies with antibodies, which are based on intrinsic immune effector functions. Such antibodies, taken in combination with each other, can have a synergistic therapeutic effect.

The combination of antibodies RX1 or its derivative, antibody Human Engineered™with other therapeutic means can have a significant effect on a patient mediated by osteoclasts disease and/or tumor growth or metastasis. For example, the antibody RX1 can be used for preparing medicines for treating a patient suffering from osteolytic disease, where treatment is specified drug is combined with treatment with antibody against RANKL, soluble RANKL receptor, other inhibitors of RANKL or bisphosphonates (e.g., Aredia; Zometa; Clodronate). Alternatively, an antibody against RANKL or a bisphosphonate can be used for preparing medicines for treating a patient suffering from osteolytic disease, where treatment is specified drug is combined with treatment with antibody RX1 or designed derived human antibodies RX1. This combination of mo is et to give a synergistic effect in the treatment of the patient. Antibody RX1 and another therapeutic agent does not have to be entered at the same time. RX1 or engineered human variant and another therapeutic agent can be introduced after 1 day, 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year or 2 years later.

The present invention also describes the use RX1 or its derivative Human Engineered™ in order to prepare medicines for the treatment of a patient suffering from osteolytic disease, where the specified drug is used to treat a patient who has already passed a course of treatment with antibody against RANKL or with bisphosphonates. The term “pretreatment” means that the patient is already undergoing treatment for 2 years, 1 year, 6 months, 3 months, 2 months, 1 month, 2 weeks, 1 week, or at least one day prior to treatment with the antibody RX1 or its variant Human Engineered™.

Antibody RX1 or its variants for Human Engineered™ can be used in combination with other anticancer therapies. For example, the antibody RX1 or engineered human variants can be used for preparing medicines for treating a patient suffering from cancer, where said treatment drug is combined with other treatment therapeutic means and/or with other treatment methods, including, but not limited to, the treatment of various chemotherapeutic agents, androgen blockers, and immune modulators (e.g., IL-2, GM-CSF, SLC) or bisphosphonates (e.g., Aredia; Zometa; Clodronate); surgery; radiation therapy; chemotherapy cytotoxic means; hormonal therapy (e.g. tamoxifen; protivoallergennoy therapy, antibody therapy (e.g., neutralizing antibodies against RANKL/RANK; neutralizing antibody against PTHrP, an antibody against Her2, an antibody against CD20, an antibody against CD40, antibody against CD22, anti-VEGF antibody, antibody against IGFR-1, an antibody against EphA2, antibody against NAAS, antibody against ™EFF2 and antibody against CAIX); therapy therapeutic protein (e.g., soluble RANKL receptor; OPG and PDGF inhibitors and DFID); the low-molecular therapy medicinal product (for example, an inhibitor of Src-kinase); treatment of kinase inhibitors of receptors of growth factors or inhibitors of RANKL; therapy oligonucleotides (e.g., antisense sequence of RANKL, RANK or PTHrP); gene therapy (for example, inhibitors of RANKL or RANK); therapy with peptides (for example, mottainai RANKL), and described here, proteins, peptides, and small molecules.

Antibody RX1 and its variants for Human Engineered™ can be used in order to manufacture the drug is tion tools for treatment of patients which took place course of treatment of the above-mentioned therapeutic agents.

The term “cytotoxic agent” means a substance that inhibits or prevents the function of cells and/or causes destruction of these cells. This term shall include radioactive isotopes (e.g.,131I125I90Y and186Re), chemotherapeutic agents, and toxins such as enzymatically active toxins derived from bacteria, fungi, plants or animals, or synthetic toxins or fragments thereof. The term “altoconsky agent” means a substance that does not inhibits or prevents the function of cells and/or causes destruction of cells. The term “altoconsky agent may also include an agent which, after activation, can be cytotoxic. Netforensics.com agents can be spheres, liposomes, matrices or particles (see, for example, the publication of patent applications U.S. 2003/0028071 and 2003/0032995 that are entered into the present description by reference). Such agents can be conjugated, linked, attached or associated with the antibody according to the invention.

The term “anti-cancer chemotherapeutic agents” include, but are not limited to, alkylating agents, such as carboplatin and cisplatin; alkylating agents containing nitrogen analogue gorcicnogo the gas, nitrosoanatabine alkylating agents, such as carmustine (BCNU); antimetabolites, such as methotrexate; polynovo acid; antimetabolites on the basis of purine analogues, mercaptopurine; antimetabolites on the basis of pyrimidine analogues such as fluorouracil (5-FU) and gemcitabine (Gemzar®); hormonal antineoplastic agents such as goserelin, leuprolide and tamoxifen; natural antineoplastic agents such as aldeslakin, interleukin-2, docetaxel, etoposide (VP-16), interferon-alpha, paclitaxel (Taxol®) and tretinoin (ATRA); natural antineoplastic antibiotics such as bleomycin, dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycin, including mitomycin C; and vinyltoluene natural antineoplastic agents such as vinblastine, vincristine, vindesine; hydroxyurea; Eagleton, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, anti-platelet factors, cyclophosphamide (Cytoxin®), sizofiran, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatin, analogs of dolastatin, such as auristatin, CPT-11 (irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin, karminomitsin, espiramicina (see, for example, a patent With The And No. 4675187), neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin (Ubenimex®), interferon-β, mepitiostane, mitobronitol, melphalan, laminine peptides, lentinan, an extract of Coriolus versicolor, tegafur/uracil, estramustin (estrogen/mechlorethamine).

In addition, other agents used as therapeutic agents for the treatment of patients with cancer are EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons α, β and γ; hormones, such as luteinizing hormone-releasing-hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors such as transforming growth factor-β (TGF-β), the fibroblast growth factor (FGF), growth factors, nerve tissue (NGF); growth hormone-releasing factor, growth hormone (GHRF), epidermal growth factor (EGF), homologous factor that activates the fibroblast growth factor (FGFHF); a growth factor for hepatocytes (HGF) and insulin-like growth factor (IGF); tumor necrosis factor-α and-β (TNF-α and-β); inhibiting invasion factor-2 (IIF-2); proteins of bone morphogenesis 1-7 (BMP 1-7); somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD); complement factors; angiogenic factors; antigenic agents; and prodrugs.

The term “prodrug” means predestin the IR or derived pharmaceutically active substances, which compared to the parent drug has less or no cytotoxicity has netforecast in relation to tumor cells and are capable of enzymatically or hydrolytically activated or to become more active Mature form. See, for example, Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Belfast (1986) and Stella et al. “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, and prodrugs of the modified D-amino acid, glycosylated prodrugs, β-lactam-containing prodrugs; prodrugs, optionally containing substituted phenoxyacetamide, or prodrug, optionally containing substituted phenylacetamide; 5-fortitudinous and other 5-ptoluidine prodrugs that can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivationally obtaining proletarienne forms for use in the present invention include, but are not limited to, chemotherapeutic agents described above.

Introduction and getting

Anti-M-CSF antibody used for the practical implementation of the method according to the invention, can be prepared as pharmaceutical compositions containing media suitable for implementing the delivery of needed medicines. Suitable carriers are any substances which, when combined with anti-M-CSF antibodies retain antitumor function of antibodies and do not react with the immune system of the individual. Examples include, but are not limited to, any of a variety of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, etc. Can be used in a variety of aqueous media such as water, buffered water, and 0.4% saline, 0.3% glycine and the like, and these carriers can include other proteins, which increases stability, such as albumin, lipoprotein, globulin, etc. subjected to weak chemical modification or the like

Therapeutic preparations of antibodies prepared by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1980)) to store them in the form liofilizovannyh drugs or aqueous solutions. Applicable to the haunted media, fillers or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chloride hexadecane; benzalkonium chloride; phenyl, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (containing less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; hepatoblastoma agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; complexes with metals (for example, complexes of Zn-protein); and/or nonionic surfactants such as tween™, Pluronic™ or polyethylene glycol.

This drug may also contain more than one active connection shown for this therapy, preferably the compound is complementary activity, which does not have a negative impact on another connection. For example, it may be desirable to use immunosuppressant. Such molecules are suitable for use in combination with other agents in amounts effective to achieve the desired effect.

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

Drugs intended forin vivothe introduction must be sterile. This may be readily accomplished by filtration through sterile filtration membranes.

The antibody is administered by any suitable method, including parenteral, subcutaneous, intraperitoneal, intra-lungs and intranasal introduction, and if necessary, it can be entered locally or in the affected area. Parenteral routes of administration include intravenous, intraarterial, intraperitoneal, nutramigen is e, cutaneous or subcutaneous administration. In addition, the antibody can be entered by periodic infusion, particularly with declining doses of the antibody. Preferably, this dose was introduced by injection, and most preferably by intravenous or subcutaneous injections, depending on whether this introduction short-term or long-term. Consider other methods of administration, including local injection, and in particular transcutaneous introduction, introduction through mucosa, rectal administration, oral administration or introduction to a specific area, for example, through a catheter placed near the desired site.

Compositions according to the invention can be prepared in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions. Compositions according to the invention can be designed for various routes of administration, such as oral administration, intranasal, rectal administration, subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection. The following dosage forms are given only for illustrative purposes and should not be construed as limiting the present invention.

Acceptable solid dosage the diversified forms for oral, transbukkalno and sublingual injection are powders, suspensions, granules, tablets, pills, capsules, gel capsules and minicapsule. These drugs can be obtained, for example, by mixing one or more compounds according to the invention and pharmaceutically acceptable salts or tautomers, and at least one additive, such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, ▫ maltitol, dextran, starch, agar, alginates, chitina, chitosans, pectins, tragacanth gum, Arabic gum, gelatin, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. To facilitate the introduction of oral forms may contain, but not necessarily, and other ingredients such as inactive diluent or oiling agents, such as magnesium stearate, preservatives such as paraben or sorbic acid, antioxidants such as ascorbic acid, tocopherol or cysteine, dezintegriruetsja agents, binders, thickeners, buffers, sweeteners, perfuming or flavoring agents. Tablets or pills may also be suitable material from known materials.

Liquid preparations for oral administration can be prepared in the form of a pharmaceutically acceptable emulsions, syrups, elixir is, suspensions and solutions, which may contain inactive diluent, such as water. Pharmaceutical compositions and medicaments can be obtained in the form of liquid suspensions or solutions using a sterile liquid, such as, but not limited to, oil, water, alcohol, and combinations thereof. In the composition for oral or parenteral administration can be added pharmaceutically acceptable surfactants, suspendresume agents and emulsifying agents.

As mentioned above, the suspension may include oil. Such oils include, but are not limited to, peanut oil, sesame oil, cotton seed, corn oil and olive oil. Drug type suspensions may also contain esters of fatty acids, such as etiloleat, isopropylmyristate, glycerides of fatty acids and acetylated glycerides of fatty acids. Drugs type suspensions may also contain alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecylamine alcohol, glycerin, and propylene glycol. Such suspensions may also contain esters, such as, but not limited to, polyethylene glycol, a hydrocarbon oil, such as mineral oil and vaseline oil, and water.

Pharmaceutical drugs and medicines is for intranasal can be prepared in the form of a spray or aerosol, containing the corresponding(s) solvent(s), and optionally, other compounds, such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations thereof. Propellant for aerosol compositions may include compressed air, nitrogen, carbon dioxide or hydrocarbon discoparade solvent.

Forms for administration by injection usually consist of aqueous suspensions or oil suspensions, which can be obtained using suitable dispersing or wetting agent and a suspending agent. Dosage forms for injection can be obtained in the liquid phase or in the form of a suspension prepared using solvent or diluent. Acceptable solvents or carriers are sterilized water, ringer's solution or isotonic aqueous saline solution. Alternatively, as solvents or suspendida agents can be used sterile oil. Preferred are non-volatile oils or fatty acids, including natural or synthetic oils, fatty acids, mono-, di - or triglycerides.

Pharmaceutical drugs and medicines for injection can be prepared in the form then SKOV, suitable for subsequent breeding respective solution described above. Examples of such powders include, but are not limited to, powders, dried by freezing, vortex mixing or spraying, amorphous powders, granules, precipitates or particles. Preparations for injection can, but not necessarily, contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations thereof.

Pharmaceutical drugs and medicines for rectal injection can be prepared in the form of suppositories, ointments, enemas, tablets or creams for the release of this compound in the small intestine, sigmoid flexure of the colon and/or rectum. Suppositories for rectal injection is prepared by mixing one or more compounds according to the invention or pharmaceutically acceptable salts or tautomers of these compounds with pharmaceutically acceptable excipients, for example, cocoa butter or poliatilenglikole that are in solid phase at normal temperatures of storage, and in the liquid phase at a temperature suitable for the release of drugs inside the body, such as in the rectum. For the preparation of compositions of the type of soft gelatin capsules or suppositories can b the th also used oil. For the preparation of type suspensions can be used water, saline, aqueous dextrose, solutions and other related sugars and glycerine, and such suspension may optionally contain suspendresume agents, such as pectins, carbomer, methylcellulose, hydroxypropylcellulose or carboxymethylcellulose, as well as buffers and preservatives.

Can be obtained drugs for prolonged release. Suitable examples of drugs for prolonged release can serve as semi-permeable matrices of solid hydrophobic polymers containing the antibody, where these matrices can be obtained in the form of molded articles, e.g. films, or microcapsules. Examples of matrices for drugs extended release are polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or polyvinyl alcohol, polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-biodegradable copolymer of ethylene and vinyl acetate, degradable copolymers of lactic acid and glycolic acid, such as the Lupron Depot™ (microspheres for injection, consisting of a copolymer of lactic acid and glycolic acid and acetate leuprolide), and poly-D-(-)3-hydroxipropionic acid. Polymers, such as copolymers of ethylene and vinyl acetate and SOP is the materials of lactic acid and glycolic acid, capable of release of molecules for over 100 days, whereas hydrogels release proteins for shorter time periods. Although encapsulated antibodies present in the body for a long period of time, however, they can denaturirate or aggregated in a humid environment at 37°C, which leads to the loss of their biological activity and possible changes in their immunogenicity. To stabilize these antibodies, depending on the specific mechanism of action, can be a rational strategy. For example, if the aggregation mechanism is the formation of intermolecular S-S-links through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization of the acid solution, regulating the moisture content, using appropriate additives, and developing appropriate formulations of polymer matrix.

In accordance with the present invention can be obtained fast compositions, compositions immediate release compositions with prolonged activity or prolonged release composition described in this application. Thus, can also be prepared in pharmaceutical compositions with controlled release or slow release drug among the STV.

Compositions according to the invention can also be included, for example, micelles or liposomes or encapsulated forms and can be entered in the form with a slow release that ensures their long-term storage and/or prolonged delivery. Therefore, these pharmaceutical drugs and medicines can be compressed into pellets or cylindrical shape and implanted intramuscularly or subcutaneously as a “depot”-preparation for injections or as implants, such as stents. In these grafts can be used known inert materials, such as silicones or biodegradable polymers.

In addition to the above representative standardized dosage forms of the present invention are considered pharmaceutically acceptable excipients or carriers, which are essentially known to specialists in this field. Such fillers and carriers are described, for example, in Remingtons Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), which is incorporated into the present description by reference.

The specific dose of the medication can be adjusted depending on the severity of the disease, age, body weight, General state of health, sex and diet, and the intervals between injection and dose the method of administration, rate of excretion of drugs and combinations used medicines. Any of the above dosage forms containing effective amounts of the medicinal product, can be obtained by routine experimentation, and therefore they are included in the scope of the present invention.

Anti-M-CSF antibodies are used as therapeutics for the treatment of metastatic cancer or osteoporosis associated with cancer metastasis, usually get, essentially, in free form, i.e. not containing other natural antibodies or other biological molecules. Preferred anti-M-CSF antibodies also have minimal toxicity when administered to a mammal suffering from a cancer metastasis and/or bone loss associated with cancer metastasis, or having a predisposition to such diseases.

Compositions according to the invention can be sterilized by standard well known methods. The obtained solutions can be packaged for immediate use or filtered under aseptic conditions and liofilizovane, and such liofilizovannye drugs before their introduction, is mixed with a sterile solvent. These compositions may contain pharmaceutically acceptable auxiliary additives, the need for the s to create conditions close to physiological conditions, such as agents for the adjustment of pH or tabularasa agents, agents, giving isotonicity and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and stabilizers (for example, 20% maltose and the like).

Anti-M-CSF antibodies according to the invention can also be entered using liposomes, which are small vesicles composed of lipids of different types of phospholipids and/or surfactant that can be used for delivery of a drug (for example, described here antibody and, optionally, a chemotherapeutic agent). Liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, and can serve as vesicles for delivery of anti-M-CSF antibodies in a particular tissue, as well as to increase the half-life of the composition. Various methods of obtaining liposomes are described, for example, in U.S. patent No. 4837028 and 5019369 that are entered into the present description by reference.

Liposomes containing the antibody can be obtained by known methods, such as methods described in the publications Thstein et al., Proc. Natl. Acad. Sci., USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci., USA, 77: 4030 (1980); and U.S. patent No. 4485045 and 4544545. Liposomes with prolonged time of a compass the AI in blood is described in U.S. patent No. 5013556. Especially effective liposomes can be prepared by evaporation with reversed phase, obtaining a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-Feh). Liposomes extruded through filters with defined pore size, resulting in a gain liposomes of the desired diameter. Fab'-fragments of the antibodies according to the invention can be conjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982), by reaction of disulfide exchange. Such liposome may contain, but not necessarily, a chemotherapeutic agent (such as doxorubicin) [see, for example, Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989)].

The concentration of anti-M-CSF antibody in the composition can vary widely, i.e. it can be approximately less than 10%, usually at least about 25%and about 75%, or 90% by weight of the composition, and can be adjusted mainly by the volume of fluid, viscosity, etc. in accordance with specifically selected by way of introduction. Existing methods of obtaining compositions for oral, local and parenteral administration are well known or obvious to specialists and are described, for example, the manual Remington's Pharmaceutical Sciences, Mack Publishing. Co., Easton, PA (1995), which is incorporated into the present description by with Alki.

Determination of the effective amount of the composition according to the invention for the treatment of cancer metastasis and/or osteoporosis associated with cancer metastasis, the patient can be carried out standard empirical methods well known in the art. For example,in vivoneutralizing activity of serum taken from the patient, which was introduced dose of anti-M-CSF antibodies, can be evaluated through analysis, which may be definedin vitrothe ability of serum to block M-CSF-induced proliferation and viability of mouse monocytes (CD11b+cells, i.e. subpopulations CD11-cells, which Express high levels of the receptor for M-CSF), as described by Cenci et al., J.Clin. Invest. 1055: 1279-87, 2000.

Compositions according to the invention is administered to a mammal, which already suffers from metastatic cancer or osteoporosis associated with cancer metastasis, or has a predisposition to such diseases, in a quantity sufficient to prevent or at least to stop the development of cancer metastases or osteoporosis associated with cancer metastasis. The amount needed to achieve these goals, called “therapeutically effective amount”. The effective amount of anti-M-CSF antibodies may vary and depend on the severity of illness is Denmark, on body weight and General health of the patient being treated, but generally, such amounts are in the range of about 1.0 μg/kg to 100 mg/kg of body weight, or from about 10 μg/kg to 30 mg/kg, but the most commonly used dose of about 1.0 μg/kg to 10 mg/kg or from about 1 μg/kg to 10 mg/kg, for example, the initial dose of the antibody candidate, which can be administered to the patient once or several times, or by continuous infusion, is from about 10 μg/kg to 5 mg/kg, or 30 mg/kg to 1 mg/kg, the dose can be injected daily or every other day, weekly, or less often, depending on the susceptibility of the disease or tolerability of this dose by the patient. Thus, it may be necessary to maintain the level of doses over a longer period of time, such as 4, 5, 6, 7, 8, 10 or 12 weeks or more until, until you obtain the desired attenuation of the symptoms, and if necessary, the dose can be adjusted. The advantage of this therapy is that it can easily be monitored using standard methods and analysis.

Single or multiple introduction of compositions can be made using the doses and mode of administration, selected by the attending physician. The appropriate dose of antibody used for prevention and or treatment of disease, will depend on the type of disease being treated and defined above, the severity and course of the disease, regardless of whether it is introduced antibody in preventive or therapeutic purposes, previous therapy, the clinical history of the patient and his susceptibility to this antibody and from doctor's appointments. The specified antibody is suitably administered to the patient within one course of treatment or within a few courses of treatment.

In any event, the composition should contain a certain amount of anti-M-CSF antibodies, providing for the delivery of this antibody in a period of time sufficient to effectively prevent or minimize the severity metastasises cancer and/or osteoporosis associated with cancer metastasis. Compositions according to the invention can be introduced separately or as a combined therapy in combination with other known therapeutic tool commonly used by specialists for the treatment of cancer metastasis and/or osteoporosis associated with cancer metastasis.

Composition antibodies can be prepared, divided into doses and implemented in accordance with well-developed standard medical practice. To implement these objectives, taking into account such factors as specific disorders is, being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of this disorder, the site to which you want the introduction of drugs, route of administration, the scheme of administration and other factors known to doctors. A therapeutically effective amount of injected antibodies can be determined taking into account the above factors and is the minimum amount necessary to prevent, improve dynamics or treatment of M-CSF-mediated disease, condition or disorder, and in particular for the destruction of cancer cells, and more particularly, for the treatment of metastatic tumors. The amount should preferably be lower than the number that has a toxic effect on the host tells the owner or a greater susceptibility to infections.

This antibody can be, but not necessarily, prepared in combination with one or more substances commonly used for the prevention or treatment considered disorders. For example, in cancer of the specified antibody can be introduced in combination with a chemotherapeutic agent or in the ADEPT system described above. The effective amount of such compounds depends on the number of antibodies at Westwego in this composition, the type of the disease, condition or disorder or applied treatments, and other factors discussed above. These substances are usually used in the same doses and in accordance with the same techniques that were described above, or about dose comprising from 1 to 99% of the commonly used doses.

In another embodiment, the present invention relates to industrial product containing substances used for the treatment of diseases, disorders or conditions described above, including the treatment of cancer. This industrial product contains the container and the liner. Suitable containers include, for example, vials, tubes, syringes, and test tubes. These containers can be made of various materials such as glass or plastic. The specified container contains a composition that is effective for the treatment of this condition, and may have a sterile access (for example, the container may be a bag of intravenous solution or vessel with stopper, protegemos needle for subcutaneous injection). The active agent in the specified composition is an antibody according to the invention. On the label affixed to the container, or in a statement attached to this container indicates that the composition is used for treatment of a specific illness who evania. Specified industrial product may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, ringer's solution and dextrose. In addition, it may include other materials necessary for commercial production and for the consumer, including other buffers, diluents, filters, needles, syringes and inserts in the package containing instructions for use.

Immunotherapy

Anti-M-CSF antibody, used to treat patients suffering from cancer, are antibodies that are able to initiate a strong immune response against the tumor, and antibodies capable of regulating cytotoxicity. In line with this, the anti-M-CSF antibodies may cause lysis of tumor cells by mechanisms of complement-mediated or antibody-dependent cellular cytotoxicity (ADCC), and for the implementation of these mechanisms requires the interaction of intact Fc portion of immunoglobulin molecules to sites of Fc-receptor of effector cells or proteins of the complement. In addition, the anti-M-CSF antibodies that exert a direct biological effect on tumor growth, can be used to implement the present invention. Possible mechanisms, which can be direct functions such cytotoxic antibodies is, are inhibition of cell growth, modulation of cell differentiation, modulation profiles of factors of tumor angiogenesis and induction of apoptosis. The mechanism by which a particular anti-M-CSF antibody exerts an anti-tumor effect can be determined using any of a number ofin vitroassays designed to determine ADCC, ADMMC, complement-mediated cell lysis, etc. and, in fact, known to specialists.

In one embodiment of the invention immunotherapy is performed with the use of antibodies that have a higher affinity to the membrane-bound form of M-CSF (M-CSFα)than the selected forms of M-CSF. So, for example, can be obtained antibodies that specifically bind to the site of cleavage of M-CSFα or at a nearby site, or with that part of M-CSFα, adjacent to the membrane. Such antibodies can also effectively inhibit the cleavage and release of soluble active part of M-CSFα.

These anti-M-CSF antibodies may be entered in their “naked” or unconjugated form, or they can be conjugated with therapeutic agents. In one embodiment of the invention the anti-M-CSF antibodies conjugated with radio-sensitizing agents. Used herein, the term “radiosensibility” means a molecule, and preferably a molecule with low molecular weight, which is administered to animals in therapeutically effective amounts to increase susceptibility radiosensibility cells of these animals to electromagnetic radiation and/or to increase the susceptibility of these animals to the treatment of diseases, by using electromagnetic radiation. Diseases that can be treated by electromagnetic radiation, are neoplastic diseases, benign and malignant tumors and cancer cells.

Used herein, the terms “electromagnetic radiation” and “radiation” includes, but is not limited to, radiation in the wave length range from 10-20up to 100 meters. In preferred embodiments of the present invention is applied gamma radiation (10-20-10-13m), x-ray radiation (10-12-10-9m), ultraviolet radiation (10-400 nm), radiation in the visible light range (400 nm-700 nm), infrared radiation (700 nm to 1.0 mm) and microwave radiation (1 mm to 30 cm).

It is known that the radio sensibilizators increase the sensitivity of cancer cells to the toxic effects of electromagnetic radiation. Currently, the radio sensibilizators activated electromagnetic x-rays are used in many treatment regimens for cancer. Examples of activated x-ray irradiation is receiving the radio sensibilizators are but not limited to, metronidazole, misonidazole, datetimeoriginal, pilonidal, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB6145, nicotinamide, 5-bromosuccinimide (BUdR), 5-iodosuccinimide (IUdR), bromosuccinimide, ftordezoksiuridin (FUdR), hydroxyurea, cisplatin and therapeutically effective analogs and derivatives.

Photodynamic therapy (PDT) of cancer involves the irradiation of visible light, where as activator of visible light is used sensitizing agent. Examples of photodynamic radio sensibilizators are, but are not limited to, hematoporphyrin derivatives, Photofrin(r), derivatives of benzoporphyrin, NPe6, tin-containing Ethiopian (SnET2), foobarbaz-α, bacteriochlorophyll-α, naphthalocyanines, phthalocyanines, zinc-containing phthalocyanine and therapeutically effective analogs and derivatives.

In another embodiment of the invention, the specified antibody can be conjugated with a receptor (such as streptavidin) for the purposes of its use prior to delivery to the tumor, where the specified conjugate antibody-receptor” is administered to the patient, after which the unbound conjugate is removed from the blood by the action of the cleansing agent and then injected ligand (e.g., avidin)that is conjugated with a cytotoxic agent (e.g. a radionuclide).

The present invention is also relates to the above described antibodies in detektirano labeled form. Antibodies can be detektirano marked with the use of radioisotopes, affinity labels (such as Biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase and the like), fluorescent, luminescent or bioluminescent labels (such as FITZ or rhodamine, and the like), paramagnetic atoms, etc. Procedures for the exercise of such labeling are well known in the art, see, for example, L.A. Sternberg et al., J. Histochem. Cytochem. 18:315 (1970); Bayer E.A. et al., Meth. Enzym. 62:308 (1979); Engval E. et al., Immunol. 109:129 (1972); J.W. Goding, J. Immunol. Meth. 13:215 (1976)).

The term “label” means detective connection or detektiruya composition, directly or indirectly conjugated with the antibody. This label itself may be detectable (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, it can catalyze chemical modification of the substrate detected compounds or compositions. Alternatively, the label itself can be netdetective, but can be a item associated with other detektivami agent (e.g., epitope tag or one of a pair of binding partners, such as Biotin-avidin, and the like). Thus, the antibody may contain a label or detected agent, which facilitates the selection of the antibodies, and methods of identifying antibodies according to the image is the shadow include the extraction of M-CSF/antibodies through interaction with the label or with detektivami agent.

Representative therapeutic immunoconjugate described here contain antibody conjugated with a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments), or a radioactive isotope (i.e radioactive conjugate). Hybrid proteins described in more detail below.

Getting immunoconjugates described in U.S. patent No. 6306393. Immunoconjugate can be obtained by indirect conjugation of a therapeutic agent component is an antibody. General methods described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer. 46:1101-1106 (1990) and Shih et al., in U.S. patent No. 5057313. This General method involves reacting the component antibodies having an oxidized carbohydrate portion with a polymer carrier, which has at least one free functional amino group, and attached to various substances such as a drug, toxin, chelate forming agent, boric component or another medication. This interaction is first leads to the formation of the communication Chippewa base (imine), which can be stabilized by restoring to the secondary amine with the formation of the final conjugate.

The polymer carrier is predpochtitelno is amylodextrin or polypeptide, having at least 50 amino acid residues, although can also be used and other essentially equivalent to the polymer carrier. In order to facilitate the introduction and delivery of medicines for effective treatment it is preferable that the end immunoconjugate was dissolved in an aqueous solution, such as serum of a mammal. For example, solubilizing functional group on the polymer carrier will improve the solubility of the final immunoconjugate in serum. In particular, a preferred carrier is amylodextrin.

The procedure for obtaining immunoconjugate with amylodextrin carrier usually start with dextranomer polymer, and preferably dextran having an average molecular weight of about 10000-100000. This acid is subjected to the reaction of interaction with the oxidant in order to implement the controlled oxidation of part of its carbohydrate rings for the formation of aldehyde groups. This oxidation is usually carried out using glycolytic chemical reagents, such as NaIO4in accordance with standard procedures.

Then the oxidized dextran is subjected to interaction with polyamines, preferably a diamine, and more preferably mono - or polyhydroxybenzenes. Suitable amines are Ethylenediamine, Propylenediamine Il is similar polyethyleneamine, Diethylenetriamine or similar polyamine, 1,3-diamino-2-hydroxypropane or other similar gidroksilirovanii diamines or polyamine etc. To ensure essentially complete conversion of the aldehyde functional groups in the group of Schiff bases using an excess of amine relative to the aldehyde groups of the dextran.

For remediation stabilization obtained intermediate Chippewa base using a reducing agent such as NaBH4, NaBH3CN or so Obtained adduct can be purified by passing it through a column of standard sizes for removal of cross-linking of dextrans.

For introduction of functional amino groups can also be applied to other standard methods of derivatization, for example the reaction of interaction with bromine cyan, and then with a diamine.

After that amylodextrin subjected to the reaction of interaction with the input derived from a particular drug, toxin, chelat forming agent, an immunomodulator, boric component or other therapeutic tool in the activated form, preferably with carboxyl-activated derivative, obtained in a standard way, for example, using dicyclohexylcarbodiimide (DCC) or its water-soluble variant, resulting in a gain staging add the CT scan.

Alternatively, polypeptide toxins, such as antiviral protein Phytolacca or A-chain of ricin and the like, can be attached to amylodextrin by glutaraldehyde condensation or by a reaction between the activated carboxyl groups on the protein with amines on amylodextrin.

Hepatoblastoma agents for radioactive metals or amplifiers magnetic resonance is well known to specialists. Typical agents are derived etilendiamintetrauksusnoy acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Usually, these hepatoblastoma agents have on their side chain groups through which such chelate forming agent can join the media. Such groups are, for example, benzylisothiocyanate whereby DTPA or EDTA can be attached to the amino group of the carrier. Alternatively, the carboxyl group or amino group on chelat forming agent can be attached to the carrier via activation or derivatization and subsequent merger in accordance with methods well known to specialists.

Boric components, such as carborane, can be attached to components-antibodies by standard methods. For example, carborane can be obtained using carboxyl functional groups, n is held on the side chains, by methods well known in the art. Joining such carboranes to the media, such as amylodextrin, can be achieved by activation of the carboxyl groups carboranes and condensation with amines to form the intermediate conjugate. Then these intermediate conjugates attached to components-antibodies with obtaining therapeutically acceptable immunoconjugate the method described below.

Instead of amylodextrin can be used polypeptide carrier, however, such a polypeptide carrier must have at least 50 amino acid residues in the chain, and preferably 100-5000 amino acid residues. At least some of the amino acid residues should be lysine residues, either glutamate or aspartate residues. To attach a drug, toxin, immunomodulator, chelat forming agent, boric component or other therapeutic agents commonly used lateral amines lysine residues and the side carboxylates glutamine and aspartate. Examples of polypeptide carriers suitable for imparting loaded media and immunoconjugate desired solubility, are polylysin, polyglutamine acid, poliasparaginovaya acid, and their copolymers and mixed polymers of these amino acids and the like, for example the EP Surinov.

Conjugation of the intermediate conjugate with the component-specific antibody is carried out by oxidation of the carbohydrate part of the component-antibodies and interaction of CARBONYLS derived aldehyde (or ketone) with the amino groups remaining in the media after joining drug, toxin, chelat forming agent, immunomodulator, boric component or another therapeutic agent. Alternatively, the intermediate conjugate can be attached to the oxidized component of the antibody by means of amine groups, which were introduced in specified intermediate conjugate after joining a therapeutic agent. The oxidation is usually carried out either chemically, for example using NaIO4or other glikoliticaskie reagent or enzyme, for example, with the use of neuraminidase and galactosidase. In the case of aminotetrazole media usually not all amines amylodextrin used to bind to a therapeutic agent. Other amines of amylodextrin are condensed with the oxidized component of the antibody with the formation of products of joining Chippewa grounds, which are then subjected to the reaction of reductive stabilization, usually in the presence of borohydride reductant.

A similar procedure is performed to obtain the other immunoconjugates according to the invention. Loaded polypeptide carriers preferably have available lysine residues for condensation with the oxidized carbohydrate component antibodies. The carboxy polypeptide on the media can be, if necessary, converted into amines, for example, through DCC activation and interaction with an excess of diamine.

Received immunoconjugate purified by standard methods such as size-exclusion chromatography on sephacryl S-300 or affinity chromatography using one or more epitopes CD84Hy.

Alternatively, immunoconjugate can be obtained by direct conjugation of the component antibodies with therapeutic tool. The General procedure is similar to the indirect method of conjugation, except that therapeutic agent is directly attached to the oxidized component of the antibody.

It should be noted that as described here, hepatoblastoma agents can be replaced with other therapeutic means. The person skilled in the art can develop such schemes conjugation without undue experimentation.

As an illustration, it may be noted that therapeutic agent can be attached to the hinge region of the restored component-antibodies through the formation of disulfide bonds. That is, for example, peptides tetanus toxoid can be constructed with a single cysteine residue, which is used to attach the peptide to the component-antibody. Alternatively, such peptides can be attached to the specified component to the antibody using heterobifunctional cross-linking of the linker, such as N-succinyl-3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). Common methods such conjugation are well known in the art. See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods,” in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995).

Conjugates of the antibody and cytotoxic agent is obtained using a variety of bifunctional binding protein agents, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-azido-compounds (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis(p-disoriented)Ethylenediamine), diisocyanates (such castaway-2,6-diisocyanate), and active bis-substituted compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, the immunotoxin ricin can be obtained, as described in the publication Vitetta et al., Science 238:1098 (1987). Representative chelat forming agent for conjugation of the radionuclide to the antibody is 1-isothiocyanatobenzene-3-methyldiethanolamine-acetic acid labeled with carbon-14 (MX-DTPA) (see, for example, WO 94/11026).

As mentioned above, the carbohydrate part of the Fc region of the antibody can be used for conjugation to a therapeutic agent. However, the Fc region may not exist, if the component antibodies immunoconjugate is a fragment of the antibody. However, you can enter the carbohydrate part of the variable region of the light chain of the antibody or antibody fragment. See, for example, Leung et al., J. Immunol. 154:5919 (1995); Hansen et al., U.S. patent No. 5443953. Then designed the carbohydrate part can be used to join a therapeutic tool.

In addition, the person skilled in the art is aware of the various possible methods of conjugation. For example, the carbohydrate part may be used for attaching polyethylene glycol to extend the half-life of intact antibodies or antigennegative fragment in the blood, lymph or other extracellular fluids. In addition, can be constructed “divalent immunoconjugate” by recognize the value of therapeutic agents to the carbohydrate portion and a free sulfhydryl group. This free sulfhydryl group may be present in the hinge region of the component antibodies.

Hybrid proteins with anti-M-CSF antibodies

In the present invention discusses the use of hybrid proteins containing one or more molecules of anti-M-CSF antibody and a molecule immunomodulator or toxin. Methods of producing hybrid proteins antibodies are well known in the art. See, for example, U.S. patent No. 6306393. Hybrid proteins with antibody-containing molecule interleukin-2, described in Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Natl. Acad. Sci., USA, 93:7826 (1996), Hank et al., Clin. Cancer. Res. 2:1951 (1996) and Hu et al., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum. Antibodies Hybridomas 6:129 (1995) described a hybrid protein comprising F(ab')2-fragment and the molecule tumor necrosis factor-alpha.

Methods of producing hybrid proteins “antibody-toxin”, in which recombinant molecule contains one or more components of the antibody and the toxin or chemotherapeutic agent, also known in the art. For example, a hybrid protein antibody-endotoxin AndPseudomonas” were described by Director, et al., Nature 339:394 (1989), Brinkmann et al., Proc. NAT'l. Acad. Sci., USA, 88:8616 (1991), Batra et al., Proc. NAT'l. Acad. Sci., USA, 89:5867 (1992), Friedman et al., J. Immunol. 150:3054 (1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J. Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996) and Schmidt et al., Int. J. Can. 65:538 (1996). Hybrid proteins “antic the lo-toxin”, containing the molecule of diphtheria toxin, have been described Kreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem. 268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995) and Vallera et al., Blood 88:2342 (1996). In the publication Deonarain et al., Tumor Targeting 1:177 (1995) described a hybrid protein “antibody-toxin containing molecule RNase, and publication Linardou et al., Cell Biophys. 24-25:243 (1994) described a hybrid protein “antibody-toxin containing such component, as Tnkase I. In hybrid protein “antibody-toxin” as the toxin part of the present gelonin, see Wang et al., Abstracts of the 209th ACS National Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. In addition, the publication Dohlsten et al., Proc. Natl. Acad. Sci., USA, 91:8945 (1994) described a hybrid protein “antibody-toxin containing staphylococcal enterotoxin-A.

Representative toxins that are commonly used to obtain such conjugates are ricin, abrin, ribonuclease, Tnkase I, staphylococcal enterotoxin-A, an antiviral protein paolacci, gelonin, diphtheria toxin, exotoxinPseudomonasand endotoxinPseudomonas. See, for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA-A Cancer Journal for Clinicians 44:43 (1994). Specialists and other known suitable toxins.

Antibodies according to the invention can be used in the method ADEPT, including conjugation, the specified antibodies with a prodrug-activating enzyme which converts a prodrug (e.g., pept dildoe chemotherapeutic agent, see WO 81/01145) in the active anti-cancer drug. See, for example, WO 88/07378 and U.S. patent No. 4975278.

The enzyme component immunoconjugate used in ADEPT, includes any enzyme capable of influencing the prodrug in such a way that the action of this prodrug is converted into the more active cytotoxic form.

Enzymes that can be used in the method according to the invention, include, but are not limited to, alkaline phosphatase, used for converting phosphate-containing prodrugs into free drugs; arylsulfatase used for converting sulfate-containing prodrugs into free drugs; sitoindosides used for converting non-toxic 5-fertilizin in anti-cancer drug, 5-fluorouracil; proteases, such as proteaseSerratiathermolysin; subtilisin; carboxypeptidase and cathepsins (such as cathepsins b and L), which are used for converting peptide-containing medicines into free drugs; D-alanismorissette used for converting prodrugs that contain D-amino acid substituents; carbohydrate-chip off the enzymes, such as β-galactosidase and neuraminidase, used for converting glycosylated prodrugs into St. the free drugs; β-lactamase used for making medicines, derivatizing β-lactams, into free drugs; and penicillin-amidase, such as penicillin-V-amidase or penicillin-G-amidase used for making medicines, derivatizing of nitrogen atoms of the amino group phenoxyacetyl or phenylacetylene groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as Azimi, can be used to convert the prodrugs according to the invention into free active drugs (see, for example, Massey, Nature 328: 457-458 (1987)). The conjugates of the antibody-Abim” can be obtained in accordance with the present description for the delivery of abzyme in the population of tumor cells.

The enzymes according to the invention can be covalently linked to antibodies by methods well known in the art, such as using heterobifunctional cross-linking agents discussed above. Alternatively, the hybrid proteins containing at least antigennegative region of the antibodies according to the invention, attached to at least a functionally active portion of an enzyme according to the invention, can be designed by methods of recombinant DNA, good is known in the art (see, for example, Neuberger et al., Nature 312: 604-608 (1984)).

Non-therapeutic use

Antibodies according to the invention can be used as agents for affinity purification of M-CSF or in diagnostic assays of protein M-CSF, for example for detecting its expression in specific cells, tissues, or serum. Such antibodies can also be used in diagnostic assaysin vivo. Usually, for these purposes, the antibody have been labelled with a radionuclide (such as111In99Tc14C,131I125I3H,32P or35S) so that the localization of this tumor can be determined using immunoscintigraphy.

Antibodies according to the invention can be used in any known analytical method such as tests for competitive binding, direct and indirect sandwich assays, such as ELISA and analyses by thus. Zola, Monoclonal Antibodes: A Manual Techniques, pp 147-158 (CRC Press, Inc. 1987). These antibodies can also be used for immunohistochemical analysis for labeling of tumor samples obtained by methods known to experts.

For convenience, the antibody according to the invention can be included in the set containing the packaged combination of reagents in predetermined amounts with instructions for diagnostic review. If the antibody is labeled Fe the COP, this kit will include substrates and cofactors required for this enzyme (e.g., a substrate precursor which represents the detected chromophore or fluorophore). In addition, may include other auxiliary components, such as stabilizers, buffers (for example, a blocking buffer or buffer for lysis), etc. Relative amounts of the various reactants can vary widely to achieve a concentration of reagents in the solution, which essentially optimizes the sensitivity of this analysis. In particular, these reagents can be prepared in the form of dry, usually liofilizovannyh, powders, including fillers, which, after dissolution, allow to obtain a reagent solution having the appropriate concentration.

The present invention is illustrated in the following examples, which should not be construed as limiting the present invention.

EXAMPLES

Example 1

This example shows that anti-M-CSF antibody RX1 and A are species-specific, and that antibodies RX1, MS and MS neutralize the activity of human M-CSF. RX1 is a commercially available antibody, on sale more than a year ago, before the filing of this application. Representative commercially available sources are, but unlimited by them, clones 116, and 21 692 mouse monoclonal antibodies against human M-CSF (Anogen); clones 21113.131, 26730 and 26786 antibodies against human M-CSF (R & D Systems, Inc.) and the M16 clone antibodies against human M-CSF (Antigenix America, Inc.).

To assess neutralizing activity RX1 and A conducted analysis on the proliferation of cell line M-NFS-60 (deposited in the American type culture collection reg. No. CRL-1838, ATSS, Rockville, MD, USA, and comes from the line myelogenous leukemia induced mice by ectopic wild-type retrovirus Cas-Br-MuLV and respond to interleukin-3 and M-CSF and contains a subset of the proto-oncogene C-myb, formed in the result of the integration of the retrovirus). For proliferation of M-NFS-60 requires the presence of M-CSF, depending on the dose. In this assay, cells M-NFS-60 was washed and were sown in medium RPMI 1640 containing 10% FBS and 3000 units/ml M-CSF and 1% Pen/Strep. Recombinant human M-CSF (final concentration 10 ng/ml) and antibody against human or murine M-CSF in various concentrations were incubated in the incubator for 1 hour at 37°C in 5% CO2. After incubation the mixture was added to the cell culture M-NFS-60 in 96-well microtiter tablets. The total volume for analysis in each well was 100 μl, and the concentration of M-CSF was 10 ng/ml, and the concentration of the antibodies indicated on the figure 5. Cells were incubated at 37°C in 5% CO 2within 72 hours, and then the cell number was determined using analysis of CellTiter Glo (Promega). The above analysis was repeated for antibodies MS and MS.

As shown in figure 5, antibodies RX1 and A against M-CSF are species-specific. Cell proliferation was assessed in the analysis of CellTiter Glo by registering the intensity of the fluorescent radiation which is linearly correlated with the number of cells. Species-specific neutralizing activity of the antibodies RX1 and A was assessed by their ability to inhibit M-NFS-60 in the presence of either human or murine M-CSF. And finally, as shown in figure 5B, antibodies MS and MS are also effective inhibitors of the activity of M-CSF.

Example 2

This example shows that the antibody RX1 at a dose of 5 mg/kg effectively inhibits osteolysis in a model of human xenotransplant. In this study, used females “Nude” mice aged 4-7 weeks with an average weight of ~20, Tumor cells (MDA-MB-231, 3×105), suspended in 10 μl of saline, were injected with the bone marrow cavity of the right tibia. Radiographs of the hind limbs was received one day after inoculation of the tumor to obtain a background picture for the detection of bone destruction caused by the injection. Mice were randomly divided into treatment groups of 10 mice per group, on the tea group, which I.P. Pavlova. were administered PBS, and the group that the I.P. Pavlova. introduced RX1 at a dose of 5 mg/kg once a week for 6 weeks. At the end of their studies again received radiographs of the hind limbs and compared them with the background picture of the damage to the bone. The degree of damage to the bones caused by tumor, was determined, as shown in figure 6. The group of animals treated with antibody RX1 at a dose of 5 mg/kg, was observed a statistically significant sustainability bones to induced tumor damage.

Example 3

This example shows that the number of metastases is reduced with the introduction of antibodies RX1 “Nude” mice bearing cancer cells MDA-MB-231 human breast cancer, at a concentration of 5 mg/kg

In this study, used females “Nude” mice aged 4-7 weeks with an average weight of ~20, Tumor cells (MDA-MB-231, 3×105), suspended in 10 μl of saline, were injected with the bone marrow cavity of the right tibia. Radiographs of the hind limbs was received one day after inoculation of the tumor to obtain a background picture for the detection of bone destruction caused by the injection. Mice were randomly assigned to different treatment groups, including the group, which I.P. Pavlova. were administered PBS, and the group that the I.P. Pavlova. introduced RX1 at a dose of 5 mg/kg once a week for 6 weeks. At the end of their studies in the mouse is each group processing took light and fixed in a solution BOINC for counting metastatic nodes in the lungs.

As shown in figure 7, the number of metastases was decreased with the introduction of antibodies RX1 at a concentration of 5 mg/kg “Nude” mice bearing cancer cells MDA-MB-231 human breast cancer.

Example 4

This example describes the process of humanization of antibodies RX1. Antibodies (5H4, MS and MS were humanitarian in accordance with the same procedures.

Designing genes for the light and heavy chains gumanitarnogo RX1

Nucleotide and amino acid sequences for murine RX1 presented in figure 4B. To construct a frame region gumanitarnogo antibodies used sequence of human antibodies identified using the database to identify the proteins present in the National Foundation for biomedical research, or using the same database. For selection sequence humanized heavy chain sequence of the heavy chain of murine RX1 was aligned with the sequence of the heavy chain of a human antibody. In each position, for humanized sequence selected amino acid human antibodies, however, if this does not fall under one of four specific categories below, then chose the amino acid murine RX1, namely, if:

(1) this position is located in the hypervariable region (CDR), as b is lo determined by Kabuto (Kabat, J. Immunol., 125, 961-969 (1980));

(2) this amino acid human antibodies are rarely found in human heavy chains in this position and the amino acid murine RX1 is the most common in the human heavy chains in this position;

(3) this provision is in close proximity to the CDR in the amino acid sequence of the heavy chain of murine RX1; or

(4) 3-dimensional modeling of murine antibody RX1 suggests that this amino acid is located in physical proximity to antigennegative area.

For selection sequence humanized light chain sequence of the light chain of the murine RX1 was aligned with the sequence of the light chain of a human antibody. In each position, for humanized sequence selected amino acid human antibodies, unless this provision also does not fall under one of the categories described above, namely:

(1) if it is a CDR;

(2) if the amino acid in this position is more typical of murine RX1 than human antibodies;

(3) if it is located near the CDR; or

(4) if there is a possibility that in three-dimensional space, this location is in close proximity to the binding region.

In fact, the following were nucleot the derivative of the sequence of genes of the heavy and light chains:

(1) the nucleotide sequence encoding the amino acid sequence, selected as described above;

(2) 5'-region of these coding sequences, that is, the nucleotide sequences encoding the leader (signal) sequence. These leader sequences were selected as typical sequence of antibodies;

(3) the 3'region of the coding sequences, that is nucleic acid sequences that correspond J5-segment of the murine light chain and J2-segment of the murine heavy chain, which are part of the murine sequence RX1. These sequences were included because they contain donor splicing signals; and

(4) each of the limit of sequences, which is aXbaI-site, providing a cleavage enzymeXbaI and the cloning vector inXbaI-site.

Designing genes humanized light and heavy chains

For the synthesis of the heavy chain were obtained for four of the oligonucleotide to DNA synthesizer, Applied Biosystems 380B. Two of these nucleotides are part of each thread of the heavy chain, and each oligonucleotide overlaps with the subsequent oligonucleotide of about 20 nucleotides that facilitates annealing. These oligonucleotides, taken together, cover the entire variable region humaniterian the heavy chain, with a few extra nucleotides at each end, what contributes to the restriction inXbaI-sites. These oligonucleotides were purified from polyacrylamide gels.

Each oligonucleotide was fosforilirovanii using ATP and polynucleotide-T4 kinase according to standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). For the annealed phosphorylated oligonucleotides these oligonucleotides were suspended together in 40 μl of TA (33 mm Tris-acetate, pH of 7.9, 66 mm potassium acetate, 10 mm magnesium acetate) at a concentration of about of 3.75 μm each, and then were heated to 95°C for 4 minutes and slowly cooled to 4°C. To obtain a full-sized gene of these oligonucleotides by synthesizing the opposite chain of each oligonucleotide were added the following components in a final volume of 100 μl:

10 álhybridisable oligonucleotides
0.16 mmeach deoxyribonucleotide
0,5 mmATP
0,5 mmDTT
100 µg/mlBSA
of 3.5 mcg/mlprotein T4 g43 (DNA polymerase)
25 mg/mlprotein g44/62 T4 (helper polymerase protein)
25 mg/mlprotein 45 (auxiliary polymerase protein)

The mixture is incubated at 37°C for 30 minutes. Then add 10 ál of DNA T4 ligase and incubation was performed for 30 minutes at 37°C. Polymerase and ligase iactiveaware by incubating the reaction mixture at 70°C for 15 minutes. For cleavage enzyme geneXbaI in the reaction mixture were added 50 μl of 2 × TA containing BSA at 200 µg/ml, and DTT at 1 mm, 43 μl of water and 50 μl of XbaI in 5 μl. The reaction mixture was incubated for 3 hours at 37°C and then purified on a gel. Then XbaI fragment was isolated from gel and cloned into the XbaI site of plasmid pUC19 by standard methods. Plasmids were purified by standard methods and sequenced dideoxy-method.

Construction of plasmids for expression of humanized light and heavy chains were made by selectingXbaI-fragments of light and heavy chains of the plasmid pUC19 in which they were built, their subsequent introduction inXbaI-site appropriate expression vector that will Express high levels of full-size heavy chain with its transfection into a suitable cell host.

Synthesis and affinity of g is monitorowania antibodies

Expression vectors were transferrable in mouse cells Sp2/0 cells, which were integrated into the plasmid were selected on selective(time marker(s)present(them) in the expression vectors by standard methods. To confirm the ability of these cells to secrete antibody, binding to M-CSF, supernatant from these cells were incubated with cells, which are known to Express M-CSF. After washing, cells were incubated with goat anti-human antibody conjugated with fluorescein, washed, and analyzed for fluorescence on a FACSCAN cytometer.

In the next experiment, the cells producing humanitariannet antibody were injected with mice and the resulting ascitic fluid was collected. Humanitariannet antibody was purified from these ascites, mainly, to a homogeneous state by passing through the affinity column with goat antibody against human immunoglobulin prepared on the media Affigel-10 (Bio-Rad Laboratories Inc., Richmond, Calif.) in accordance with standard procedures. To determine the affinity of gumanitarnogo antibodies compared with the affinity of the original murine antibody RX1 conducted an experiment on competitive binding methods known to experts.

Example 4A

This example describes the cloning and expression of antibodies RX1 Human Enginered™, as well as purification of such antibodies and tested for binding activity. Antibodies Human Engineered™, (5H4, MS and MS were obtained similar methods.

Designing sequences of Human Engineered™

Getting variable domains of the antibody is Human Engineered™ was described in Studnicka [see, for example, Studnicka et al., patent No. 5766886; Studnicka et al., Protein Engineering 7:805-814 (1994)] as a method of reducing immunogenicity while retaining binding activity of the antibody molecules. In accordance with this method, each amino acid variable region was established risk replacement. Amino acid substitutions were divided into three risk categories: (1) replacement of low risk, in which there is the highest probability of reducing immunogenicity with the least disruption of binding to the antigen; (2) replacement of moderate risk, which also lead to reduced immunogenicity, but to increase the likelihood of violations of binding to the antigen or protein folding; (3) replacement of high risk residues important for binding or for the preservation of antibody structures that create the highest risk of negative effects on the binding of antigen or laying protein. Usually it is considered that due to the three-dimensional structural configuration prolinol modification prolinol will lead to replacements, Myung is our least moderate risk, even if the change in this position is usually considered the replacement of low risk. Preferred modifications are replacement, but may also be made insertions and deletions. In figures 4B and 4C shows the degree of risk attributed to each amino acid residue of the light and heavy chains of murine RX1, respectively, and divided into categories of modifications of high, moderate or low risk.

Variable region light and heavy chains of the murine antibody RX1 represent the field of Human Engineered™, obtained using this method. Amino acid residues that are candidates for modification in accordance with this method and are in the “low risk”, were identified by aligning the amino acid sequences of murine variable regions with the sequence of the human variable regions. This can be used any human variable region, including a separate sequence of VH or VL or human consensus sequence of VH or VL. Amino acid residues can be modified in any position to be “low risk” or in the “low risk”. For sequences of the heavy chain Human Engineered™ “low risk”, presented in figures 19A-B, as the matrix is the objects of study were the human consensus sequence Vh2 (Kabata), and in every position “low risk”, in which mouse and human amino acid residues differed from each other, have introduced amino acid modification, namely murine residue was replaced by the human residue. For sequences of the light chain of the Human Engineered™ “low risk”, presented in figures 20A-b, was used as a matrix human consensus sequence of the Kappa-3 (Kabuto), and each provision of “low risk”, in which mouse and human amino acid residues differed from each other, have introduced amino acid modification, namely murine residue was replaced by the human residue. All was done on 16 amino acid modifications at low risk in the light chain and 8 modifications of low risk in the heavy chain.

Similarly, amino acid residues that are candidates for modification in accordance with this method and are in the “low and moderate risk”, were identified by aligning the amino acid sequences of murine variable regions with the sequence of the human variable regions. Amino acid residues can be modified in any position low or moderate risk” or all the provisions of the “low and moderate risk”. For the sequence of the heavy chain uman Engineered™, shown in figures 19A-b, was used as a matrix human consensus sequence Vh2 (Kabuto), and each provision of low or moderate risk, in which mouse and human amino acid residues differed from each other, have introduced amino acid modification, namely murine residue was replaced by the human residue. For the sequence of the light chain of the Human Engineered™shown in figures 20A-b, was used as a matrix human consensus sequence of the Kappa-3 (Kabuto), and each provision of low or moderate risk, in which mouse and human amino acid residues differed from each other, have introduced amino acid modification, namely murine residue was replaced by the human residue. All was done 19 amino acid modifications of low and moderate risk in the light chain and 12 modifications of low and moderate risk in the heavy chain.

As shown in figures 21A-B, was also obtained sequence of the light chain of the alternative low-risk”, in which the modification at position 54 is again restored by replacing the murine residue. As shown in figures 21A-B, was also obtained sequence of the light chain of the alternative low + moderate risk”, in which modifications in the provisions 54-56 again vosstanavlivat and by replacing the murine residues.

Finally, the sequence V-region light chain Human Engineered™ low + moderate risk” was also obtained using sequence VK6 human germline subgroup 2-1-(1) A14 as a matrix, as shown in figures 22A-C.

The present invention also addresses other amino acids 41-43 (NGS), is shown in figure 4A, which are the site of glycosylation. An alternative can be saved in only one or two of the amino acids 41-43 (e.g., NG).

Obtain expression vectors for permanent growth cell line

DNA fragments encoding each of the above sequences of V-region heavy and light chain, together with derived from the antibody signal sequence, was designed by the methods of nucleotide synthesis. DNA encoding each of the above-described amino acid sequences of the V-region light chain, embedded in the vector rmhr containing human constant region light chain Kappa. DNA encoding each of the above-described amino acid sequences of the V-region heavy chain, embedded in the vector rmhr containing human constant region of the heavy chain gamma-2. Were designed additional vectors containing the amino acid sequence of V-region heavy chain connec is installed to human constant regions of the gamma-1 (cDNA) and gamma 4 (genomic and cDNA), having the sequence shown in figures 29A, 29b and 30. All of these vectors containing the hCMV promoter and 3'-noncoding region of the mouse light chain Kappa, as well as the selective marker genes, such asneoorhisfor selection of transfectants resistant to G418 or histidine, respectively. Vectors containing light and heavy chain described in tables 2 and 3, respectively.

Table 2
Vectors, permanent expressing single genes
light chain Kappa
PlasmidsV-regionSelective marker
rmhsLow + moderate risk (Kabat)neo
rmhsLow risk (Kabat)neo
rmhsLow risk (Kabat) - R54-Sneo
rmhsLow + moderate risk (Kabat)- RAT54, 55, 56 - SISneo
rmhsLow + moderate risk (germline who INIA) neo

Table 3
Vectors, permanent expressing single genes heavy chain
PlasmidsV-regionC-scopeSelective marker
rmhsLow + moderate risk (Kabat)gamma-2neo
rmhsLow risk (Kabat)gamma-2neo
rmhsLow risk (Kabat)gamma 1neo
rmhsLow + moderate risk (Kabat)gamma 1neo
rmhsLow + moderate risk (Kabat)gamma 4 (genomic)neo
rmhsLow + moderate risk (Kabat)gamma (cDNA) neo

Then were constructed vectors containing the desired genes light + heavy chain Human Engineered™ (gamma-1, gamma-2 or gamma-4). These “consisting of 2 genes” vectors contain genes encoding each chain of the antibody, i.e. heavy and light chain, and under the control of the hCMV promoter, donor splicing CMV, acceptor splicing of SV40 16S and 3'-noncoding DNA murine light chain Kappa, including the polyA site. These vectors also contain a selective marker gene, such asneoorhisand the gene for resistance to ampicillin. The vectors containing the genes for the heavy and light chains described in table 4. Can be constructed vectors containing two copies of the genes for the light and heavy chain vectors of the four genes).

Table 4
Vectors, permanent expressing two genes
Heavy chain
PlasmidsLight chain KappaV-regionC-scopeSelective marker
rmhsLow risk (Kabat)Low is the risk (Kabat) gamma-2neo
rmhsLow risk (Kabat)Low risk (Kabat)gamma-2his
rmhsLow + moderate risk (Kabat)Low + moderate risk (Kabat)gamma-2neo
rmhsLow risk (Kabat)Low + moderate risk (Kabat)gamma-2neo
rmhsLow + moderate risk (Kabat)Low risk (Kabat)gamma-2neo
rmhsLow risk (Kabat)- R54-SLow + moderate risk (Kabat)gamma-2neo
rmhsLow + moderate risk (Kabat)- RAT54, 55, 56 - SISLow + moderate risk (Kabat) gamma-2neo
rmhsLow risk (Kabat)- R54 - SLow risk (Kabat)gamma-2neo
rmhsLow + moderate risk (Kabat)- RAT54, 55, 56 - SISLow risk (Kabat)gamma-2neo
rmhsLow + moderate risk (germ line)Low + moderate risk (Kabat)gamma-2neo
rmhsLow + moderate risk (germ line)Low + moderate risk (Kabat)gamma-2his
rmhsLow + moderate risk (germ line)Low risk (Kabat)gamma-2neo
rmhsLow + moderate risk (germ line)Low risk(Kabat) gamma 1neo
rmhsLow + moderate risk (germ line)Low + moderate risk (Kabat)gamma 1neo
rmhsLow + moderate risk (germ line)Low + moderate risk (Kabat)gamma 1his
rmhsLow + moderate risk (germ line)Low + moderate risk (Kabat)gamma 4 (cDNA)neo
rmhsLow + moderate risk (germ line)Low + moderate risk (Kabat)gamma 4 (genomic)neo

Obtain expression vectors for temporal expression

The vectors containing the genes for light or heavy chain, described above, were also constructed for the time of transfection. These vectors were similar to those described above vectors for permanent transfection, the and except instead of genesneoorhisthey contain geneoriP virus Epstein-Barr replication in HEK293 cells that Express a nuclear antigen of Epstein-Barr. These vectors for temporary transfection are described in tables 5 and 6.

Table 5
Vectors for temporal expression of light chain Kappa
PlasmidsV-region
rmhsLow + moderate risk (Kabat)
rmhsLow risk (Kabat)
rmhsLow + moderate risk (Kabat)- RAT54, 55, 56 - SIS
rmhsLow risk (Kabat) - R54-S
rmhsLow + moderate risk (germ line)

Table 6
Vectors for temporal expression of the heavy chain
PlasmidsV-regionC-scope
rmhsLow + the unintended risk (Kabat) gamma-2
rmhsLow risk (Kabat)gamma-2
rmhsLow risk (Kabat)gamma 1
rmhsLow risk (Kabat)gamma 4 (genomic)
rmhsLow + moderate risk (Kabat)gamma 1

Transient expression RX1 Human Engineered™ cells NAKE

Separate vectors, each of which contains a geneoriP virus Epstein-Barr genes for light or heavy chain, described above, were subjected to temporary transfection into cells NICE. Cells subjected to transfection, were left for incubation over a period of time up to 10 days, after which the supernatant was isolated and the antibody was purified by chromatography on protein A. the Protein obtained by the time of transfection cells E described below in table 7.

Table 7
Antibodies RX1 Human Engineered™
Light chainagela chain
AntibodyPlasmidProteinPlasmidProtein
heRX1-1.G2rmhsLow risk (Kabat)rmhsLow risk (Kabat)
heRX1-2.G2rmhsLow risk (Kabat)rmhsLow + moderate risk (Kabat)
heRX1-3.G2rmhsLow + moderate risk (Kabat)rmhsLow risk (Kabat)
heRX1-4.G2rmhsLow + moderate risk (Kabat)rmhsLow + moderate risk (Kabat)
heRX1-5.G2rmhsLow risk (Kabat)- R54 - SrmhsLow risk (Kabat)
heRX1-6.G2rmhsLow + moderate what about the risk (Kabat)-R54-S rmhsLow risk (Kabat)
heRX1-7.G2rmhsLow + moderate risk (Kabat)- RAT54, 55, 56 - SISrmhsLow risk (Kabat)
heRX1-8.G2rmhsLow + moderate risk (Kabat)- RAT54, 55, 56 - SISrmhsLow + moderate risk (Kabat)
heRX1-9.G2rmhsLow risk + moderate risk (germ line)rmhsLow risk (Kabat)
heRX1-10.G2rmhsLow risk + moderate risk (germ line)rmhsLow + moderate risk (Kabat)
heRX1-1.G1rmhsLow risk (germ line)rmhsLow risk (Kabat)
heRX1-10.G1rmhsLow risk + mind the military risk (germ line) rmhsLow + moderate risk (Kabat)
heRX1-9.G4rmhsLow risk + moderate risk (germ line)rmhsLow risk (Kabat)

The permanent cultivation of transfected cells Cho-K1

The above-described vectors (table 4), containing one copy each of the combined genes of the light and heavy chains, was transferrable in Ex-Cell-302-adapted cells Cho-K1. Cells Cho-K1, adapted for growth in suspension culture in medium Ex-Cell 302, usually subjected to electroporation 40 μg of linearized vector. Alternatively, the linearized DNA can be conjugated to a linear polyethylenimine (PAYS) and used for transfection. Cells were planted in 96-well plates, containing medium Ex-Cell 302, which has been added 1% FBS and G418. Clones were skanirovali in 96-well tablets and the best of the clones, ~10% from each transfection, was transferred to a 24-hole tablets containing medium Ex-Cell 302.

Test on productivity was carried out in 24-hole tablets in the medium Ex-Cell 302 for cultures grown for 7 and 14 days, and after this time supernatant cultures were tested for levels of secreted antibodies in ELIS-analysis of immunoglobulin IgG.

The best clone was transferred into a shaker flask containing the medium Ex-Cell 302. Because the cells were adapted to growth in suspension culture, the testing of these clones in shaker flasks was carried out in the medium Ex-Cell 302. These cells were cultured for a period of time up to 10 days in 125-ml Erlenmeyer flasks containing 25 ml of medium. During the incubation period, the flasks were opened at least every other day for gas exchange and at the end of the incubation period the levels of immunoglobulin polypeptide in the culture medium were determined using an ELISA analysis of IgG. After many successive transpency the same cell lines two or three multicomponent transcriptional vectors obtained clones and cell lines, which saw a further increase in the levels of production of immunoglobulin, preferably up to 300 mcg/ml or more.

Clean

Can be developed a method of separation of immunoglobulin polypeptides of the vectors and all cell lines according to the invention. In accordance with methods well known in the art, cells are removed by filtration after completion of the cultivation. The filtrate is loaded onto a column of protein A (with a lot of deletions. through the column, if necessary). Then the column is washed and expressed, and the Secretary is directed to the polypeptides of the immunoglobulin elute from the column. For the production of antibodies, protein a pool was kept at a low pH (pH 3, a minimum of 30 minutes and a maximum of one hour) for stage inactivation of the virus. Then for additional purification of the product is carried out stage absorption and cation exchange. The eluate from the absorption separating column is passed through a Seitz filter (filter filter virus) for additional cleaning may present viral particles. Then the filtrate is additionally cleaned by passing through the anion-exchange column, with which this product is not bound. Finally, the cleaning procedure is completed by transferring the product into a buffer for drugs through diafiltration. Retentate adjusted to a protein concentration of at least 1 mg/ml and add stabilizer.

Activity linking

Estimated M-CSF-binding activity of recombinant antibodies Human Engineered™. Protein was extracted from supernatants culture in shaker flasks by passing through a column of protein And subsequent determination of the concentration of a280. Analyses linking was performed as described above in example 1, or as described below in example 12. Tablets Immulon II was previously senzibilizirani antigen sM-CSF, which was pre-dissolved in a sensitizing solution of PBS for its immobilization on the microtiter plate. Then we use the and various test concentrations of M-CSF, components from 0.25 to 20 μg/ml in a volume of 50 μl/well, and incubated at 4°C over night. Then the tablets three times washed with PBS-0.05% tween. Blocking was performed by adding a solution of 1% BSA in PBS-0.05% of the room followed by a 30 minute incubation at 37°C. Cultivation of immunoglobulin polypeptides were prepared in a solution of 1% BSA in PBS-0.05% of the twin. As a result, received a 2 - or 3-fold serial dilution was added to wells (100 μl/well) with two or three repetitions. After 90 minutes incubation at 37°C. the microplate 3 times washed with PBS-0.05% tween. For the manifestation of the signal in each well was added a second goat antibody against human IgG (gamma - or Fc-specific)conjugated with peroxidase and incubated for 60 minutes at 37°C, followed by addition of OPD at 0.4 mg/ml in citrate buffer + 0,012% H2O2. After analysis for 5-10 minutes at room temperature, it was stopped by adding 100 μl of 1M H2SO4and the tablets were read at 490 nm. Were used goat antibody against human IgG (gamma-specific) and goat antibody against human IgG (Fc-specific).

Example 5

The following example describes a method of treatment of a person using M-CSF-specific antibody, such as antibody, derived from the RX1 or competing with the RX1, including the traveler originating from RX1 antibody Human Engineered™ with a modified or unmodified constant region is IgG1 or IgG4. A similar procedure can also be implemented for antibodies originating from MC1 or MS, or antibodies that compete with MS or MS. The expected effective dose is in the range from 2 μg/kg to 10 mg/kg. This assessment was made of the following considerations, supported by experimental data.

The measured level of M-CSF in human plasma (in healthy patients and in patients with breast cancer) was approximately 1 ng/ml M-CSF-neutralizing antibody RX1 had a measured concentration EC50equal to 2 ng/ml to 1 ng/ml human M-CSF. In line with this, it was suggested that the effective concentration of antibodies in human plasma in 10-50000 times its EC50then there is 20 ng/ml to 100 μg/ml antibody in human plasma. On the basis of RK-studies, to achieve this concentration in a human patient, namely to achieve the antibody concentration of 20 ng/ml-100 μg/ml in plasma, required dose of 2 mg/kg 10 mg/kg

Example 6

This example describes the procedure for evaluation of anticancer activity of monoclonal anti-M-CSF antibodies in the model of subcutaneously implanted tumors. In the above example 2, it was shown that the processing of monoclonal anti-M-CSF antibody leads to a significant inhibition of tumor growth in the bone marrow. This study aims calopresti, could this antibody can inhibit the growth of tumors in soft tissue.

In this research, we used female mice nu/nu at the age of 10 weeks with an average weight of ~20, Before the study, the mice were left for at least 7 days for acclimatization. On day 0, the right side “Nude” mice were subcutaneously injected with cells SW620 colon cancer man in an amount of 5×106cells/mouse in 100 μl. When tumor volume reached 100-200 mm3(usually 1 week after tumor inoculation), mice were randomly divided into 5 groups of 10 mice per group, which was introduced:

1) PBS

2) RX1

3) A

4) control Ab isotypes mIgG1 + rIgG1

5) A+RX1

Mice intravenously injected these antibodies at 10 mg/kg once weekly for 4 weeks. When tumor volume reached 2000 mm3the investigation was completed. Alternatively, animals were also killed in those cases, if the surface is ulcerated tumor exceeded 30% of the total surface area of the tumor, there was a significant decrease in body weight (>20%)was observed dehydration and painful condition. All mice took the whole blood and the population of monocytes was analyzed as a potential surrogate marker. Growth/tumour size was determined in 2-factor analysis. Tumor volume was calculated based on the measured width and length of the tumor. As expected,the growth of the tumor in the soft tissue is inhibited, as it was shown by the result of the above experiment.

Example 7

The following procedure describes how to evaluate the effectiveness of combination therapy for treatment and prevention of severe osteolytic diseases associated with cancer metastasis.

The plan of the experiment. The study described above in example 5 was performed essentially in the same way, but with the following exceptions. In addition to the antibody or combination of antibodies used for introduction of the treatment groups described below, animals were subjected to one of the following additional treatments, such as:

1. Treatment with bisphosphonates (e.g., Aredia; Zometa; Clodronate).

2. A surgical operation.

3. Radiation therapy.

4. The chemotherapy.

5. Hormonal therapy (e.g. tamoxifen; therapy antiandrogens).

6. The antibody therapy (for example, RANKL/RANK-neutralizing antibodies; PTHrP neutralizing antibody).

7. Therapy therapeutic protein (e.g., soluble RANKL receptor; OPG and PDGF inhibitors and MMP).

8. Therapy low molecular weight drug (e.g., an inhibitor of Src-kinase).

9. Therapy oligonucleotides (for example, antimuslim sequences of RANKL, RANK or PTHrP).

10. Gene therapy (for example, inhibitors of RANKL or RANK).

11. Therapy peptides (for example, muteena the RANKL).

Group processing is presented below. The above additional processing described below as “plus therapy X”:

1. Only PBS.

2. Only therapy X.

3. Handling mouse IgG1 isotype control.

4. Handling mouse IgG1 isotype control.

5. Processing only antibody RX1 against human M-CSF.

6. Processing only mouse IgG1 A against murine M-CSF.

7. Processing a combination of rat IgG1 and murine IgG1 control isotypes.

8. Treatment combination RX1 and A.

9. Handling mouse IgG1 isotype control + therapy X.

10. Handling mouse IgG1 isotype control + therapy X.

11. Treatment with antibody RX1 against human M-CSF therapy + X.

12. Handling mouse IgG1 A against murine M-CSF therapy + X.

13. Processing a combination of rat IgG1 and murine IgG1 control isotypes + therapy X.

14. Treatment combination RX1 and A + therapy X.

The dose: each animal was injected with 0.1-30 mg/kg of each antibody. The preferred dose is 10 mg/kg of Introduction can be carried out intravenously (i.v.), intraperitoneally (I.P. Pavlova.) or subcutaneously (s.c.). It is preferable intraperitoneal administration. Processing starts from the day after the injection of tumor cells, as described above in example 5.

Measurement: To assess the severity of osteolysis in different groups of processing, for each of the th mouse from the day after the injection of tumor cells received basic scintigrams on Faxitron. Scintigraphy on Faxitron was also conducted at the end of the study (8 weeks). Simultaneously measured the growth of tumors using the Xenogen system, because tumor cells stably Express luciferase. As expected, combination therapy, performed for the treatment and prevention of severe osteolytic diseases associated with cancer metastasis, gave the best results compared to treatments by using only one antibody.

Example 8

The following example describes a Protocol for evaluating the ability of M-CSF-specific antibodies to contact, for example, breast cancer cells (cell line MDA231) or cancer multiple myeloma cells (cell line ARH77) cell sorting device with activation of fluorescence.

First, the cells twice washed with PBS (without Ca2+, Mg2+). In each 10-cm tablet was added 2 ml of 3 mm EDTA, and the plates were incubated at 37°C for 2-3 minutes until such time as the cells did not acquire a round shape and did not begin to separate from the tablet. Then added 10 ml of buffer A (PBS + 5% FBS) and the contents stirred. After that, the cells were besieged and resuspendable about 5×106cells/ml in PBS + 5% FBS, and then placed in a test tube at 100 μl/sample.

On e is th stage was added to 0.1-10 μg/ml of the “first” antibody (antibody against M-CSF or control antibodies at the indicated concentrations). Breeding, if necessary, was performed in 5% FBS/PBS. The mixture is then incubated for 30 minutes at 4°C. After incubation the cells 3 times washed by centrifugation at 400 g for 5 minutes and resuspendable in PBS.

FITZ - or PV-labeled anti-IgG antibody (0.25 microgram/sample) were diluted in 1% BSA/PBS at optimal dilution and cells resuspendable in this solution and incubated for 30 minutes at 4°C. Then the cells 3 times washed as described above. After washing, cells these cells resuspendable with 0.5 ml/sample PI-PBS (if it was necessary to identify dead cells from live cells). These cells can also be fixed for subsequent analysis (cells, if they are fixed in 0.1% formaldehyde, can be stored for approximately 3 days). Then these cells were analyzed by FACS with activation of fluorescence in accordance with standard procedures.

As shown in figures 8A and 8B, M-CSF-specific antibody RX1 was associated with cell cancer line MDA231 breast cancer or a cancer cell line multiple myeloma ARH77 at various indicated concentrations of antibodies.

Example 9

In the following example it is shown that M-CSF is prevalent on the surface of some cancer cells. Immunohistochemical staining of M-CSF was performed using M-CSF-specific antic the La RX1 as follows:

At the beginning of the experiment, slides were heated in an oven at 55-60°C for 1 hour and left to cool for 2-3 minutes. Thus, we used the following parameters dewaxing and re-hydration:

A.Treatment with xylene:3×5 minutes
b.Processing 100% alcohol reagent:2×5 minutes
S.Processing 95% alcohol reagent:2×4 minutes
d.Processing 75% alcohol reagent:2×3 minutes
E.Processing 50% alcohol reagent:1×3 minutes
g.Processing deionized H2O:2-3 quick wash

Before the implementation stage blocking peroxide conducted the restoration of the antigen using reagent 1 × Biogenex Citra Plus. The first solution was heated in a microwave oven at full power until boiling. After boiling solution mode is the works of the microwave oven quickly set on power level 2 in this mode, the solution was stood still 13 minutes and then it was left to cool before handling. Stage blocking peroxide were performed as described below. Slides were immersed in 3% H2O2(25 ml 30% - 250 ml deionized H2O) and left for 10 minutes at room temperature. Then slides 2 times washed with deionized H2O and washed with 1 × PBS, 2×2 minutes.

The procedure of blocking Avidya/Biotin was carried out as follows. Slides were placed in a metal rack in a horizontal position. For marking fabrics used blue pencil RAR (hydrophobic marker for slides). Then add 2 drops avidin Zymed (reagent a), sufficient to cover the tissue, and these slides were incubated at room temperature for 10 minutes. After incubation slides were washed as follows:

2×3 minutes, washed in 1 × PBS;

added 2 drops of Biotin Zymed (reagent) at room temperature for 10 minutes.

2×3 minutes, washed in 1 × PBS.

The procedure of blocking the protein was carried out as follows. First was added 10% serum [to a final concentration of 2%], containing the second antibody. Then concentrated blocking solution (Power Block) BioGenex diluted 1 × deionized H2O. the Stand-up of slides for 8 minutes immersed in blocking Astor Power Block at room temperature and slides were washed 2 times in 1X PBS.

To add “first” antibody (RX1) glass slides were placed in a metal rack in a horizontal position. Then add the antibody to cover each slice (~350 ml), and this antibody was applied using a pipette tip (if necessary), without affecting the fabric. Then slides were incubated for 1 hour at room temperature. After incubation slides 3 times washed with 1 × PBS, each time for 3-5 minutes. At this stage the cuts inflicted reagent BioGenex Multi-Link and the mixture is incubated for 10-11 minutes at room temperature. After this, sections were washed for 3 minutes each time.

Tagging was carried out by applying HRP-label BioGenex on the slices, which are then incubated at room temperature for 10-11 minutes and 3 times washed with 1 × PBS, for 3 minutes. Then these sections were added to the substrate H2O2BioGenex (1 drop of AES per 2.5 ml H2O2), and then incubated at room temperature for 10 minutes. Then the slices several times washed with geonosians H2A. Phase contrast staining was carried out as follows. The sections were stained with hematoxylin for 1 minute at room temperature. Then the slices twice washed with H2O, and incubated in 1 x PBS for 1 minute. After that, the sections were thoroughly washed in H2O to remove PBS. Sections p is aparatow on slides were prepared by application of drops of reagent Super Mount BioGenex, and then air dried over night at room temperature.

As shown in figure 9, M-CSF prevailed on the surface of some cancer cells. Cuts for cancer cells of these types was evaluated by the following point system.

0staining is absent
1coloration similar to the background colour
2positive but weak staining
3positive and significant staining
4positive and strong staining

Example 10

The following example describes the procedure for obtaining antibodies MS and MS. MS and MS represent two mouse monoclonal antibodies that neutralize human anti-M-CSF antibody and contact with human M-CSF. Amino acid sequences of these antibodies are shown in figures 14 and 15, respectively. These sequences have been identified in several stages, including (a) immunization of mice Balb/C recombinant human M-CSF; (b) screening for positive clones that produce antibodies, binding the stories with human M-CSF in the ELISA format; (C) subclavian positive clones to generate stable hybridoma clones; (d) scale cultivation of cells for producing large quantities of antibodies; (e) purification and characterization of antibodies in assays for affinity, binding to cells and neutralizing activity, as described in the previous examples.

In figures 16A and 16B shows an alignment of sequences of the CDRs of the heavy and light chains, respectively, for antibodies RX1, (5H4, MS and MS.

Humanized variants and variants of Human Engineered™ received, as described above in the examples.

Example 11

This example shows that anti-M-CSF antibody RX1 and (5H4, as well as their Fab-fragments have different neutralizing activities. The following example also shows that antibodies RX1, (5H4 and MS have different affiniscape binding to M-CSF. This example also demonstrated that the affinity of the above intact antibodies exceed the affinity of Fab fragments of the above-mentioned antibodies.

Neutralizing activity of intact antibodies RX1 and (5H4 compared with the activity of Fab-fragments of RX1 and (5H4 was determined by evaluating the M-CSF-dependent cell proliferation in the presence of various concentrations of the antibody. Cell proliferation was assessed using a chemiluminescent dye. As shown in figure 17, the intact RX1 the highest activity, and the activity of the Fab-fragment RX1 decreased and was similar to the activity of the antibodies (5H4 and Fab-fragment (5H4.

Binding properties of the above-mentioned antibodies were analyzed using BIAcore analysis. To determine the relative affinely binding antibodies RX1, (5H4 and MS with M-CSF, Fc fragment of rabbit antimelanoma antibody was immobilized on a biosensor chip SM by linking with amine. Then the above-mentioned antibody was immobilized on a biosensor chip SM with artemisinin Fc-fragment at a concentration of 1.5 μg/ml for 3 minutes at 2 µl/min M-CSF was applied to the modified biosensor surface at various concentrations (Rmax ~ 15). The test antibody and the antigen was diluted in 0,01M HEPES, pH 7.4, 0,15M NaCl, 3 mm EDTA, 0.005% of a surfactant P20 (HBS-EP). All experiments were performed at 25°C. Kinetic constants and affinity constants were determined using the computer program Biaevaluation (Biacore) with the selection of models of interaction/total match 1:1. As shown below in table 8, RX1 was associated with M-CSF with the highest affinity compared to (5H4 and MS.

RX1
Table 8
Ka (M-1· with-1)Kd (-1)KD (nm)
1,6462,7-40,16
(5H45,9451,77-33,0
MS? 7.04 baby mortality51,93-40,27

To determine the relative differences between the binding affinity of the intact mAb and Fab fragments RX1, (5H4 and MS was used alternative configuration Biacore analyses. More specifically, M-CSF was immobilized on a biosensor chip SM by linking with amine. 0.5 μg/ml M-CSF in 10 mm sodium acetate, pH 4.0, were injected with 1 μl/min for 5 minutes before reaching RL=6-12 EN. The test antibody (or Fab-fragment) was applied to the modified biosensor surface at various concentrations. Test antibodies were diluted in 0,01M HEPES, pH 7.4, 0,15M NaCl, 3 mm EDTA, 0.005% of a surfactant P20 (HBS-EP) and all experiments were performed at 25°C. Kinetic constants and affinity constants were determined using the computer program Biaevaluation (Biacore) with the selection of models of interaction/total match 1:1. As shown below in table 9, RX1 was associated with M-CSF with the highest affinity in comparison with the other tested the mi antibodies. Fab-fragment RX1 was associated with M-CSF with much lower affinity than holoprotein RX1.

Table 9
Ka (M-1· with-1)Kd (-1)KD (nm)
rRX1 (mouse)2,3452,35-41,0
Fab rRX1 (mouse)2,8153,03-310,8
(5H41,2751,26-39,9
Fab (5H42,0452,85-314,0

Data affinity binding and neutralization showed that the neutralizing activity of antibodies RX1 is due mainly to its very high affinity for M-CSF and that this high affinity can be at least partially due to the ability of both branches of the specified antibodies to simultaneously contact the dimer M-CSF.

Example 12

In digislide the General sample was identified linear epitope (i.e. amino acid sequence) to M-CSF, recognized by antibodies RX1, (5H4 and MS.

First strategy was developed epitope mapping to determine which epitopes on M-CSF, i.e. linear or konformacionnye are recognized by antibodies RX1, (5H4 and MS. In line with this, the anti-M-CSF antibody against M-CSF was tested with 0.1 ág M-CSF in reducing, as well as in non conditions. Each antibody recognizes only unrestored form M-CSF, suggesting that recognized epitopes in nature epitopes are discontinuous type.

Then, for each antibody was determined linear epitope of M-CSF. In particular, we have obtained membrane SPOT (Sigma Genosys), where interest is the sequence of the fragment of M-CSF, overlapping with 10-dimensional peptides synthesized with a shift of one amino acid was loaded onto cellulose membrane media. Then the membrane was probed above antibodies and identified reactive SPOT. After this peptide sequence identified by its corresponding localization on the membrane and overlapping amino acids in positively reacting the peptides identified as the epitope. As shown in figure 18, the antibody RX1 is associated with other linear epitope that is not with the epitope bound (5H4 and MS, and cards is the formation of this epitope was pointing to another location on M-CSF. RX1 binds to a linear epitope having the sequence RFRDNTPN (SEQ ID NO: 120) or RFRDNTAN (SEQ ID NO: 121), amino acids 98-105 of M-CSF in the figure 12. (5H4 binds to a linear epitope having the sequence ITFEFVDQE (SEQ ID NO: 122), amino acids 65-73 M-CSF in the figure 12. MS associated with two linear epitope having the sequence of (1) ITFEFVDQE (SEQ ID NO: 122), amino acids 65-73 M-CSF in the figure 12 and (2) FYETPLQ (SEQ ID NO: 123), amino acids 138-144 M-CSF in figure 12.

Example 13

Was determined the binding affinity of variants of the Human Engineered™ antibodies RX1, obtained as described above in example 4A. This example shows that the variants of Human Engineered™ antibodies RX1 with constant regions of different IgG subclasses are associated with M-CSF with different affiniscapein vitro. To determine the relative differences between appendectomy binding of intact antibodies using Biacore analysis, M-CSF was immobilized on a biosensor chip SM by linking with amine. 0.5 μg/ml M-CSF in 10 mm sodium acetate, pH 4.0, were injected with 1 μl/min for 5 minutes before reaching RL=6-12 EN. Test antibody or its Fab fragments were applied to a modified biosensor surface at various concentrations, ranging from 100 nm to 1.5 nm in 2-fold dilutions. Test antibodies were diluted in 0,01M HEPES, pH 7.4, 0,15M NaCl, 3 mm EDTA, 0.005% of a surfactant P20 (HBS-EP) and all of the experience is imenti was carried out at 25°C. Experiments for each concentration and buffer control was performed with three repetitions, and the data were collected within 3 minutes of the Association and 8 minutes of dissociation. Kinetic constants and affinity constants were determined using the computer program Biaevaluation (Biacore) with the selection of models of interaction/total match 1:1. As shown below in table 10, heRX1-1.G1 and heRX1-1.G4 was associated with M-CSF with affinity, which was almost similar to the affinity of binding murine RX1 with M-CSF.

Table 10
AntibodyKa (M-1·with-1)Kd (-1)KD (nm)
murine RX1, n=21(2,23±0,35)×105(1,56±0,67)×10-40,7±0,27
heRX1-1.G2, n=5(2,36±0,18)×105(1,37±0,24)×10-35,9±1,4
heRX1-10.G2, n=2(1,73±0,29)×105(1,1±0,11)×10-36,3±1,7
heRX1-1.G1of 2.50×105 of 2.38×10-40,95
heRX1-1.G42,07×105of 2.93×10-41,42

In contrast, table 11 shows that although there are some variations in the affinity of binding, however, all designs gamma-2 was found at least 7-fold decrease in the affinity of binding compared to the affinity of the parent murine antibody.

Table 11
AntibodyKa (M-1· with-1)Kd (-1)KD (nm)
murine RX1, n=21(2,23±0,35)×105(1,56±0,67)×10-40,7±0,27
heRX1-1.G2, n=5(2,36±0,18)×105(1,37±0,24)×10-35,9±1,4
heRX1-2.G2to 2.18×105of 1.65×10-37,6
heRX1-3.G2a 2.01×105 of 1.18×10-35,9
heRX1-4.G2of 2.38×1051,08×10-34,6
heRX1-5.G2of 1.75×105of 1.29×10-37,4
heRX1-6.G2of 1.88×105for 1.49×10-37,9
heRX1-7.G2of 1.57×105for 1.49×10-39,5
heRX1-8.G2of 1.52×105to 1.48×10-39,8
heRX1-9.G22×105the 1.44×10-37,2
heRX1-10.G2, n=2(1,73±0,29)×105(1,1±0,11)×10-36,3±1,7

Example 14

This example shows that the variants of Human Engineered™ antibodies RX1 with constant regions of different IgG subclasses have different neutralizing activitiesin vitro. To assess the neutralizing activity of the anti-M-CSF antibodies were analyzed for proliferation of the cell line M-NFS-60 (deposited in the American type culture collection reg. No. CRL-1838, ATSS, Rockville, MD, USA, and comes from the line myelogenous leukemia induced mice by ectopic wild-type retrovirus Cas-Br-MuLV. This cell line responds to interleukin-3 and M-CSF and contains truncated protooncogene C-myb, formed in the result of the integration of the retrovirus). For proliferation of M-NFS-60 requires the presence of M-CSF, depending on the dose. In this assay, cells M-NFS-60 was washed and were sown on Wednesday RPIM 1640 containing 10% FBS and 1% Pen/Strep. Recombinant human M-CSF (final concentration 10 ng/ml, equivalent to 3000 u/ml M-CSF activity) were incubated with various concentrations of antibodies in the range from 1 μg/ml to 0.5 ng/ml (at serial 2-fold dilution) in the incubator for 1 hour at 37°C in 5% CO2. After incubation the mixture was added to the cell culture M-NFS-60 in 96-well microtiter tablets. The total volume for analysis in each well was 100 μl, and the concentration of M-CSF was 10 ng/ml, and the concentration of the antibodies listed below. Cells were incubated at 37°C in 5% CO2within 72 hours, and then the cell number was determined using analysis of CellTiter Glo (Promega). Each antibody was tested with three repetitions, and this test was repeated on the following day (for all six tests for each antibody). IC50each antibody was analyzed by constructing a curve.

This analysis is carried and using human M-CSF in serum, in MDA231-conditioned medium (containing M-CSF), and using M-CSF abacadabra monkey serum and recombinant M-CSF abacadabra monkeys. The results are shown in figure 25 (recombinant M-CSF), figure 26 (human M-CSF in serum) and figure 27 (MDA231-air-conditioned environment) and were obtained by reading the fluorescence intensity in the analysis of CellTiter Glo, and these results are linearly correlated with the number of cells. The neutralizing activity of these antibodies is represented as the degree of inhibition of cell proliferation of M-NFS-60.

These results showed that the concentration of the IC50for heRX1-1-IgG1 and heRX1-1-IgG4 was almost similar concentration IC50recombinant parental murine antibody RX1 and the IC50heRX1-1-IgG2 was 2 or 4 times higher.

In the following table 12 shows the relative concentration of the IC50(expressed as the reduction IC50for different designs IgG2, obtained as described above in example 4A. Of these designs are the least reduction IC50found design heRX1-1.G2 and heRX1-10.G2.

Table 12
AntibodyThe reduction IC50
heRX1-1.G2 2,8×
heRX1-2.G23,1×
heRX1-3.G25,3×
heRX1-4.G24,6×
heRX1-5.G26,5×
heRX1-6.G25,9×
heRX1-7.G26,1×
heRX1-8.G25,9×
heRX1-9.G23,6×
heRX1-10.G22.2 x
heRX1-1.G1not declined
heRX1-1.G4not declined
heRX1-10.G1not declined
heRX1-10.G4not declined

Example 15

This example shows that the variants of Human Engineered™ antibodies RX1 with constant regions of different IgG subclasses have different TRAR activities inin vitrothe analysis osteoclastogenesis.

CD34+cells from human bone marrow (Biowhittaker, catalog No. 2M-101A, 3×105cells/vessel) induced for differentiation in osteocel the texts described in this experimental conditions. On day 1, CD34+cells were thawed from one of the frozen vessel in 10 ml of medium (alpha-MEM with 10% FCS, 1 × Pen/Strep and 1 × Fungizone). These cells were once washed and resuspendable in 2 ml of medium, and then were placed in a 96-well plate at 100 μl/well. On day 2, without removing the original medium in each well was added 50 μl of 4 × CSF-1 to a final concentration of 30 ng/ml and 50 μl of 4 × RANKL (sRANKL, Chemicon catalog # GF091, 10 µg/packaging) to a final concentration of 100 ng/ml On day 7 in each well was added 50 μl of 5 × RANKL to a final concentration of 100 ng/ml On the 17th day the cells were stained on TRAR activity (set for coloring on TRAR-activity containing acid phosphatase leukocyte, Sigma catalog # 387-A) and evaluated under a microscope.

On the 2nd day of analysis was added M-CSF-neutralizing antibodies. These antibodies inhibited the differentiation of osteoclasts, depending on the dose, as shown in figure 28. Inhibitory activity of the antibodies in the analysis for the differentiation of osteoclasts expressed as the absence of visible osteoclasts on the 17th day of analysis.

Example 16

Were further characterized antibodies Human Engineered™ RX1 with constant regions of different IgG subclasses.

Complexes “antigen-antibody”, which was a complex of various variants of Human Engineered™ antibodies RX1 with M-CSF, investigated by combining M-CSF and antibodies in the buffer in equal molar is otnoshenijah the subsequent evaluation of the size of the complex antigen-antibody using exclusion chromatography or light scattering. The results showed that the murine parent RX1 forms 1:1 complexes with M-CSF of approximately 200 kDa. Antibodies heRX1-9.G2 or heRX1-10.G2 form a 2:1 or 2:2-complexes with M-CSF of approximately 400 kDa. heRX1-10.G2 forms large aggregates with a lattice structure with M-CSF larger than 2×106Yes. Design of IgG1 and IgG4 were formed small complexes, similar to the complexes formed by the murine parent RX1.

Electrophoresis under denaturing pampering and non PAG with LTOs held for heRX1-1.G4, showed that the variant IgG4, as expected, was formed poloitical.

All of the above U.S. patents, published patent applications U.S. patent application U.S., foreign patents, foreign patent applications and non-patent publications cited in this application and/or listed in the beginning of this application, in its entirety introduced into the present description by reference.

Based on the above it is apparent that although the present invention is described in specific embodiments, its implementation, are presented for purposes of illustration, however, it can be made various modifications not beyond being and scope of the invention.

1. No mouse anti-M-CSF-antibody, which contains an amino acid sequence that is at least 90% identical to the consequences of the successive SEQ ID NO: 24, and which preserves Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

2. The antibody according to claim 1, which is more than 75% competes with a monoclonal antibody RX1 for binding to M-CSF, where the specified monoclonal antibody RX1 contains amino acid sequences of the heavy and light chains, are presented in SEQ ID NO: 2 and 4, respectively.

3. The antibody according to claim 1, which contains SEQ ID NO: 24.

4. The antibody according to claim 1, which further comprises at least one of the sequences SEQ ID NO: 18, 21, 29, 32 and 36.

5. The antibody according to claim 1, which further comprises at least two of the sequences SEQ ID NO: 18, 21, 29, 32 and 36.

6. The antibody according to claim 1, which further comprises one or more of the sequences SEQ ID NO: 16, 19, 22, 27, 30 and 34.

7. The antibody according to claim 1, which further comprises one or more of the sequences SEQ ID NO: 17, 20, 23, 28, 31 and 35.

8. The antibody according to claim 1, which further comprises one or more of the sequences SEQ ID NO: 18, 21, 25, 29, 32 and 37.

9. The antibody according to claim 1, which further comprises one or more of the consensus CDR presented in SEQ ID NO: 18, 21, 26, 29, 33 and 38.

10. No mouse anti-M-CSF is an antibody that contains at least 1 of the sequences SEQ ID NO: 18, 21, 24, 29, 32 and 36, and which Sokh is anal K dthe affinity (equilibrium constant of dissociation) with respect to M-CSF SEQ ID NO: 9, is equal to at least 10-8M or greater affinity.

11. The antibody of claim 10, which contains at least 2 of the sequences SEQ ID NO: 18, 21, 24, 29, 32 and 36.

12. The antibody of claim 10, which contains at least 3 of the sequences SEQ ID NO: 18, 21, 24, 29, 32 and 36.

13. The antibody of claim 10, which contains at least 4 of the sequences SEQ ID NO: 18, 21, 24, 29, 32 and 36.

14. The antibody of claim 10, which contains at least 5 of the sequences SEQ ID NO: 18, 21, 24, 29, 32 and 36.

15. The antibody of claim 10, which contains all the sequences SEQ ID NO: 18, 21, 24, 29, 32 and 36.

16. The antibody according to item 12, in which at least one amino acid in the CDR replaced by the corresponding residue of the corresponding CDR of another anti-M-CSF antibodies.

17. No mouse anti-M-CSF-antibody containing the amino acid sequence of the variable region of the light chain that is at least 65% homologous to the amino acid sequence represented in SEQ ID NO: 4, where the specified amino acid sequence of the variable region of the light chain contains SEQ ID NO: 29, 32 and 36, and the amino acid sequence of the variable region of the heavy chain that is at least 65% homologous to the amino acid sequence represented in SEQ ID NO: 2, where aminomalononitrile variable regions of the heavy chain contains SEQ ID NO: 18, 21 and 24, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

18. Antibody on 17 containing the constant region sequences of human antibodies and one or more variable frame regions of the heavy and light chain sequences of a human antibody.

19. The antibody according p, where the sequence of the human antibody is a separate human sequence, a human consensus sequence, a separate sequence of human germline or the consensus sequence of the human germ line.

20. The antibody according to 17, which contains a fragment of the constant region of IgG1.

21. The antibody according to claim 20, containing a mutation in the constant region of IgG1, which reduces antibody-dependent cellular cytotoxicity or complement-dependent cytotoxic activity.

22. The antibody according to 17, which contains a fragment of the constant region of IgG4.

23. The antibody according to item 22, which contains a mutation in the constant region of IgG4, which reduces the formation of poloitical.

24. No mouse anti-M-CSF-antibody containing the amino acid sequence of the variable region of the heavy chain, which at IU is at 65% homologous amino acid sequence, presented in SEQ ID NO: 2, where the specified amino acid sequence of the variable region of the heavy chain contains SEQ ID NO: 18, 21 and 24, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

25. The antibody according to paragraph 24, containing the constant region sequences of human antibodies and one or more variable frame regions of the heavy and light chain sequences of a human antibody.

26. The antibody according A.25, where the sequence of the human antibody is a separate human sequence, a human consensus sequence, a separate sequence of human germline or the consensus sequence of the human germ line.

27. The antibody according to paragraph 24, which contains a fragment of the constant region of IgG1.

28. The antibody according to paragraph 24, which contains a fragment of the constant region of IgG4.

29. No mouse anti-M-CSF-antibody containing the variable region of the heavy chain, which contains the amino acid sequence selected from the group consisting of
a)
XVXLXEXGXXXXXXXXXLXLXCXVXDYSITSDYAWNW1XQXXXXXLXWMGYIS
YSGSTSXNXXLXXXIXIXRXXXXXXFXLXLXXVXXXDXAXYYCASFDYAHAMDY
WGXGTXVXVXX,
b)
DVXLXEXGPXXVXPXXXLXLXCXVTDYSITSDYAWNWIRQXPXXKLEWMGYIS
YSSYNPSLKXRIXIXRXTXXNXFXLXLXXVXXXDXATYYCASFDYAHAMYWGX
GTXVXVXX,
c)
XVQLQESGPGLVKPSQXLSLTCTVXDYSITSDYAWNWIRQFPGXXLEWMGYISY
SGSTSYNPSLKSMXIXRDTSKNQFXLQLNSVTXXDTAXYYCASFDYAHAMDYW
GQGTXVTVSS,
and (d)
DVQLQESGPGLVKPSQXLSLTCTVTDYSITSDYAWNWIRQFPGXKLEWMGYISY
SGSTSYNPSLKSRIX1XRDTSKNQFXLQLNSVTXXDTATYYCASFDYAIIAMDYW
GQGTXVTVSS,
where X represents any amino acid, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

30. No mouse anti-M-CSF-antibody containing the variable region of the heavy chain, which contains the amino acid sequence:
DVQLQESGPGLVKPSQTLSLTCTVTDYSITSDYAWNWIRQFPGKKLEWMGYISY
SGSTSYNPSLKSRITISRDTSKNQFSLQLNSVtaadtatyycasfdyajieamdyw
GQGTTVTVSS,
and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

31. No mouse anti-M-CSF-antibody containing the variable region of the heavy chain, which contains the amino acid sequence:
QVQLQESGPGLVKPSQTLSLTCTVSDYSITSDYAWNWIRQFPGKGLEWMGYISY
SGSTSYNPSLKSRITISPvDTSKNQFSLQLNSvtaadtavyycasfdyahamdyw
GQGTTVTVSS,
and where the specified antibody retains the Kdaffinity (constant
the equilibrium dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

32. No mouse anti-M-CSF-antibody containing the variable region of light chain, which contains amino acid series is here, selected from the group consisting of
a) XIXLXQXXXXXXVXXXXXVXFXCXAXQSIGTSIHWYXQXXXXXPXLL1KYASEX
XXXIXXXFXGXGXGXXFXLXIXXVXXXDXADYYCQQINSWPTTFGXGTXLXXXX
X,
b)
XIXLXQXPXXLXVXPXXXVXFXCXASQSIGTSIHWYQQXTXXSPRLLIKYASEXIS
XI PXRFXGXGXGXXFXLXIXXVXXXDXADYYCQQINSWPTTFGXGTXLXXXXX,
c)
XIXLTQSPXXLSVSPGERVXFSCRASQSIGTSIHWYQQXTXXXPRLLIKYASEXXX
GIP XRFSGSGSGTDFTLXIXXVESEDXADYYCQQINSWPTTFGXGTKLEIKRX,
d)
XIXLTQSPXXLSVSPGERVXFSCRASQSIGTSIHWYQQXTXXSPRLLIKYASEXISG
IPX RFSGSGSGTDFTLXIXXVESEDXADYYCQQINSWPTTFGXGTKLEIKRX, and
e)
XIXLTQSPXXLSVSPGERVXFSCRASQSIGTSIHWYQQXTXXXPRLLIKYASESISG
IPX RFSGSGSGTDFTLXIXXVESEDXADYYCQQINSWPTTFGXGTKLEIKRX,
where X represents any amino acid, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

33. No mouse anti-M-CSF-antibody containing the variable region of light chain, which contains the amino acid sequence:
EIVLTQSPGTLSVSPGERVTFSCPvASQSIGTsihwyqqktgqaprllikyasesis
GIPD RFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQGTKLEIJCRT,
and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

34. No mouse anti-M-CSF-antibody containing the variable region of light chain, which contains the amino acid sequence:
EIVLTQSPGTLSVSPGERVTFSCRASQSIGTSIHWYQQKTGQAPRLLIKYASERAT
GIP DRFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQGTKLEIKRT,
and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) in respect to the tion to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

35. No mouse anti-M-CSF-antibody containing the variable region of light chain, which contains the amino acid sequence:
EIVLTQSPGTLSVSPGERVTFSCRASQSIGTSIHWYQQKTGQSPRLLIKYASERISG
IPD RFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQGTKLEIKRT,
and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

36. The antibody according to any one of p-35, where at least one X represents the same amino acid as the amino acid in the same position in SEQ ID NO: 2 or 4, in accordance with the numbering Kabata.

37. The antibody according to any one of p-35, where at least one X is a conservative substitution of amino acids present in the same position in SEQ ID NO: 2 or 4, in accordance with the numbering Kabata.

38. The antibody according to any one of p-35, where at least one X represents a non-conservative substitution of amino acids present in the same position in SEQ ID NO: 2 or 4, in accordance with the numbering Kabata.

39. The antibody according to any one of p-35, where at least one X is an amino acid present in the same position in which it is present in the sequence of human antibodies, in accordance with the numbering Kabata.

40. The antibody according to any one of p-5, where at least one X is an amino acid present in the same position in which it is present in the consensus sequences of human antibodies, in accordance with the numbering Kabata.

41. The antibody according to § 39, where the sequence of the human antibody is a human consensus sequence, the sequence of human germline, a consensus sequence of human germline or any of the sequences of human antibodies in accordance with the numbering Kabata.

42. Anti-M-CSF-antibody containing any one of the sequences of the heavy chain, represented in SEQ ID NO: 41, 43, 114, 116, or 119, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

43. The antibody according to § 42 containing the sequence of the heavy chain, represented in SEQ ID NO: 114, and the sequence of the variable region of the light chain, represented in SEQ ID NO: 47.

44. The antibody according to § 42 containing the sequence of the heavy chain, represented in SEQ ID NO: 116, and the sequence of the variable region of the light chain, represented in SEQ ID NO: 47.

45. The antibody according to § 42 containing the sequence of the heavy chain, represented in SEQ D NO: 119, and the sequence of the variable region of the light chain, represented in SEQ ID NO: 47.

46. Anti-M-CSF-antibody containing any one of the sequences of the variable regions of the light chain, represented in SEQ ID NO: 45, 47, 48, 51, 53 or 136, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

47. No mouse anti-M-CSF-antibody containing the amino acid sequence of the variable region of the heavy chain that contains any two of the sequences SEQ ID NO: 18, 21 and 24 and which is at least 65% identical to the amino acid sequence of variable region of the heavy chain, represented in SEQ ID NO: 41 or 43, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

48. The antibody according p containing the amino acid sequence of the variable region of the heavy chain that is at least 80% identical to the amino acid sequence of variable region of the heavy chain, represented in SEQ ID NO: 41 or 43.

49. No mouse anti-M-CSF-antibody containing the amino acid sequence of the variable region of the light chain, which contains any two of the village is edutella SEQ ID NO: 29, 32 and 36 and which is at least 65% identical to the amino acid sequence of variable region of the light chain, represented in SEQ ID NO: 45, 47, 48, 51 or 53, and where the specified antibody retains the Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity.

50. The antibody according to § 49, containing the amino acid sequence of the variable region of the light chain that is at least 80% identical to the amino acid sequence of variable region of the light chain, represented in SEQ ID NO: 45, 47, 48, 51 or 53.

51. The antibody according to any one of claims 1 to 35 or 42-50, which is a chimeric antibody, humanitariannet antibody, a human engineered antibody, a human antibody or single-chain antibody.

52. The antibody according to any one of claims 1 to 35 or 42-50, which is an IgG antibody.

53. The antibody according to any one of claims 1 to 35 or 42-50, which is a Fab fragment, F(ab')2 fragment, an Fv fragment or single-chain Fv fragment.

54. Anti-M-CSF-antibody containing a heavy chain according to any one of p-31, 42 or 47-48 and light chain according to any one of p-35, 46 or 49-50.

55. The antibody according to item 54, which is an IgG antibody.

56. The antibody according to item 54, which is a Fab fragment, F(ab')2 fragment, an Fv fragment or single-chain Fv-fragments is.

57. The antibody according to any one of claims 1 to 35 or 42-50, which has Kdaffinity equal to at least 10-9.

58. The antibody according to any one of claims 1 to 35 or 42-50 conjugated to a toxin.

59. The antibody according to item 54, conjugated to a toxin.

60. The antibody according to item 53, conjugated to a toxin.

61. The antibody according to any one of claims 1 to 35 or 42-50, which binds to M-CSF for the prevention of osteoporosis in a mammal suffering from a disease that causes or stimulates osteolysis, where the specified antibody effectively prevents osteoporosis associated with disease.

62. The antibody according to any one of claims 1 to 35 or 42-50, which binds to M-CSF for the treatment of a mammal suffering from a disease causing or stimulating osteolysis, where the specified antibody promotes effective to reduce the severity of osteoporosis associated with the disease.

63. The antibody according to item 62, where the disease is selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrinopathies (including hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce status (including rickets/disorder, scurvy, malnutrition), chronic diseases (including malabsorption syndromes, chronic pacec the th failure (including renal osteodystrophy), chronic liver disease (including hepatic osteodystrophy), diseases associated with medicines (including glucocorticoids (induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (including imperfect osteogenesis, homocystinuria), cancer, osteoporosis, osteoporosis, inflammation associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

64. The antibody according to any one of claims 1 to 35 or 42-50, which binds to M-CSF for the prevention or treatment of cancer metastases in bone, where the metastatic cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina or cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; or skin cancer, including the Aya malignant melanoma or squamous cell carcinoma.

65. The selected nucleic acid containing a nucleic acid sequence encoding the antibody according to any one of claims 1 to 35, 42-50, 53 or 54.

66. The selected nucleic acid encoding the antibody that contains any two of the sequences SEQ ID NO: 18, 21 and 24 and which preserves Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10~8 M or greater affinity, where this nucleic acid contains a nucleic acid sequence that is at least 65% identical to the nucleotide sequence of the heavy chain, represented in SEQ ID NO: 1 or SEQ ID NO: 40 or 42.

67. The selected nucleic acid p containing a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of the heavy chain, represented in SEQ ID NO: 1 or SEQ ID NO: 40 or 42.

68. The selected nucleic acid encoding the antibody that contains any two of the sequences SEQ ID NO: 29, 32 and 36 and which preserves Kdthe affinity (equilibrium constant of dissociation) with respect to M-CSF (SEQ ID NO: 9), equal to at least 10-8M or greater affinity, where this nucleic acid contains a nucleic acid sequence that is at least 65% identical to the nucleotide sequence of egcoa circuit, presented in SEQ ID NO: 3 or SEQ ID NO: 44, 46, 52, 135, or 137.

69. The selected nucleic acid p containing a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of the light chain, represented in SEQ ID NO: 3 or SEQ ID NO: 44, 46, 52, 135, or 137.

70. The expression vector containing the selected nucleic acid according to any one of p-69.

71. The vector according to item 70, where the specified selected nucleic acid is functionally linked to a regulatory sequence.

72. A host cell for production of anti-M-CSF antibodies containing the vector according to any one of p-71 or nucleic acid according to any one of p-69.

73. The method of obtaining anti-M-CSF-antibody, involving the cultivation of a host cell according to item 72, so that it expressives selected nucleic acid produced antibodies.

74. The method according to p, which additionally provides for the extraction of antibodies from the culture of the host cell.

75. A dedicated anti-M-CSF-antibody obtained by the method according to p.

76. Hybridoma, secreting the antibody according to any one of claims 1 to 64, selected from the group consisting of hybridoma deposited in ATSC number of Deposit of the MOUTH-6263, and hybridoma deposited in ATSC number of Deposit of the MOUTH-6264.

77. Pharmaceutical composition for treating a mammal suffering from Zab is levanim, causing or stimulating osteolysis containing any one of the antibodies according to claims 1 to 35, 42-50, 53 or 54, and a pharmaceutically acceptable carrier, excipient or diluent.

78. The pharmaceutical composition according p, optionally containing a second therapeutic agent.

79. The pharmaceutical composition according p, where the specified second therapeutic agent is a cancer chemotherapeutic agent.

80. The pharmaceutical composition according p, where the second therapeutic agent is another antibody.

81. Pharmaceutical composition comprising any one of the antibodies according to claims 1 to 35, 42-50, 53 or 54 and a bisphosphonate.

82. The pharmaceutical composition according p, where the bisphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate.

83. The method of prevention or attenuation of osteoporosis, involving the administration to a mammal in need, a therapeutically effective amount of the antibody according to any one of claims 1 to 35 or 42-50 in amounts effective to prevent or ameliorate osteoporosis associated with the disease.

84. The method according to p, where the disease is selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrinopathies (including hypercorticism is m, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce status (including rickets/disorder, scurvy, malnutrition), chronic diseases (including malabsorption syndromes, chronic renal failure (including renal osteodystrophy), chronic liver disease (including hepatic osteodystrophy)), diseases associated with medicines (including glucocorticoids (induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (including imperfect osteogenesis, homocystinuria), cancer, osteoporosis, osteoporosis, inflammation associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

85. The method according to p, which additionally provides for the introduction of a second therapeutic agent.

86. The method according to p, where the second therapeutic agent is a cancer chemotherapeutic agent.

87. The method according to p, where the antibody is effective to reduce the dose of the second therapeutic agent required to achieve a therapeutic effect.

88. The method according to p, where the antibody and the second therapeutic agent is administered in synergistically effective amounts.

89. The method according to p, where the antibody and the anti-Christ. Iakovou chemotherapeutic agent is administered in synergistically effective amounts.

90. The method according to p, where the antibody is administered at a dose of from about 2 μg/kg to 30 mg/kg of body weight.

91. The method according to p, where the antibody is administered at a dose of from about 0.1 to 30 mg/kg of body weight.

92. The method according to p, where the antibody is administered at a dose of from about 0.1 to 10 mg/kg of body weight.

93. The method according to p, where the specified mammal previously exposed to treatment a second therapeutic agent.

94. The method according to p, where the second therapeutic agent is another, non-M-CSF, colony stimulating factor, or an antibody against RANKL, or soluble RANKL receptor.

95. The method according to p, where the second therapeutic agent is a bisphosphonate.

96. The method according to p, where the bisphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate.

97. A method of treating a mammal suffering from a disease causing or stimulating osteolysis, introducing a specified mammal a therapeutically effective amount of the antibody according to any one of claims 1 to 35 or 42-50 in amounts effective to prevent or diminish the severity of osteoporosis associated with the disease.

98. The method according to p, where the antibody is a Fab fragment, F(ab')2 fragment, an Fv fragment or single-chain Fv fragment.

99. The method according to p, where the mammal not treated with bisphosphonates.

100. The way PR is the prevention or treatment of cancer metastases in bone, introducing a mammal exposed to the risk of or suffering from cancer with metastases, a therapeutically effective amount of the antibody according to any one of claims 1 to 35 or 42-50.

101. The method according to item 100, where the antibody is a Fab fragment, F(ab')2 fragment, an Fv fragment or single-chain Fv fragment.

102. The method according to item 100, where the specified cancer cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina or cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; or skin cancer, including malignant melanoma or squamous cell carcinoma.

103. Method of cancer treatment involving the administration to a mammal in need this, therapeutically effective amounts of antibodies according to any one of claims 1 to 35 or 42-50.

104. The method according to p, which is more envisages the introduction of a second therapeutic agent.

105. The method according to p, where the second therapeutic agent is another, non-M-CSF, colony stimulating factor, or an antibody against RANKL, or soluble RANKL receptor.

106. The method according to p, where the second therapeutic agent is a bisphosphonate.

107. The method according to p, where the bisphosphonates is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate or ibandronate.

108. The method according to p, where the antibody is effective to reduce the dose of the second therapeutic agent required to achieve a therapeutic effect.

109. The method according to p, where the antibody and the second therapeutic agent is administered in synergistically effective amounts.

110. The method according to p, where the antibody and the second therapeutic agent is administered in synergistically effective amounts.

111. The method according to p, where the antibody and the bisphosphonate is administered in synergistically effective amounts.

112. The method according to p, where the antibody and the bisphosphonate is administered in synergistically effective amounts.

113. The method according to any of PP-112, where the specified mammal is man.

114. The method according to any of PP-112, where the aforementioned antibody is administered in an amount effective for inhibiting proliferation and/or differentiation of osteoclasts induced by tumor cells.

115. The method according to p, where the antibody is administered in a dose of the ome from 0.1 to 30 mg/kg of body weight.

116. The method according to p, where the antibody is administered at a dose of from about 0.1 to 10 mg/kg of body weight.

117. The method according to p, where the specified mammal previously exposed to treatment a second therapeutic agent.

118. The method according to p, where the second therapeutic agent is a cancer chemotherapeutic agent.

119. Set containing a therapeutically effective amount of the antibody according to any one of claims 1 to 35 or 42-50, packaged in a container such as a jar or bottle, and optionally containing a label attached to the container or sub-container, where the label includes a description of the contents of the container, indications and/or instructions for using the contents of the container to prevent or mitigate osteoporosis.

120. Set p containing the dose of the antibody effective to reduce the dose of the second therapeutic agent required to achieve a therapeutic effect.

121. Set p containing a synergistic dose of antibodies.

122. Set p containing the dose of the antibody component of from about 2 μg/kg to 30 mg/kg of body weight.

123. Set p containing the dose of the antibody component of from about 0.1 to 30 mg/kg of body weight.

124. Set p containing the dose of the antibody component of from about 0.1 to 10 mg/kg of body weight.

125. Set containing a therapeutically effective amount is about antibody according to any one of claims 1 to 35 or 42-50, Packed in a container such as a jar or bottle, and optionally containing a label attached to the container or sub-container, where the label includes a description of the contents of the container, indications and/or instructions for using the contents of the container for the treatment of a patient suffering from disease causing or stimulating osteolysis.

126. Set p, including instructions for its use to treat a patient who has not been subjected to treatment with bisphosphonates.

127. Set p, where the disease is selected from the group consisting of metabolic bone diseases associated with relatively increased activity of osteoclasts, including endocrinopathies (including hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, scarce status (including rickets/disorder, scurvy, malnutrition), chronic diseases (including malabsorption syndromes, chronic renal failure (including renal osteodystrophy), chronic liver disease (including hepatic osteodystrophy)), diseases associated with medicines (including glucocorticoids (induced by glucocorticoid osteoporosis), heparin, alcohol), hereditary diseases (including imperfect osteogenesis, homocystinuria, cancer, osteoporosis, osteoporosis, inflammation associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia, and/or Paget's disease.

128. Set containing a therapeutically effective amount of the antibody according to any one of claims 1 to 35 or 42-50, packaged in a container such as a jar or bottle, and optionally containing a label attached to the container or sub-container, where the label includes a description of the contents of the container, indications and/or instructions for using the contents of the container to prevent or treat metastatic cancer to bone.

129. Set p, where the specified metastatic cancer is breast cancer, lung cancer, kidney cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, malignant disease of blood cells, including leukemia and lymphoma; head and neck cancer; cancer of the gastrointestinal tract, including esophageal cancer, stomach cancer, colon cancer, cancer of the small intestine, cancer of the colon, colorectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignant diseases of the female genital organs, including carcinoma of the ovary, endometrial cancer, cancer of the vagina or cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, OS is asarama; or skin cancer, including malignant melanoma or squamous cell carcinoma.

130. Set containing a therapeutically effective amount of the antibody according to any one of claims 1 to 35 or 42-50, packaged in a container such as a jar or bottle, and optionally containing a label attached to the container or sub-container, where the label includes a description of the contents of the container, indications and/or instructions for using the contents of the container to treat cancer.

131. Set p containing the dose of the antibody effective to inhibit proliferation and/or differentiation of osteoclasts induced by tumor cells.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method of producing an antigen-binding VH domain wherein an elongated CDR3 loop similar to a camel loop is absent, involves transformation of a mammal cell by a heavy-chain VH heterolocus. The locus contains a gene coding a variable area, at least containing one segment of VH gene, one segment of D gene not being camel's, one segment of J gene not being camel's, one invariable heavy chain region provided that any gene coding the invariable regions does not code a Sn1 functional domain. The segments of V, D and J genes are capable to recombination and formation of the VDJ coding sequence. The transformed cell is capable to express a heavy chain antibody only containing an antigen-binding VH domain and the invariable constant effector region without the functional domain Sn1. The cell is used for producing a transgene animal to be immunised with an antigen of concern. Further the cells or tissues expressing specific antibodies to the heavy chain antigen of concern are recovered; nucleic acid coding the VH domain of the specific heavy chain antibody only is recovered; and the specified antigen-binding VH domain is expressed. The specified VH domains can be used for production of fused proteins or binding complexes of monovalent, bivalent or polyvalent polypeptide.

EFFECT: invention allows producing antigen-specific human antibodies of any class, exhibiting high affinity.

11 cl, 26 dwg, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L.4 A2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: invention enables to obtain monoclonal antibodies which are specific and do not compete with each other for antigen epitopes and which, when used together in a "sandwich" format immunoenzymometric system, ensure high reliability of results for exposing the matrix protein VP40 of the Ebola virus.

5 cl, 3 dwg, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 1B2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the neucleoprotein of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Rattus Norvegicus 7B11 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the nucleoprotein of Ebola virus, Zaire subtype (Mainga strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain). The invention describes monoclonal antibodies 1B2 which are produced by the strain of hybrid animal cells Mus musculus L. 1B2, which relate to the subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain, and monoclonal antibodies 7B11 which are produced by the strain of hybrid animal cells Rattus Norvegicus 7B 11 related to the subclass of immunoglobulins IgG. The antibodies are used together in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: use of the invention enables to obtain results during "ВЭ" laboratory reseach and when designing a test system for highly reliable exposure of an antigen.

5 cl, 3 dwg, 2 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 7D8, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain) and a strain of hybrid animal cells Mus musculus L. 7H10 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain). Monoclonal antibodies 7D8 which are produced by the strain of hybrid animal cells Mus musculus L. 7D8 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7H10 which are produced by the strain of hybrid animal cells Mus musculus L. 7H10 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7D8 and antibodies 7D8 and 7H10 are used together in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain).

EFFECT: use of the invention increases reliability of enzyme immunoassay.

5 cl, 3 dwg,1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of Marburg haemorrhagic fever. A strain of hybrid animal cells Mus musculus L. 3F9 is formed, which is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR. The hybridoma strain produces monoclonal antibodies which are specific to the VP35 protein of the Marburg virus (Popp strain) (hereinafter MCA). MCA 3F9 produced by hybrid animal cells Mus musculus L. 3F9 relate to a subclass of immunoglobulins IgGI, having a heavy 55 kDa and a light 25 kDa chain and having a unique feature of detecting the VP35 protein of the Marburg virus (Popp strain) in a "sandwich" immunoenzymometric system format owing to antigen "capture" properties and simultaneously be an indicator, labeled biotin. The antigen epitope for MCA 3F9 produced by the 3F9 hybridoma is localised between 252 and 278 aminoacid residues.

EFFECT: invention enables to obtain MCA with specificity to VP35 protein of the Marburg virus (Popp strain), suitable for immunodiagnosis of Marburg haemorrhagic fever.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of human cytomegalovirus. The strain of hybrid animal cells Mus musculus L.5F10 is obtained by merging mouse myeloma cells p3-X63/Ag8.653 (NS/1) with mouse spleen cells BALBc, immunised by an affinity purified recombinant protein pp65. The hybridoma strain is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR and is used as a producer of monoclonal antibodies for detecting the pp65 protein of human cytomegalovirus.

EFFECT: invention enables to widening of range of strains of hybrid cells Mus musculus L - producers of MCA for detecting the pp65 protein of human cytomegalovirus and production of domestic diagnostic test-systems for detecting cytomegaly.

2 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention can be used for production of monoclonal antibodies (MCAs) to heat shock protein 70 (HSP 70). A hybridoma strain is made by immunisation of BALB/c mice with bovine HSP 70 within 78 days. For the third days, splenocytes of immune mice (108 cells) are hybridised with murine myeloma cells P3-X63 Ag/8-653 (107 cells). A fusion agent is polyethylene glycol of molecular weight 4000 (Merk, Germany). The hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma. Hybridoma 6G2 is deposited in the microorganism collections of "ГНТТ ПМБ" under No. H-2. MCA.

EFFECT: produced hybridoma under the invention is more evident to be detected as HSP 70 on the cell surfaces, and change of endocellular HSP 70 level when exposed to the stress factors.

4 dwg, 1 tbl, 6 ex

FIELD: pharmacology.

SUBSTANCE: invention can be used to identify a pseudotuberculosis agent in bacterial cultures, a biological material and environmental objects by applying the indirect hemagglutination test. Substance of the invention consists in development of a new diagnosticum that represents formalinised sheep's erythrocytes sensitised with monoclonal antibodies to lipopolysaccharide antigen of cold version Yersinia pseudotuberculosis serotype I (strain 164/84 serovariant I) and frozen-dried in a protective medium. Shelf life of the preparation is 2 years.

EFFECT: diagnosticum provides high sensitivity, specificity to the UHAT in detecting Yersinia pseudotuberculosis serotype I.

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology. Hybridoma strain is prepared by immunisation of BALB/c mice with bovine HSP 70 within 78 days. On the third day, splenocyte hybridisation of immune mice (10 cells) with murine myeloma cells P3-X63 Ag/8-653 (107 cells) is carried out. As a fusion agent, polyethylene glycol of molecular weight 4000 (Merk, Germany) is applied. Hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma.

EFFECT: invention can be used for preparing monoclonal antibodies (MCA) to heat shock protein 70 (HSP 70).

4 dwg, 1 tbl, 6 ex

FIELD: veterinary.

SUBSTANCE: obtained is strain 8C12 of inter-species hybrid cells of mouse Mus musculus and sheep Ovis aries - producent of monoclonal antibodies of sheep to glycoproteidal antigen of virus of cattle (C) leucosis. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 72. Strain is permanent hybrid line of cells and possesses high level of production of monoclonal antibodies of sheep. Antibody titers in native culture liquid constitute 1:32-1:64 in immuno-enzymatic analysys (IEA). Monoclonal antibodies are specific to general antigen determinant of glycoproteids of C leucosis - external gp51 and transmembranous gp30. When used in IEA for detection of antibodies in blood serum and milk of C infected with leucosis virus, antibodies provide strong selective binding with solid-phase carrier and optimal space orientation of glycoproteidal antigen.

EFFECT: strain 8C12 can be used in production of immuno-enzymatic test-system for diagnostics of cattle leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms and, as a result, reduce incidence of leucosis in cattle.

1 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biochemistry, specifically to obtaining a hybrid, and can be used for obtaining a strain-producer of monoclonal antibodies to the MUCI human antigen. Using monoclonal antibody technology, a strain of hybrid cultured animal cells "ВКПМ Н-105" - producer of clinical rat monoclonal antibodies, specific for hypoglycosylated and deglycosylated isoforms of tumours associated with the human MUCI antigen can be obtained.

EFFECT: possibility of identifying clinical isoforms of MUCI antigen using antibodies, produced by an obtained strain, the antibodies of which can be used determining concentration of MUCI in blood plasma of a person previously diagnosed with tumours.

1 dwg, 3 ex

FIELD: medicine, peptides, biochemistry, pharmacy.

SUBSTANCE: invention relates to modification of glycosylation of proteins for preparing polypeptides with improved therapeutic indices including antibodies with enhanced antibody-dependent cellular cytotoxicity. For preparing indicated polypeptides cell-host is used that is modified with nucleic acid encoding enzyme β-1,4-N-acetylglucosaminyltransferase III (GnTIII). Prepared polypeptide represents, in particular, IgG or its fragment. Invention discloses a method for preparing polypeptide and antibodies or its fragment and a fusion protein prepared by indicated method. Invention describes a pharmaceutical composition used for increasing Fc-mediated cellular cytotoxicity and comprising antibody and carrier, and its using in cancer treatment, and a method for treatment of disease associated with elevated amount or production of B cells using indicated antibody, in particular, against CD20, and representing antibody IDEC-C2B8 in the preferable variant. Invention provides preparing polypeptide and antibody possessing the enhanced Fc-mediated cellular cytotoxicity that decrease the content of B cells in a patient body.

EFFECT: improved preparing method, valuable medicinal properties of polypeptide and antibodies.

38 cl, 21 dwg, 4 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention relates to antibodies showing specificity to anomalous processed form of human tau protein that differs by conformation from the normal tau protein and doesn't bind with normal tau protein. Also, invention relates to conformational distinctive tau proteins ("tauones") and diagnostic and therapeutic aspects related to Alzheimer's disease and related taupathies. Proposed antibodies are produced by hybridomas DC-11 or Dc-11/1 deposited in ECACC at numbers 00082215 and 00082216. Also, invention described truncated forms of human tau protein that are truncated by N- and/or C-end and comprise amino acid residues from amino acid 300 to amino acid 400 in the longest isoform of human tau protein (441 amino acids residues). Above mentioned truncated forms of human tau protein can be recognized specifically by antibodies described above. Also, invention describes a method for assay of truncated forms of tau protein in a patient biological sample using a set comprising a proposed antibody and suitable container. Using the proposed invention provides a suitable target for medicinal preparations with early therapeutic effect used in Alzheimer's disease and other taupathies.

EFFECT: valuable medicinal properties of proteins.

11 cl, 15 dwg, 10 ex

FIELD: biotechnology.

SUBSTANCE: claimed method includes injection of vector construct pX-RSVhGH encoding human growth hormone or pX-Ins, encoding human insulin into parenchyma of 8-9-week rabbit males. Transformation effectiveness of gene constructs is determined by immunohystochemical kit Novocastra (Sigma, USA).

EFFECT: method for genetic transformation of rabbit testicle stem cells with increased effectiveness.

3 dwg, 1 ex

FIELD: biotechnology; morphologic investigation of cultivated cells.

SUBSTANCE: primary culture of fibroplasts is subject to influence of electromagnet radiation during 5 minutes continuously with pulse frequency of 73+- Hz and with pulse amplitude of 50V and higher.

EFFECT: improved effectives of growth; higher density of fibroplasts in culture.

1 tbl

FIELD: biotechnology and virology.

SUBSTANCE: strain of hybrid culturing Rattus norvegicus 122H9 cells is disclosed. Said strain is obtained by fusion of rat myeloma cells 210RC.Y3-Ag1.2.3 with rat spleen cells LOU, immunized with purified ectromelia virus of K-1 strain. Said strain is producer of cross-reactive neutralizing monoclonal antibodies against pathogenic for human orthopoxviruses.

EFFECT: vaccines for prophylaxis and therapy of diseases associated with pathogenic for human orthopoxviruses.

2 tbl, 5 ex

The invention relates to medicine and relates to monoclonal antibodies against VASP (vasodilator-stimulated the phosphoprotein), which bind VASP as antigen only when VASP is phosphorylated in the form hybridoma cells for their preparation and to the use of antibodies or fragments of antibodies as diagnostic and/or therapeutic agents

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

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