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
This application claims the priority of provisional patent application U.S. No. 60/558290, filed March 31, 2004, this application claims priority to this provisional application under 35 U.S.C. § 119, the contents of which are incorporated here by reference.
The technical field to which the invention relates
The present invention relates to humanized anti-TGF-beta antibodies and methods for their preparation and their use in methods for treatment related TGF-beta violations. Antibodies are suitable for immunoaffinity cleanings, immunoassays, imaging in vivo, radioreceptor analysis and treatments, where it is desirable to have an antagonistic effect on the activity of TGF-beta, in particular the activity of TGF-beta.
The level of technology
Transforming factor-beta growth (TGF-beta) is a multifunctional cytokine, originally named for its ability to transform normal fibroblasts into cells capable of anchor-independent growth. TGF-beta, mainly produced hematopoietic and tumor cells, may regulate, i.e., to stimulate or inhibit the growth and differentiation of cells from various normal tissues and tumors (Sporn et al., Science, 233: 532 (1986)) and to stimulate the formation and development of the discrepancies between the different stromal elements. An overview on TGF-beta and its functions, see Sporn et al., J. Cell Biol., 105: 1039-1045 (1987) and Sporn and Roberts, Nature, 332: 217-219 (1988).
It is known that they are involved in many proliferative and non-proliferation cellular processes such as proliferation and differentiation of cells, embryonic development, formation of extracellular matrix, bone growth, wound healing, hematomas and immune and inflammatory responses. Pircher et al., Biochem Biophys. Res. Commun., 136: 30-37 (1986); Wakefield et al., Growth Factors, 1: 203-218 (1989); Roberts and Sporn, pp 419-472 in Handbook of Experimental Pharmacology, eds M.B.Sporn & A.B.Roberts (Springer, Heidelberg, 1990); Massague et al., Annual Rev. Cell Biol., 6: 597-646 (1990); Singer and Clark, New Eng. J. Med., 341: 738-745 (1999). Also TGF-beta is used to treat and prevent diseases of the intestinal mucosa. WO 2001/24813.
Of particular interest from the point of view of immunology is high immunosuppressive activity of TGF-beta, which includes inhibition of lymphokine-activated killer cells (LAK) and cytotoxic T lymphocytes (CTL) (Ranges et al., J. Exp. Med., 166: 991 (1987); Espevik et al., J. Immunol., 140: 2312 (1988); Grimm et al., Cancer Immunol. Immunother., 27: 53 (1988); Kasid et al., J. Immunol., 141: 690 (1988); Mule et al., Cancer Immunol. Immunother., 26: 95 (1988)), inhibition of lymphopoiesis In cells and the expression of light chain Kappa (Lee et al., J. Exp. Med., 166: 1290 (1987)), negative regulation of hematopoiesis (Hino et al., Br. J. Haematol., 70: 143 (1988); Sing et al., Blood, 72: 1504 (1988)), the lower regulation of expression of HLA-DR on tumor cells (Czrniecki et al., J. Immunol., 140: 4217 (1988); Zuber et al., Eur. J. Immunol., 18: 1623 (1988)) and inhibition of proliferation of antigen-activated b-lymphocytes in response to growth factor b-cells (Petit-Koskas et al., Eur. J. Immunol., 18: 111 (1988)). Based on the observation that many human tumor (deMartin et al., EMBO J. 6: 3673 (1987); Kuppner et al., Int. J. Cancer, 42: 562 (1988)), and many lines of tumor cells (Derynck et al., Cancer Res., 47: 707 (1987); Roberts et al., Br. J. Cancer, 57: 594 (1988)) produce TGF-beta, you can assume about a possible mechanism for these tumors to escape from the normal immunological control. Based on this negative immunomodulation in combination with the observation that some transformed cell lines lose their ability to respond to TGF-beta autocrine manner (Wakefield et al., J. Cell Biol., 105: 965 (1987); McMahon et al., Cancer Res., 46: 4665 (1986)), and that TGF-beta stimulates the formation of stroma and reduces immune control of tumor, it is possible to assume about attractive models revoke control and proliferation of tumors (Roberts et al., Br. J. Cancer, above).
In addition, U.S. patent No. 5824297 and 5262319 disclosed method of inhibiting cytotoxic poisoning of normal cells by introducing TGF-beta, such as TGF-beta.
Currently identified at least five forms of TGF-beta, TGF-beta, TGF-beta2, TGF-beta, TGF-beta and TGF-beta. Known appropriate ways to clear and this family of TGF-beta from various species such as human, mouse, green monkey, pig, cattle, chicken and frog, and from various sources of the body, such as bone, platelets, or placenta, to generate a recombinant cell culture, and to determine its activity. See, for example, Derynck et al., Nature, 316: 701-705 (1985); the publication of the European patent No. 200341, published on 10 December 1986, 169016, published January 22, 1986, 268561, published may 25, 1988, and 267463, published may 18, 1988; U.S. patent No. 4774322; Cheifetz et al., 48: 409-415 (1987); Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); Dijke et al., Proc. Natl. Acad. Sci. (U.S.A.), 85: 4715-4719 (1988); Derynck et al., J. Biol. Chem., 261: 4377-4379 (1986); Sharples et al., DNA 6: 239-244 (1987); Derynck et al., Nucl. Acids Res., 15: 3188-3189 (1987); Derynck et al., Nucl. Acids Res., 15: 3187 (1987); Derynck et al., EMBO J. 7: 3737-3743 (1988)); Seyedin et al., J. Biol. Chem., 261: 5693-5695 (1986); Madisen et al., DNA, 7: 1-8 (1988) and Hanks et al., Proc. Natl. Acad. Sci. (U.S.A.), 85: 79-82 (1988), the entire contents of which are incorporated herein in summary form for details.
An activated form of TGF-beta is glycosilated formed by dimerization of the C-terminal 112 amino acids 390-amino acid precursor (Derynck et al., Nature, above). TGF-beta2 has a predecessor of 414 amino acids and it is also processed in glycosilated of the C-terminal 112 amino acids, which has about 70% homology with the active form of TGF-beta (Marquardt et al., J. Biol. Chem., 262: 12127 (1987)). TGF-beta2 was isolated from porcine platelets (Seyedin et al., J. Biol. Chem., 262: 1946-199 (1987)) and cells of human glioblastoma (Wrann et al., EMBO J. 6: 1633 (1987)) and was cloned recombinant human TGF-beta2 (deMartin et al., above). Cloned recombinant TGF-beta (Derynck et al., Nature, above) and expressed in the ovary cells of Chinese hamsters (Gentry et al., Mol. Cell Biol., 7: 3418-3427 (1987)). See U.S. patent No. 4774322; 4843063 and 4848063 relating to CIF-A and CIF-B, currently named respectively TGF-beta and 2. Ellingsworth et al., J. Biol. Chem., 261: 12362-12367 (1986). Despite having 14 amino acid differences in the first 36 amino acid residues of the two forms (TGF-beta and TGF-beta2) their biological activity is similar. Cheifetz et al., Cell 48: 409-415 (1987); Seyedin et al., J. Biol. Chem., 262, above.
TGF-beta, TGF-beta and TGF-beta, which are the most recent open forms of TGF-beta, were identified by screening cDNA libraries. None of these three suspected protein has not been isolated from natural sources, although, in Northern-blotting was shown the expression of corresponding mRNA. Were cloned human and porcine TGF-beta, and they were described as homodimer and expressed in the ovary cells of Chinese hamsters (Derynck et al., EMBO J. 7: 3737-3743 (1988); ten Dijke et al., Proc. Natl. Acad. Sci. USA, 85: 4715 (1988); U.S. patent No. 4886747). See also WO 1992/00318 related protein TGF-beta and antibodies. TGF-beta different from TGF-beta2 is 27 conservative substitutions and TGF-beta mainly 22 conservative substitutions. Generatrice belong to the 3-D structure. Schlunegger and Grutter, Nature, 358: 430-434 (1992).
TGF-beta and TGF-beta cloned from cDNA library of chicken chondrocytes (Jakowlew et al., Molec. Endocrinol., 2: 1186-1195 (1988)) and from a cDNA library of frog oocytes. A cDNA library of frog oocytes can be screened using a probe derived from one or more sequences of another type TGF-beta. mRNA TGF-beta detected in chicken embryo chondrocytes, but in smaller numbers compared to the mRNA of TGF-beta in the developing embryo or chick embryo fibroblasts. mRNA TGF-beta expressed in frog embryos at the stage preceding narula, and in cells of the Xenopus tadpole (CTS).
Recombinant production of TGF-beta, TGF-beta2 and TGF-beta described in U.S. patent No. 5061786, 5268455 and 5801231. See also U.S. patent No. 5120535 by TGF-beta2, used for the treatment of hormone-dependent carcinoma and for the production of antibodies. Was identified heterodimer TGF-beta and TGF-beta2, called TGF-beta and have shown its application, as disclosed in U.S. patent No. 4931548 and 5304541, the latter also disclosed antibody to him. In WO 1990/00900, filed July 20, 1989, discloses the treatment of inflammatory disorders of homodimeric TGF-beta and-beta2 and heterodimers TGF-beta. In U.S. patent No. 5462925 revealed heterodimer TGF-beta2 and TGF-beta. In U.S. patent No. 5780436 opening is as small peptide mimetics TGF-beta.
Increased activity of TGF-beta is involved in the development of a large number of pathological conditions, including, but not limited to: (i) fibrosis, scarring, and adhesion during wound healing; (ii) fibrotic lung disease, liver and kidney; (iii) atherosclerosis and arteriosclerosis; (iv) some types of malignant tumors, including malignant prostate tumors, neuroendocrine tumors of the digestive system, tumors of the cervix, glioblastomas and swelling of the stomach; (v) a disorder, the disorder of vessels, nephropathy; (vi) multiple sclerosis; (vii) a viral infection, such as hepatitis C and HIV; and viii) immunological and inflammatory disorders and deficiencies, such as rheumatoid arthritis. Modulation of immune and inflammatory reactions induced by TGF-beta includes: (i) inhibition of proliferation of all subgroups of T cells; (ii) inhibiting effects on the proliferation and function of b-lymphocytes; (iii) - reducing regulation of activity of natural killer cells and the response of T cells; (iv) regulation of cytokine production by immune cells; (v) regulating the functions of macrophages and (vi) the recruitment and activation of leukocytes.
Specifically in relation to malignant tumors known that the members of the family of TGF-beta have a number of biological activities related to the oncogenic potential (including angiogenesis) and Metastasio the interview. TGF-beta inhibits proliferation of many cell types, including endothelial cells of the capillaries and smooth muscle cells. TGF-beta reduces the expression of integrin (alfaretta, Alfama and v3 involved in migration of endothelial cells). Integrins are involved in the migration of all cells, including those relating to metastasis. TGF-beta reduces the expression of matrix metalloproteinases required for angiogenesis, and metastasis. TGF-beta induces an inhibitor of plasminogen activator, which inhibits the cascade of proteases required for angiogenesis and metastasis. TGF-beta induces normal cells to suppress the transformed cells. See, for example, Yingling et al., Nature Reviews, 3 (12): 1011-1022 (2004), where it is revealed that deregulation of TGF-beta is involved in the pathogenesis of various diseases, including malignant tumor and fibrosis, and presents the rationale for consideration of inhibitors of signaling pathways involving TGF-beta as drugs for the treatment of malignant tumors, biomarkers/diagnostics, developed small molecule inhibitors, and their development is used purposeful model of drug discovery. A very important early diagnosis of malignant tumors (Ruth et al., Nature Reviews Cancer, 3: 243-252 (2003)), and research what is the pathogenesis of metastasis of tumors. Fidler, Nature Reviews Cancer, 3: 453-458 (2003).
TGF-beta showed himself to be a major modulator of angiogenesis in the regulation of proliferation, migration of endothelial cells, metabolism of extracellular matrix (ECM) and the expression of adhesion molecules. It is an effective inhibitor of the growth of normal epithelial breast cells and the number of lines of tumor cells of the breast. It turned out that TGF-beta exerts pleiotropic effects in the oncogenesis of breast tumors contextual manner, i.e. it inhibits carcinogenesis at an early stage through direct inhibition of angiogenesis and growth of tumor cells. However, the overproduction of TGF-beta in advanced tumor may exacerbate the progression of the disease through indirect stimulation of angiogenesis and immune suppression. Antigen cell membrane CD 105 (endoglin) is associated with TGF-beta and TGF-beta and preferably is expressed in angiogenic vascular endothelial cells. Reducing the CD 105 in HUVEC leads to the suppression of angiogenesis in vitro and high cell death in the presence of TGF-beta. Mouse with no CD 105 killed in the womb in violation of the vascular network, indicating the important role of the CD 105 in the development of blood vessels. Li et al., Microsc. Res. Tech., 52: 437-449 (2001). Abnormal angiogenesis, but not broken hematopoi observed in mice with deficiency of the receptor for the TGF-beta type I. Larsson et al., EMBO J., 20(7): 1663-1673 (2001). In addition, the lack of receptor TGF-beta type II leads to defects in hematopoiesis and vasculogenesis yolk SAC. Oshima et al., Developmental Biology, 179 (1): 297-302 (1996). In addition, abnormalities of the heart and liver and decreased sensitivity to transforming factor-beta 2 growth was observed in embryos deficient receptor TGF-beta type III. Stenvers et al., Mol. Cell Biol., 23(12): 4371-4385 (2003). In addition, directional gap murine gene TGF-beta leads to the development of multifocal inflammatory disease Shull et al., Nature, 359 (6397): 693-699 (1992). It was shown that multifocal inflammation with early-onset mice lack TGF-beta is mediated by lymphocytes. Diebold et al., Proc Natl. Acad. Sci. (USA), 92 (26): 12215-12219 (1995).
The most important non-proliferation function of TGF-beta, is the increase of extracellular matrix. Although, this is mainly achieved through increased transcription of collagen and fibronectin, inhibition of proteases from degrading matrix also contributes to its stability. Degradation of the extracellular matrix is suppressed by reducing the secretion of proteases and simultaneously increasing concentrations of protease inhibitors.
In WO 1984/001106 describes TGF-beta and its application for stimulation of cell proliferation and tissue repair, wound healing and treatment of herbs is. In U.S. patent No. 4806523 revealed TGF-beta and TGF-beta2, both have anti-inflammatory activity, and they are inhibitors of mitogen-stimulated proliferation of T cells and activated b cells. It is also reported that TGF-beta is localized at the centers of hematopoiesis and lymphopoiesis, and that TGF-beta may, therefore, be suitable for the treatment of indications associated with impaired function or dysfunction of hematopoiesis and lymphopoiesis.
It was shown that TGF-beta2 is the predominant isoform TGF-beta in the neural retina, pigmented choroid and vitreous body of the human eye (Pfeffer et al., Exp. Eye Res., 59: 323-333 (1994)), and it was found in ocular fluids of a person in samples from the eye after cataract extraction with intraocular lens implantation. Jampel et al., Current Eye Research, 9: 963-969 (1990). Normal pigment epithelial cells from the retina of a person predominantly secrete TGF-beta2. Kvanta, Ophthalmic Res., 26: 361-367 (1994).
Other diseases that could potentially be treated with antibodies against TGF-beta, include respiratory distress syndrome adults, cirrhosis, post-myocardial infarction, restenosis after angioplasty, keloid scars and scleroderma. Increased expression of TGF-beta2 in osteoporosis (Erlenbacher et al., J. Cell Biol., 132: 195-210 (1996)), this means that the fence is evanie potentially can be treated with antibodies against TGF-beta2.
As a result of participation TGF-beta in the development of a large number of pathological conditions, there is great interest in developing inhibitors of TGF-beta. Many assumptions for inhibitors of TGF-beta based on antibodies.
This is an urgent task to select part of an antibody specific for TGF-beta are the same species. Animals do not normally produced antibodies to their own antigens, this phenomenon was called tolerance (Nossal, Science, 245: 147-153 (1989). Basically vaccination self-antigen does not lead to the production of circulating antibodies. Therefore, it is difficult to induce the formation of antibodies in humans to self-antigen in man. In addition, there are also ethical issues in vaccination of people. When the induction of antibodies, non-human specific to TGF-beta, there are some problems. TGF-beta is an immunosuppressive molecule and additionally have a high conservatism sequence between human and mouse molecules of TGF-beta. Mouse and human TGF-beta differ by only one amino acid residue, the substitution of alanine (a human) to serine (mouse) in a deep rest. Derynck et al., J. Biol. Chem., 261: 4377-4379 (1986). Mouse and human TGF-beta2 differ in only three amino acid residues; residue 59 (T mouse, 's the human who th); the residue 60 (mouse, R human) and the residue 94 (N mouse, human). This makes it difficult for the induction of antibodies in mice against human TGF-beta. In addition, any induced antibodies may be directed only against a limited number of epitopes.
Were obtained monoclonal antibodies against TGF-beta by immunization of chickens and the immortalization of b-cells, used, for example, to diagnose and passive treatment of the disease, as described in U.S. patent No. 6143559.
Were induced polyclonal antibodies, to bind to the human TGF-beta and human TGF-beta2, against both neutralizing and non-neutralizing epitopes in rabbits (Danielpour et al., Growth Factors, 2: 61-71 (1989); Roberts et al., Growth Factors, 3: 277-286 (1990)), chickens (R&D Systems, Minneapolis) and turkeys (Danielpour et al., J. Cell Physiol., 138: 79-86 (1989); Danielpour and Sporn, J. Cell Biochem., 13B: 84 (1989)).
Peptides representing partial or complete sequences of TGF-beta, also used as immunogens to obtain a neutralizing polyclonal antisera in rabbits. Ellingsworth et al., J. Biol. Chem., 261: 12362 (1986); Ellingsworth et al., Cell Immunol., 114: 41 (1988); Border et al., Nature, 346: 371-374 (1990); Flanders, Biochemistry 27: 739-746 (1988); Flanders et al., Growth Factors, 3: 45-52 (1990); Flanders et al., Development 113: 183 to 191 (1991). In addition, there are limited reports about the allocation of mouse monoclonal antibodies against TGF-beta. After immunization with bovine TGF-beta2 (identify the major human TGF-beta2) identified three non-neutralizing monoclonal antibodies which were specific for TGF-beta2 and one neutralizing antibody specific for TGF-beta and TGF-beta2. Dasch et al., J. Immunol., 142: 1536-1541 (1989). In another message after immunization of human TGF-beta allocated neutralizing antibodies specific for either TGF-beta or cross-reactive with TGF-beta, TGF-beta2 and TGF-beta. Lucas et al., J. Immunol., 145: 1415-1422 (1990). It was shown that the polyclonal anticavity to human and porcine TGF-beta (Keski-Oja et al., Cancer Res., 47: 6451-6458 (1987)) and porcine TGF-beta2 (Rosa et al., Science, 239: 783-785 (1988)) will neutralize the biological activity, respectively TGF-beta and TGF-beta2. Rabbit anti-TGF-beta serum described by Roberts et al., Proc. Natl. Acad. Sci. USA, 83: 4167-4171 (1986). In addition, were obtained RIA against TGF-beta using rabbit antisera to quantify the released protein during platelet aggregation. Assoian and Sporn, J. Cell Biol., 102: 12178-1223 (1986).
Industrial is available neutralizing mouse monoclonal antibody, which binds to the isoform TGF-beta2 and TGF-beta, production Genzyme Diagnostics. Industrial available mouse monoclonal antibody against human TGF-beta production R&D Systems. This antibody only in a low degree neutralizes TGF-beta in the neutralization test. Were also obtained by neutralizing mouse monoclonal antibodies from the mouse is, immunized human peptides, TGF-beta having the amino acid positions 48-60 (antibody reacted with TGF-beta, TGF-beta2 and TGF-beta) and amino acid position 86-101 (antibody specific for TGF-beta). Hoefer and Anderer, Cancer Immunol. Immunother., 41: 302-308 (1995).
Using ragovoy technologies antibodies (WO 1992/01047; WO 1993/19172; WO 1992/20791; WO 1993/06213 and WO 1993/11236) was offered the opportunity to highlight human antibodies against human TGF-beta. Describes the selection of antibodies from the spectrum segments antibodies located in the phage. Griffiths et al., EMBO J. 12: 725-734 (1993); Nissim et al., EMBO J. 13: 692-698 (1994); Griffiths et al., 13: 3245-3260 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA, 90: 10003-10007 (1993) and WO 1993/11236. In addition, Tempest et al., Immunotechnology, 2: 306 (1996) describes human antibodies specific for human TGF-beta, obtained from libraries of phage display.
In WO 1997/13844 revealed the selection of human antibodies specific for human TGF-beta, and human antibodies specific for human TGF-beta2. It describes the antibody with the VH domain 31G9 and variants of the domain, more specifically, the antibody CS37, which includes domain VH 31G9 with VL CS37 and variants of this domain, including antibodies, which are: (i) compete with CS37 in ELISA for binding to TGF-beta; (ii) is preferably associated with TGF-beta compared with TGF-beta and (iii) neutralizing TGF-beta.
U.S. patent No. 6492497 about novan on the identification of antibodies, related to CS37, but which unexpectedly possess a predominant properties in relation to binding and neutralization of TGF-beta. They are not bound or not neutralize TGF-beta2 or TGF-beta. The epitope for these antibodies is in the C-terminal region of TGF-beta (residues 83-112) and includes the loop comprising residues 92-98 TGF-beta, also known as "the finger" ("finger") 2, an area that was identified as interacting with the receptor for TGF-beta.
Monoclonal antibody against human TGF-beta, which is highly specific and can be used to diagnose tumors and affinity chromatography, as disclosed in the patent application of Japan 95068278 B2, published on July 26, 1995
The use of TGF-beta and its antagonists to modulate blood pressure and to treat accordingly hypertension and hypotension disclosed in WO 1991/19513.
In WO 1991/15223 disclosed purified factor suppression of respiratory attack, which can be incubated with Turkey anti-TGF-beta antibody that specifically binds to TGF-beta. Antibody completely neutralized the activity of TGF-beta on activated macrophages, but had no effect on the factor of suppression of respiratory attack on the macrophage.
Suppressing the activity of TGF-beta and accumulation of extracellular matrix in the diagnosis Leche and the Institute of fibrotic diseases, such as glomerulonephritis, contacts with suppressor activity of ECM products, such as anti-TGF-beta antibody disclosed in WO 1991/04748 and WO 1993/10808. Also disclosed antibodies against a linear peptide of TGF-beta, and cells producing antibodies.
In U.S. patent No. 5888705 disclosed method of induction of cell proliferation in the pancreas of the adult person or of their differentiation by contacting primary cultures of such cells with a single growth factor, hepatocyte or in combination with anti-TGF-beta antibodies.
In WO 2001/66140 disclosed the use of antagonists of TGF-beta, such as antibodies, for the treatment or prevention of loss of renal function.
In WO 2000/40227 disclosed methods of treating conditions associated with the accumulation of excess extracellular matrix using tools that inhibit TGF-beta, such as antibodies.
Antibodies to TGF-beta are disclosed as means of weakening the apoptosis of renal tubules with unilateral occlusion of the ureter, Miyajima et al., Kidney International., 58: 2301-2313 (2000).
Prolonged prophylaxis renal failure, redundant gene expression matrix and increase glomerular mesangial matrix in the processing of monoclonal anti-TGF-beta antibody in mice db/db diabetic disclosed Ziyadeh et al., Proc. Natl. Acad. Sci. USA, 97(14): 8015-8020 (2000).
Described good re is the query result of treatment with neutralizing anti-TGF-beta antibodies in experimental kidney disease in diabetes, Han and Ziyadeh, Peritoneal dialysis international, 19 Suppl 2: S234-237 (1999). It was found that TGF-beta is a key mediator in the development of hyperglycemia and renal disease in diabetes. Sharma and Ziyadeh, Diabetes, 44 (10) p 1139-46 (1995). The use of TGF-beta in diabetic nephropathy disclosed Border et al., Diabetes Metab. Rev., 12/4: 309-339 (1996).
In U.S. patent No. 5662904 describes a composition for use in the treatment of wounds to suppress the formation of runaway tissue. An example of such composition is an antibody neutralizing the growth factor, such as antibodies against TGF-beta, TGF-beta2 and PDGF.
In U.S. patent No. 5972335 disclosed compositions containing at least two antibodies, for use in stimulating wound healing in fibrotic diseases, where the first antibody is specific for a single epitope in TGF-beta and the second antibody specific for a single epitope in TGF-beta2.
In U.S. patent No. 5958411 disclosed methods of treatment of diseases of the Central nervous system by the introduction of a neutralizing anti-TGF-beta antibodies.
In U.S. patent No. 5616561 disclosed is a method of treatment of tissue damage caused by irradiation, using antagonists of TGF-beta, such as antibodies.
In U.S. patent No. 6500920 disclosed peptide of 10-25 amino acids containing amino acids 49-58 TGF-beta2, where the peptide is able to inhibit the specific binding of TGF-beta to its receptor TGF-beta in the cell.
New antibodies for the treatment of disorders associated with TGF-beta, for example, osteoporosis, AIDS, cancers, etc. are disclosed in WO 1992/00330 and U.S. patent No. 5262319. Such antibodies bind to human TGF-beta and do not show cross-reactivity with TGF-beta and TGF-beta2.
In U.S. patent No. 6509318 disclosed a family of small peptides with inhibitory against TGF-beta activity for applications such as suppression of the formation of runaway tissue during wound healing.
The use of compounds (e.g., antibodies), which can inhibit the biological activity of TGF-beta on pre-damaged neurons for the treatment of cerebral disorders, such as cerebral ischemia, revelation is about in WO 2000/13705.
Monoclonal antibody that recognizes all three isoforms of TGF-beta, which can inhibit the biological activity of TGF-beta on pre-damaged neurons for the treatment of cerebral disorders, disclosed in WO 2000/54804. This antibody was used for neutralization of endogenous TGF-beta during the main period of ontogenetic cell death ciliary node (CG) and spinal sites (DRG), and motor neurons of the spinal cord in chicken embryos.
Diagnosis or prediction of the likelihood of susceptible to resistant to tamoxifen or tamoxifen breast tumors using antibodies specific for angiogenic factors or receptors, such as an antibody specific to TGF-beta, disclosed in WO 2000/34788.
In EP 945464 B1 disclosed to members of specific binding with human TGF-beta, i.e. the members of the specific binding containing human domains bind an antigen-antibody specific for human TGF-beta, which preferably specifically associated with the isoforms of TGF-beta2 and TGF-beta, or both, compared with TGF-beta. Members with specific binding can be extracted and used for the treatment of diseases, in particular, fibrotic diseases, and immune/inflammatory diseases.
It was shown, Thu the antibodies against TGF-beta are effective in the treatment of glomerulonephritis (Border et al., Diabetes Metab. Rev., above); neuronal scarring (Logan et al., Eur. J. Neurosci., 6: 355-363 (1994); WO 1993/19783); scarring of the skin (Shah et al., Lancet, 339: 213-214 (1992); Shah et al., J. Cell Science, 107: 1137-1157 (1994); Shah et al., J. Cell Science, 985-1002 (1995); WO 1992/17206; fibrosis of the lungs (Giri et al., Thorax, 48: 959-966 (1993)); damage to the arteries (Wolf et al., J. Clin. Invest., 93: 1172-1178 (1994)); and rheumatoid arthritis (Wahl et al., J. Exp. Medicine, 177: 225-230 (1993)). It has been suggested that TGF-beta acts antagonistically against TGF-beta and TGF-beta2 in scarring of the skin (Shah et al., 1995, above).
Arteaga et al., J. Clin. Invest., 92: 2569-2576 (1993) reveal that anti-TGF-beta antibodies inhibit carcinogenesis tumor breast cells and increase the activity of murine natural killer cells in the spleen.
Antifibrosis tools for wound healing and treatment of fibrotic disorders, including anti-TGF-beta antibodies described in WO 1993/19769.
The specific sequence of anti-TGF-beta2-antibodies are described in EP 853661 B1.
Other applications where antibodies against TGF-beta showed promising therapeutic efficiency include the use of antibodies against TGF-beta for the treatment of eye diseases, including fibrosis of the eye, including proliferative retinopathy (Pena et al., Invest. Ophthalmology Vis. Sci., 35: 2804-2808 (1994)), cataract prevention (WO 1995/13827), retinal detachment and oranyone after surgery for glaucoma (Khaw et al., Eye, 8: 188-196 (1994)). 'connor et al., J. Clin Invet., 83: 1661-1666 (1989) showed that significantly higher concentrations of TGF-beta2 were aspirate vitreous from patients with intraocular fibrosis associated with proliferative retinopathy compared with patients with uncomplicated retinal detachment without fibrosis eyes and that the biological activity of this TGF-beta2 can be neutralized by antibodies against TGF-beta2.
The use of antibodies against TGF-beta for the treatment of diseases has been the subject of patent applications for fibrotic diseases (WO 1991/04748); diseases of deficiency of macrophages (WO 1993/14782); macrophage pathogenic infections (WO 1993/17708; U.S. patent No. 5730976); and vascular diseases (WO 1993/21945).
Processed TGF-beta antibody composition of stem cells that can survive for 14 days in vitro or ex vivo, and rapid repopulation of the hematopoietic system in vivo, described in WO 2000/43499.
Scrip 2580 p 14, 4 October 2000 reported that Cambridge Antibody Technology (CAT) and Genzyme collaborated to develop human monoclonal antibodies against TGF-beta. The SAT has two full human TGF-beta antibody, CAT-152 and CAT-192, Genzyme - 1D11, mouse poly-specific monoclonal antibody that neutralizes TGF-beta, TGF-beta2 and TGF-beta, and he was recognized as a potential effective treatment for diffuse sclerodermia has developed a human analogue of 1D11 using phage display technology resulting in. Several other clinical indications for treatment with anti-TGF-beta antibodies, including ophthalmic indications, scarring after surgery, fibrosis of the main organs, such as lungs, kidneys and liver, and some malignant tumors will also be discussed along with treatment of malignant brain tumors by inhibition of growth by TGF-beta2. CAT-152 (anti-TGF-beta2) is in phase II clinical trials for the prevention of postoperative scarring in patients undergoing surgery for glaucoma, and for CAT-192 (anti-TGF-beta) has completed phase I clinical trials. See also "Trends in Antibody Research: The Monoclonal Elite", Tim Searle, Bioventure View 1510, p. 14, October 1, 2000
Method of quantitative determination of TGF-beta using anti-TGF-beta antibodies disclosed in WO 1995/19987. A new test for the determination of active TGF-beta in the sample with the use of eukaryotic cells, which contain TGF-beta reaction expressing vector, described in WO 2000/00641. This test involves determination of the concentrations of the isoforms of TGF-beta in the sample, where the frozen slices pre-incubated with neutralizing antibody isoforms of anti-TGF-beta. The immunoassays based on TGF-beta using TGF-beta antibodies are described, for example, in Japan patent 2126157 and the patent application of Japan 92041307, published July 7, 1992/p>
Darland and D'amore, J. Clin. Invest., 103: 157-158 (1999) reveal that the development of vessels proceeds from the stage, depending on the growth factor when the loss factor of survival leads to apoptosis. Stabilization of vessels observed at the coating moralnymi cells, local activation of TGF-beta and production of the basal membrane. Several questions arise regarding the role of growth factors to formed blood vessels, including VEGF and TGF-beta. Benjamin et al., J. Clin. Invest., 103: 159-165 (1999) revealed a selective disappearance of undeveloped blood vessels in the developed human tumors after removal of VEGF.
Methods of obtaining chimeric and humanized antibodies is described in, and other sources in this area, including, for example, U.S. patent No. 6235883 full of monoclonal antibodies against receptor human epidermal growth factor; ER 184187 on mouse-human chimeric antibody; EP 844306 method for producing a recombinant antibodies using ragovoy technology; U.S. patent No. 5859205 for obtaining CDR-transplanted antibody, preferably a humanized antibody containing the non-human donor and a human acceptor or frame sections, EP 120694; EP 125023; EP 171496; EP 173494; EP 239400; WO 1989/07452; WO 1990/07861 and WO 1986/01533 on methods of producing humanized antibodies; application for U.S. patent No. 2003/0039649 on SuperG monitorowanym antibody; application for U.S. patent No. 2003/0039645 on humanized antibodies with specificity for human TNF-alpha; EP 239400 on recombinant antibodies and their receipt; WO 1991/09967 on humanized antibody; WO 1992/01047 for obtaining antibodies; WO 1992/22653 on methods of producing humanized antibodies; WO 1993/11161 by polyvalent antigennegative proteins; WO 1994/13804 by polyvalent antigennegative proteins; WO 2000/66631 for members with specific binding to TGF-beta; and Henry "Special Delivery: Alternative methods for delivering drugs improve performance, convenience and patient compliance". C&EN, p. 49-65 (2000). See also U.S. patent No. 6140471 and 5969108, and 5872215, and 5871907, and 5858657, and 5837242, and 5733743; EP 1024191; EP 774511; WO 1997/13844; EP 656941 and 605522, and WO 1994/13804; EP 589877; EP 585287; WO 1993/19172; EP 540586; WO 1993/06213; WO 1992/20791; WO 1992/01787 and WO 1992/01047. In addition, in WO 2004/065417 revealed various changes in antibodies and antigenspecific fragments to improve yield. See also U.S. patent 20050049403.
There is a need for regulation of molecules of TGF-beta to prevent their negative effect in the development of diseases, such as presented above. Also there is a need to provide monoclonal antibodies with high affinity, which are specifically associated with TGF-beta and neutralize the activity of TGF-beta in such a way that they act as antagonists of TGF-beta. The apparent lack of regulation of TGF-beta in cells is Oh tumors in combination with suppression of immune function and induced TGF-beta education stroma makes the potential impact of antagonists of TGF-beta attractive option for the treatment of malignant tumors. In addition, TGF-beta antibodies are suitable for the production of diagnostic tests and immunoaffinity cleanup.
In the first aspect of the present invention relates to humanitarianlaw the antibody that binds to TGF-beta containing the variable domain of the heavy chain (VH), which includes the remains of inhuman hypervariable region included in the human domain (VH), where the specified variable domain contains a replacement frame region (FR) in the sequence SEQ ID NO: 6 in a position selected from the group consisting of 48, 49, 67, 69, 71, 73 and 78 when using the numbering system presented Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes, Bethesda, MD (1991). The antibody includes an intact IgG1 antibody or antibody fragment, such as Fab.
Preferably humanitariannet antibody includes replacement FR at positions 49, 67 and 71, where more preferably in position 49 alanine substituted for glycine at position 67 phenylalanine replaced with alanine and at position 71 arginine is replaced by alanine.
In another preferred embodiment, humanitariannet antibody includes replacement FR in regulations 48, 49 and 71, where more preferably at position 48 valine is replaced by isoleucine, at position 49 alanine substituted for glycine in position 71 arginine samananda alanine.
Also preferably, humanitariannet antibody includes replacement FR at positions 49, 69 and 71, where more preferably in position 49 alanine substituted for glycine at position 69 isoleucine replaced with leucine and at position 71 arginine is replaced by alanine. In another aspect of an additional replacement FR is in position 73, more preferably at position 73 arginine is replaced by lysine.
In another preferred embodiment, humanitariannet antibody includes replacement FR at positions 49, 71 and 73, more preferably at position 49 alanine substituted for glycine at position 71 arginine is replaced with alanine and at position 73 asparagine is replaced by lysine.
In another preferred embodiment, humanitariannet antibody includes replacement FR at positions 49, 71 and 78, more preferably at position 49 alanine substituted for glycine at position 71 arginine is replaced with alanine and at position 78 leucine is replaced by alanine.
In another preferred embodiment, any of the antibodies described above, contains the remains of determining the complementarity of the plot (CDR) of the variable domain of the light chain (VL) RASQSVLYSSNQKNYLA (sequence SEQ ID NO: 36); WASTRES (sequence SEQ ID NO: 38) and HQYLSSDT (sequence SEQ ID NO: 40) or contains the remains of CDR domain VLwhere the first CDR (CDR L1) RewriteRule in the follower of the ity locus Kappa human germ line L8/L9: RASQGISSYLA (sequence SEQ ID NO: 37) and/or the second CDR (CDR L2) RewriteRule in the sequence of the Kappa locus of the human germ line L8/L9/L14/L15: YASSLQS (sequence SEQ ID NO: 39). In another preferred embodiment, any of the antibodies described above, contains the remains of determining the complementarity of the plot variable domain of the heavy chain (VH) GYAFTNYLIE (sequence SEQ ID NO: 41); VNNPGSGGSNYNEKFKG (sequence SEQ ID NO: 42), or VINPGSGGSNYNEKFKG (sequence SEQ ID NO: 43) and SGGFYFDY (sequence SEQ ID NO: 44).
The invention relates to any of the antibodies described above, anywherefrom with a cytotoxic agent or not anywherefrom so. In addition, the invention relates to compositions containing such antibodies, and the media.
In an additional embodiment, the invention relates to a selected nucleic acid that encodes humanitariannet antibody, a vector that includes the specified nucleic acid and cell host containing such nucleic acid.
Additionally, the invention relates to a method for gumanitarnogo antibodies involving culturing a host cell containing nucleic acid encoding the antibody, so that the nucleic acid is expressed and produced antibody, and preferably stands out from the culture of host cells, preferably from the culture medium host cells. The cell master can be cotranslational with a vector containing a nucleic Ki the lot, encoding a variable domain of the heavy chain, and with a vector containing a nucleic acid encoding a variable domain of the light chain.
In yet another embodiment, the invention relates to a method of treating disorders associated with TGF-beta in a mammal, preferably a Primate, and more preferably a human, involving the administration to a mammal an effective amount of gumanitarnogo antibodies. The method may additionally include an introduction to the mammal an effective amount of a therapeutic agent other than humanitariannet antibody, such as chemotherapy, antiangiogenic or cytotoxic agent, or a cytokine.
In yet another embodiment, the invention relates to a method for detection of TGF-beta in a sample from an organism, which involves contacting gumanitarnogo antibodies with a sample from an organism and determining whether the associated antibody to TGF-beta.
The invention also relates to a product containing container containing humanitariannet antibody and user manual with instructions for the treatment of disorders associated with TGF-beta in a mammal, preferably human, with antibody. This product can also include a container containing a therapeutic agent other than humanitariannet EN is Italo, where there are instructions for the user with guidance on the treatment of impaired antibody in combination with the tool.
In another embodiment, this invention relates to a method of treating a malignant tumor in a mammal, involving the administration to a mammal an effective amount of antibodies against TGF-beta and antibody that binds to vascular endothelial growth factor. Preferably the mammal is a human. In another embodiment, the antibody against TGF-beta is associated with one or more of the following: TGF-beta, TGF-beta2 and TGF-beta. In an additional embodiment, the antibody binds to TGF-beta or TGF-beta and TGF-beta2.
Brief description of figures
On figa and 1B presents the comparison of amino acid sequences of the variable domain of the light chain (VL) (figa) and the variable domain of the heavy chain (Vn) (pigv) mouse monoclonal antibody 2G7 (respectively the sequences SEQ ID NO: 1 and 2); domain VLand Vngumanitarnogo option hux (V5H.V5L) (respectively the sequences SEQ ID NO: 3 and 4) and human VLand Vnthe consensus skeleton plots (hum κ1, subgroup I light chain Kappa; humIII, subgroup III heavy chain) (respectively the sequences SEQ ID NO: 5 and 6). C is ezDock indicate differences between humanized hux and mouse monoclonal antibody 2G7 or between humanized hux and human consensus frame sections. Underlined region complementarity determining (CDR), and the CDR of the actual sequence of the human germ line are below the consensus skeleton plots for comparison of the sequence SEQ ID NO: 7-11).
Figure 2 presents the DNA sequence (the sequence SEQ ID NO: 12-17), encoding different areas CDR (sequence SEQ ID NO: 18-23).
Figure 3 presents the amino acid sequence 709.landH.IgG1 (sequence SEQ ID NO: 24); H2NI.V5L (sequence SEQ ID NO: 25); V11H.V11L (sequence SEQ ID NO: 26); V5H.V5L (sequence SEQ ID NO: 27); chimL.chim.H (sequence SEQ ID NO: 28) and V5H.g1L2 (sequence SEQ ID NO: 29).
Figure 4 presents nukleinovokisly sequence with and without signal sequences, coding sequences, is shown in figure 3 (a sequence of SEQ ID NO: 30-35).
Figure 5 presents the curves of binding of the variant humanized IgG antibody 2G7 with TGF-beta.
Figure 6 presents the sequence of the plasmid pDR1 (sequence SEQ ID NO: 45, 5391 P.N.) for expression of immunoglobulin light chains as described in example 2. Plasmid pDR1 contains sequences encoding irrelevant antibody, i.e. light chain gumanitarnogo anti-CD3 antibodies (Shalaby et al., J. Exp. Med., 175: 217-225 (1992)), start - and stop-codons, which are indicated in the frame and underlined
Figure 7 presents the sequence of the plasmid pDR2 (sequence SEQ ID NO: 46, 6135 P.N.) for expression of immunoglobulin heavy chains as described in example 2. Plasmid pDR2 contains sequences encoding irrelevant antibody, i.e. a heavy chain gumanitarnogo anti-CD3 antibodies (Shalaby et al., above), start - and stop-codons, which are marked in bold and underlined.
On Fig presents curves of the binding of humanized antibodies revertants germline 2G7 with TGF-beta.
Figure 9 presents the results of the block in the test proliferation of murine mesangial cells using antibody 2G7 against TGF-beta and several variants of humanized antibodies against TGF-beta.
On figa-10C shows the neutralization of TGF-beta at three different concentrations, respectively, in the test fibroblasts T three humanitarianism antibodies against TGF-beta (H2NI.V5L, H2NI.g1L2 and V5H.g1L2) and mouse antibody 2G7.
On figa-11C shows the neutralization of TGF-beta2 in three different concentrations, respectively, in the test fibroblasts T four antibodies presented on figure 10.
On figa-12C shows the neutralization of TGF-beta at three different concentrations, respectively, in the test fibroblasts T four antibodies presented on figure 10.
On figa-13C n the cauldron neutralization of TGF-beta, -beta2 and-beta at a concentration of 2 ng/ml, respectively in the test fibroblasts T four antibodies presented on figure 10.
On figa-14D shows the neutralization of the three isoforms of TGF-beta in the test fibroblasts T humanized antibody H2NI.V5L (figa), humanized antibody H2NI.g1L2 (pigv), mouse antibody 2G7 (figs) and humanized antibody V5H.g1L2 (fig.14D).
On figa and 15V presents the results of ELISA for production and inhibition of TGF-beta in normal and neoplastic epithelial cells. On figa shows the production of TGF-beta in vitro of normal epithelial cells (EPC) (C) and tumor cells EPC (model T). On FIGU shows the effect of anti-TGF-beta antibody 2G7 in vivo on the level of TGF-beta in serum in tumor cells EPC in comparison with the control IgG antibody (isotype IgG, which is antidilutive antibody) in normal and tumor cells EPC. The same control IgG was used for the other figures, and Fig used control IgG, which may or may not be the same.
On figa and 16B shows the effect of anti-TGF-beta antibody 2G7 secondary tumors of the lung compared with control IgG. On figa presents the scoring of histological studies with graduation and the number of affected shares to control the CSOs IgG antibodies and anti-TGF-beta antibody 2G7, and figv presents tissue mass in grams and percentage of body weight for the same control and anti-TGF-beta antibody 2G7.
On Fig presents the effect of anti-TGF-beta antibody 2G7 to quantify uCT lung tumors compared with control IgG-volume tumors and number of tumors.
On figa and 18V presents the effect of anti-TGF-beta antibody 2G7 and chemotherapy on the model of mammary tumor T in comparison with the control IgG. On figa shows tumor volume as a function of time after injection of control IgG with saline, control IgG and docetaxel (TAXOTERE®) and anti-TGF-beta antibody 2G7 and docetaxel (TAXOTERE®). On FIGU presents a mass of tissue of brain, lung, spleen and tumors for the two controls (IgG with and without docetaxel (TAXOTERE®)and anti-TGF-beta antibodies with docetaxel (TAXOTERE®).
On Fig presents concentrations of VEGF (PCG/ml) in plasma in mice without tumors (norm) or in mice with breast tumors T treated with control IgG antibody (control) or anti-TGF-beta antibody 2G7).
On figa and 20B presents the effect of TGF-beta antibody 2G7 on another model of breast cancer PymT. On figa shows tumor volume as a function of tumor growth in the day for TGF-beta antibodies and control IgG antibodies, and figv presents a mass of tissue for anti-TGF-beta antibodies and to ntrolling IgG antibodies.
On figa and 21B shows a model of murine B16 melanoma and the effect of TGF-beta antibody 2G7 and control IgG antibodies. On figa presents the percentage of mice with lung tumors in relation to the surface and pathology for the control and TGF-beta antibodies, and figv presents the number of tumors is released to the surface and ARTICLE for the control and TGF-beta antibodies.
On Fig presents the effect of TGF-beta antibody 2G7 on the B16 tumor growth (volume) within 14 days after vaccination compared with the control IgG.
On Fig presents the effect of TGF-beta antibody 2G7 on the number of visible lung metastases B16 (E6) compared with the control IgG.
On Fig presents the effect of TGF-beta antibody 2G7 on the B16 tumor growth (volume) within 17 days after inoculation compared to the control IgG.
On Fig presents the effect of TGF-beta antibody 2G7 on the mass of the primary tumor B16 compared with control IgG.
On Fig presents the effect of TGF-beta antibody 2G7 on the number of visible metastases of B16 (E7) compared with the control IgG.
On Fig presents the effect of TGF-beta antibody 2G7, mouse monoclonal anti-VEGF antibody A and a combination of two antibodies on tumor volume in the transplant of human lung tumor cells Calu-6 in mice in comparison with control IgG for a period of time up to 42 days. Processing different what Redstone started 2 days.
On Fig presents masses of tumors to experience with Calu-6, shown in Fig, for the three types of treatments antibodies and control IgG.
A detailed description of the preferred embodiments
The terms "TGF-beta and transforming growth factor-beta" are used herein interchangeably, and they belong to the family of molecules, described above, which contain full-length, the natural amino acid sequence of any of the factors TGF-beta from human rights, including the latent form and is associated or not associated complex of precursor and Mature TGF-beta ("latent TGF-beta). When referring here to such TGF-beta should be understood that this is an appeal to any of the identified currently forms, including TGF-beta, TGF-beta2, TGF-beta, TGF-beta and TGF-beta and their latent variants, and human TGF-beta, which will be defined in the future, including polypeptides derived from the sequence of any known TGF-beta and which, at least about 75%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% and even more preferably at least about 95% homologous to the sequence. Particular those who mines "TGF-beta", "TGF-beta2 and TGF-beta"and "TGF-beta" and "TGF-beta" refers to the factors TGF-beta, which is defined in the literature, for example, Derynck et al., Nature, above, Seyedin et al., J. Biol. Chem., 262, above and deMartin et al., above. The term "TGF-β" refers to a gene codereuse human TGF-beta. The preferred factor TGF-beta is natural, the human sequence of TGF-beta.
The members of the family of TGF-beta are defined as those having nine cysteine residues in the Mature part of the molecules that share at least 65% homology with other known sequences of TGF-beta in the Mature region, and which may compete for the same receptor. In addition, it was found that all of them are encoded in the form of larger predecessor, which has a region with high homology near N-Terminus and conservative in relation to three cysteine residues in the field of the predecessor, which is removed later during processing. In addition, it was found that TGF-beta has a website processing of four or five amino acids.
The polypeptide with the "natural sequence" is one that has the same amino acid sequence as the polypeptide (for example, TGF-beta), as in nature. Such polypeptides with "natural sequence" can be distinguished from a natural source or can be obtained recombi is based or synthetic means. Thus, a polypeptide with "natural sequence" may have the amino acid sequence of natural human polypeptide, mouse polypeptide or polypeptide from any other species of mammals.
The term "variant amino acid sequences" refers to polypeptides having amino acid sequences that are somewhat different from the polypeptide with the natural sequence. Generally, amino acid sequence variants will possess at least 70% homology with the polypeptide with the natural sequence or its region, which is compared with the variant, and preferably they will be at least about 80%, more preferably at least about 90%, and even more preferably at least about 95% homologous with that polypeptide with the natural sequence or area. Variants of the amino acid sequence contains substitutions, deletions and/or insertions at certain positions in the natural amino acid sequence.
"Homology" is defined as the percentage of residues in the variant amino acid sequence that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. IU the odes and computer program for aligning well known in this field. One such computer program is a "Align 2", owned by Genentech, Inc., which was filed with the documentation for a user in the United States Copyright office, Washington, DC 20559, December 10, 1991
The term "antibody" is used here in its broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies (e.g., bivalent antibodies)obtained at least two intact antibodies, and antibody fragments, provided that they exhibit the desired biological activity.
The term "monoclonal antibody"in the sense in which it is used here, refers to an antibody obtained from a population of mostly homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to the preparations of polyclonal antibodies, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinants. In addition to their specificity, monoclonal antibodies have the advantage, namely, h is about them can be synthesized without admixtures of other antibodies. The definition of "monoclonal" indicates the character of the antibody that is derived mainly from a homogeneous population of antibodies, and is not limited to the production of the antibody by any particular method. For example, monoclonal antibodies for use in 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" also can be isolated from phage libraries of antibodies using, for example, methods described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991).
Monoclonal antibodies here specifically include "chimeric" antibodies in which a plot heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. patent No. 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Interested hee is Ernie antibodies here include "primaryservername" antibodies, containing antigennegative sequence of the variable domain obtained from a Primate, non-human (e.g., lower old world monkeys, the highest APE and so on), and the sequence of the human constant region.
"Fragments of antibodies include the site of the intact antibody, preferably containing antigennegative or variable region. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')2and Fv; dyatel; linear antibodies; single-stranded molecules of antibodies and multivalent antibodies derived from a fragment(s) of the antibody.
"Intact" antibody is such that contains antigennegative variable region and the constant domain of the light chain (CL) and the constant domains of the heavy chain CH1, CH2 and CH3. The constant domains may represent a constant domains with the natural sequence (e.g., human constant domains with the natural sequence or amino acid sequence variants. Preferably, the intact antibody has one or more effector functions.
"Effector functions" antibodies refer to such biological activities, which are inherent in the Fc region (Fc region natural sequence or variant Fc region amino acid sequentially the tee) antibodies. Examples of effector functions of antibodies include C1q binding; complementability cytotoxicity; the binding of the Fc receptor; mediated antibody-dependent cell cytotoxicity (ADCC); phagocytosis; lower regulation of cell surface receptors (e.g., receptor b-cell; BCR), etc
Depending on the amino acid sequence of the constant domain of their heavy chains of intact antibodies can be divided into different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into "subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Stuck-at domains of the heavy chain, which correspond to the different classes of antibodies, denote, respectively, α, δ, ε, γ and µ. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.
"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to mediated cell reaction in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer cells (NK), neutrophils, and macrophages) recognize bound antibody on the target cell and then cause lysis of the target cells. Primary cells for mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes, Express the comfort FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-92 (1991). To assess ADCC activity for molecules of interest can be put to the test ADCC in vitro, such as described in U.S. patent No. 5500362 or 5821337. Suitable effector cells for the production of such tests include the mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additionally, ADCC activity of the interesting molecules can be assessed in vivo, e.g., in animal models, such as disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95: 652-656 (1998).
"Human effector cells are leukocytes which Express one or more FcR and perform effector functions. Preferably, the cells Express at least FcγRIII and perform effector function is ADCC. Examples of human leukocytes which mediate ADCC include mononuclear cells of peripheral blood (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils, while PBMC and NK-cells are preferred. Effector cells can be isolated from their natural source, for example, from blood or PBMC, as described here.
The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the region is part of an Fc antibody. Preferred FcR is human FcR with the natural sequence. Moreover, a preferred FcR is one that binds to IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and alternative splanirowannya forms of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains a motif-based activation of tyrosine immunoreceptor (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an inhibition motif-based tyrosine immunoreceptor (ITIM) in its cytoplasmic domain (see review Daëron, Annu. Rev. Immunol., 15: 203-234 (1997)). Review FcR look at Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-492 (1991); Capel et al., Immunomethods, 4: 25-34 (1994) and de Haas et al., J. Clin. Med., 126: 330-41 (1995). Other FcR, including those that will be opened in the future, unite here, the term "FcR". The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol., 117: 587 (1976) and Kim et al., J. Immunol., 24: 249 (1994).
"Complement-dependent cytotoxicity" or "CDC" refers to the ability of the molecule to lyse target in the presence of the comp is amenta. Path activation of complement is initiated by binding of the first component complemental system (C1q) with a molecule (e.g. antibody), forming a complex with a cognate antigen. To assess activation of the complement fixation test can be performed by the CDC, for example, described in Gazzano-three-bet et al., J. Immunol. Methods, 202: 163 (1996).
"Natural antibodies" are usually heterotetrameric glycoproteins weight of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different isotypes of immunoglobulins. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH), followed by more constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. Constant domain of the light chain is built with the first constant domain of the heavy chain variable domain and the light chain is built from the variable domain of the heavy chain. Believe that certain amino acid residues form a boundary surface between the variable domains of the light chain and heavy chain.
The term "variable" refers to the fact that certain sections of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed in the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in the variable domains of the light chain and heavy chain. More highly conservative areas of variable domains are called the frame sections (FR). Each variable domain of the natural heavy and light chains comprises four FR, basically taking a configuration of a β-fold, connected by three hypervariable regions, which when combined form a loop, and in some cases form part of the structure of β-folds. Hypervariable region in each chain are located together in close proximity by the FR and hypervariable regions of the other chain, contribute to the education antigennegative site of antibodies (see Kabat et al., above). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in the manifestation of antibody-dependent cellular cytotoxicity (ADCC).
The term "hypervariable region is here", in the sense in which it is used here, refers to the amino acid residues of an antibody which are responsible for binding to the antigen. Typically, a hypervariable region contains amino acid residues from a "complementarity determining plot" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al., above) and/or residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)). The remains of the "frame area" or "FR" are variable domain residues other than residues hypervariable region, as defined here.
Cleavage of antibodies with papain leads to the formation of two identical antigenspecific fragments, called " fragments "Fab", each with one antigennegative site, and a residual fragment "Fc", whose name reflects its ability to easy crystallization. Treatment with pepsin gives the fragment F(ab')2that contains two antigenspecific site and it is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antipersonnel and antigens the binding site. This region consists of a dimer variable domain of one heavy chain and one light chain in a tight non-covalent linkages. She has such a configuration that the three hypervariable region of each variable domain interact with the formation antigennegative site on the surface of the dimer VH-VL. Together the six hypervariable region give the antibody antigennegative specificity. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although with low affinity compared with the whole binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fragments, Fab' differ from Fab fragments by the addition of a few residues at the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteine residues of the hinge region of the antibody. Fab'-SH denotes here Fab'in which the residue of cysteine constant domains has at least one free Tilney group. Fragments of antibodies F(ab')2were initially obtained in the form of pairs of fragments, Fab', which are hinged cysteine between them. Also known other chemical combinations of fragments of antibodies.
"Easily the e chains" of antibodies from any vertebrate species can be divided into two distinct types, called Kappa (κ) and lambda (λ), based on amino acid sequences of their constant domains.
Antibody fragments "single-chain Fv" or "scFv" contain domains VHand VLantibodies, where these domains are in the same polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between domains VHand VLthat enables the scFv to form the desired structure for antigen binding. A review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, p. 269-315 (1994). Described fragments of scFv anti-TGF-beta antibodies in WO 1993/16185; U.S. patent No. 5571894 and U.S. patent No. 5587458.
The term "diately" refers to small fragments of the antibodies with two antihistamine sites, where these fragments include the variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). When using a linker that is too short to connect the two domains on the same chain, the domains are forced to connect with complementary domains of another chain and form two antigenspecific site. Diately described in more detail, for example, in EP 404097; WO 1993/11161 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
"Humanized" forms of non-human antibodies (e.g., rodents) are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. Basically, humanized antibodies are human immunoglobulins (recipient antibody)in which residues of the hypervariable region of the recipient are replaced by residues from a hypervariable region species, non-human (donor antibody)such as mouse, rat, rabbit, or Primate, non-human, having the desired specificity, affinity and efficacy. In some cases, remnants of the area frame plot (FR) of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. Data modification is performed to further improve the efficiency of antibody. As a rule, humanitariannet antibody will contain mostly, at least one, and typically two, variable domain, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin or all or substantially all of the FR, which are part of a human immunoglobulin sequence. Humanitariannet antibody also not necessarily will contain at least the constant plot the region of immunoglobulin (Fc), typically, a human immunoglobulin. For more details, see Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).
"Isolated" antibody is such that were identified and/or separated from a component of its natural environment. Contaminant components of its natural environment are substances which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, the implementation of the antibody will be purified 1) to more than about 95% by weight of the antibodies according to the Lowry method, and most preferably more than about 99% by weight, 2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence when using small plastic Cup sequencing machine or 3) to homogeneity in the formulation of SDS-PAGE in reducing or non conditions using Kumasi blue or preferably with silver staining. The selected antibody includes the antibody in situ within recombinant cells, since at least one component of the natural environment antibodies will be missing. As a rule, however, isolated antibody will be wearable is toulene, at least one stage of purification.
An antibody "which binds" with the antigen of interest, i.e. the antigen TGF-beta, is such, is able to bind to the antigen with sufficient affinity such that the antibody was suitable as a therapeutic agent for targeting a cell expressing the antigen. In cases where the antibody is such that binds to TGF-beta, as a rule, it is preferable to contact TGF-beta in contrast to other members of the superfamily of TGF-beta, and can be such that substantially does not undergo cross-interaction with other proteins of this family, such as BMP, activin, etc. In such scenarios exercise the degree of antibody binding sites with data that is not related to TGF-beta proteins, will be less than 10% according to the analysis cell sorting with excitation fluorescence (FACS) or radioimmunoprecipitation (RIA).
Antibody having a "biological property" labeled antibodies, such as monoclonal antibody labeled 2G7, is such, that has one or more biological property of the antibodies, which distinguishes it from other antibodies that bind to the same antigen (e.g., TGF-beta). For example, the R, the antibody with the biological property 2G7 can block the activation of TGF-beta and/or to contact the same epitope in the extracellular domain of TGF-beta, which is associated with 2G7.
Unless otherwise stated, the expression "monoclonal antibody 2G7" refers to an antibody that has antigennegative residues, or isolated from a mouse antibody 2G7 of the examples presented below. For example, monoclonal antibody 2G7 may submit a mouse monoclonal antibody 2G7 or its variant, such as humanitariannet antibody 2G7, with antihistamine amino acid residues of murine monoclonal antibody 2G7. An example of humanized antibody 2G7 shown in example 2 below. Unless otherwise stated, the expression "rhuMAb 2G7", in the sense in which this term is used here, refers to an antibody comprising the variable sequence of the light chain (VLand variable sequence of the heavy chain (VHrespectively the sequences SEQ ID NOS: 1 and 2 fused to the sequences of the constant region of the light and heavy chain of human immunoglobulin (not-a allotype), not necessarily expressed in cells of the Chinese hamster ovary (Cho).
"Growth inhibitory agent", in the sense in which this term is used here, refers to a compound or composition and, which inhibits cell growth, especially expressing TGF-beta tumor cells in vitro or in vivo. Thus, the growth inhibitory agent can be present such that substantially reduces the percentage expressing TGF-beta cells in S-phase. Examples of inhibiting the growth of funds include funds that are blocking the flow cell cycle (other than S phase), such funds, which induce a delay in G1-phase and delay in M-phase. Classical M-blockers phase include Vincas (vincristine and vinblastine), taxanes and inhibitors of topo II, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Such funds, which inhibit G1-phase, also cause a delay in S-phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and Ara-C. further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13.
Examples of "inhibiting the growth" antibodies are those that bind to TGF-beta and inhibit the growth of tumor cells with overexpression of TGF-beta. Preferred inhibiting the growth of anti-TGF-beta antibodies inhibit the growth of tumor cells of breast cancer SK-BR-3 cell cultures more than n is 20%, and preferably more than 50% (e.g., in the range from about 50% to about 100%) at the antibody concentration of about 0.5 to 30 μg/ml, when the inhibition of growth is determined six days after exposure to the antibodies on the cells SK-BR-3 (see U.S. patent No. 5677171 dated October 14, 1997). Test of inhibition of growth of cells SK-BR-3 are described in more detail in this patent.
An antibody that "induces cell death", is such, that makes a viable cell to become nonviable. Typically, the cell is one which expresses the receptor, TGF-beta, especially when the cell sverkhekspressiya receptor TGF-beta. Preferably the cell is a tumor cell, for example, the cell tumors of breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder. In vitro cell can be a cell SK-BR-3, BT, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3. Cell death in vitro can be determined in the absence of complement and immune effector cells in order to distinguish cell death induced antibody-dependent cretaceouspaleogene cytotoxicity (ADCC) or complementability cytotoxicity (CDC). So, test to determine cell death can be done using inactiv the trated heat treatment serum (i.e. in the absence of complement), and in the absence of immune effector cells. To determine whether the antibody is able to induce cell death, we can estimate the loss of membrane integrity by the absorption of iodide of propecia (PI), Trypanosoma blue (see Moore et al., Cytotechnology, 17: 1-11 (1995)) or 7AAD compared with untreated cells. Preferred inducing cell death of antibodies are those that induce PI uptake in the test assess the absorption of PI, the cells WT (see below).
An antibody that "induces apoptosis" is such, which induces programmed cell death that is determined by binding of annexin V, fragmentation of DNA, the shrinkage of the cells, the expansion of the endoplasmic reticulum, fragmentation of cells and/or formation of membrane vesicles (called apoptotic bodies). Typically, the cell is such that sverkhekspressiya receptor TGF-beta. Preferably the cell is a tumor cell, such as cell tumors of breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder. In vitro cell can be a cell SK-BR-3, BT, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3. There are various methods of evaluation of cellular events associated with apoptosis. the example you can determine the translocation of phosphatidylserine (PS) in the binding of annexin; DNA fragmentation can be evaluated by gradation DNA; and condensation cores/chromatin along with DNA fragmentation can be evaluated by the increase in the number hypodiploidy cells. Preferably the antibody that induces apoptosis, is such that about 2 to 50 times, preferably about 5-50 times and most preferably about 10-50 times more than that induces the binding of annexin compared with untreated cells in the test determine the binding of annexin when using cells WT (see below). In some cases proapoptotic antibody will be such, which additionally blocks the binding of TGF-beta (e.g., antibody 2G7); i.e., the antibody shares biological properties with the antibody against TGF-beta. In other cases, the antibody is such that substantially does not block TGF-beta. In addition, the antibody may be such that inducyruya apoptosis does not induce a significant reduction in the percentage of cells in S-phase (e.g., such that only causes about 0-10% decrease of the percentage of such cells compared with control).
"Epitope 2G7" represents a region in the extracellular domain of TGF-beta, which binds the antibody 2G7 (ATSS HB 10240). For screening the ha antibody, associated with the epitope 2G7, you can put a generic test of cross-blocking, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).
"Antibody against TGF-beta" refers to an antibody that binds to any isoform TGF-beta, preferably binds to TGF-beta, TGF-beta2 or TGF-beta, or any combination thereof, more preferably, at least with TGF-beta, or at least with TGF-beta2 and most preferably TGF-beta or TGF-beta together with TGF-beta2. Optionally, the antibody can bind at least with TGF-beta.
"Treatment" refers to therapeutic and prophylactic measures. Those who were in need of treatment include those that already have violations, as well as those in which the violation must be prevented. Therefore, the mammal undergoing treatment, can be defined as a violation of, or which is or sensitive to disturbance.
"Mammal" for purposes of treatment refers to the animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, horses, cats, cows, etc. Preferably the mammal is a Primate such as a monkey, monkey or higher h is, the rights, for example, most preferably a human.
"Violations related to TGF-beta" or "associated with TGF-beta violation" refers to a disorder, disease or condition will have a positive impact treatment with antibody against TGF-beta. They include chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder. Violations for this treatment include diseases characterized by accumulation of extracellular matrix, diseases caused by circulating TGF-beta or activated TGF-beta in local, state, caused by suppression of the immune system due to endogenous production of TGF-beta, acute human immunodeficiency as a result of severe injuries, burns and diseases, such as viral or bacterial diseases, many organ systems in the production or very high production of TGF-beta, and producing TGF-beta tumor. Non-limiting examples include neuronal, glial, astatically, hypothalamus and other glandular, macrophagal, epithelial, stromal and blastocoele disorders, fibrosis, scarring, tissue damage caused by radiation, and adhesion during wound healing and fibrotic skin disorders, such as scleroderma, TKA is Evie scars due to pathology of the Central nervous system, skin scars, keloid scars, neural scarring and fibrotic diseases of the abdominal cavity, lungs, liver and kidneys, such as chronic fibrosis of the liver, acute liver damage, interstitial fibrosis of the lungs and kidneys, and liver cirrhosis, cystic fibrosis, cardiovascular disorders, for example, cardiac fibrosis, damage to the arteries, such as atherosclerosis and arteriosclerosis, benign and malignant tumors, some types of leukemia, not suppressed TGF-beta and malignancies (e.g., sarcoma, carcinoma and melanoma), including malignant prostate tumors, ovarian cancer, malignant melanoma, breast cancer, lung, colon, rectum, colon, rectum or cervix, and metastasis of tumors and neuroendocrine tumors of the digestive tract and glioblastoma, disorder, disorder of vessels, nephropathy, multiple sclerosis, infections, such as macrophage pathogenic infections and viral infections such as hepatitis C and AIDS, immunological, angiogenic and inflammatory disorders and deficient conditions, such as rheumatoid arthritis, eye disorders, especially related to fibrosis of the eye, including proliferative retinopathy, retinal detachment and drainage after surgery for glaucoma, such as neural katchatka, pigmented vascular the membrane of the retina and the vitreous body of the human eye, and cataracts, osteoporosis, respiratory distress syndrome of adults, post-myocardial infarction, restenosis after angioplasty, glomerulonephritis, diabetes-related condition, such as hyperglycemia, diabetes, kidney disease in diabetes, nephropathy in diabetes, neuropathy in diabetes or retinopathy, and diseases associated with deficiency of macrophages.
Preferably the violation is fibrosis, damage to the arteries, infection, rheumatoid arthritis, diabetes or a diabetic condition, or a malignant tumor, such as expressing TGF-beta, more preferably, when the tumor is characterized by increased activation of TGF-beta. This tumor may Express high amounts of TGF-beta, or an alternative to it is not characterized by overexpression of TGF-beta.
The term "effective amount" refers to the amount of a drug effective for treating diseases or disorders in a mammal. In the case of a malignant tumor a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., to slow to some extent and preferably stop) infiltration of tumor cells into peripheral organs; inhibit (i.e., slow to some flat and preferably stop) metastasis of tumors. to inhibit, to some extent, tumor growth; and/or alleviate to some extent one or more symptoms associated with a tumor. To some extent, the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. In the treatment of malignant tumors efficiency can, for example, be determined by assessing the time to disease progression (TTP) and/or determining the speed of response (RR).
The terms "malignant disease" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of malignant diseases include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy. More specific examples of such malignant tumors include squamous cell malignant tumor (e.g., epithelial squamous cell tumor), malignant tumor of the lung, including small cell tumor of the lung, non-small cell malignant tumor of the lung, adenocarcinoma of the lung and squamous carcinoma of the lung, a tumor of the peritoneum, hepatocellular tumor, a stomach tumor, including tumors of the organisation is offering the gastrointestinal tract, malignant tumor of the pancreas, glioblastoma, cervical tumor of the uterus, ovarian tumor, liver tumor, malignant bladder cancer, hepatoma, malignant tumor of the breast, a tumor of the colon, tumor of the rectum, swelling of the colon, rectum, carcinoma of the endometrium or uterine carcinoma of the salivary glands, malignant kidney tumor, prostate tumor, a tumor of the vulva, swelling of the thyroid gland, liver tumor, carcinoma of the anus, swelling of the penis and swelling of the head and neck.
"Expressing TGF-beta tumor" is one that produces sufficient quantities of TGF-beta on the surface of its cells so that the anti-TGF-beta antibody can contact her and have a therapeutic effect against the tumor.
The tumor, "characterized by an increased activation of the receptor TGF-beta is one in which the degree of activation of the receptor for TGF-beta in cancer cells significantly exceeds the level of activation of this receptor in nonneoplastic cell of the same tissue type. This increased activation may result from overexpression of the receptor for TGF-beta and/or higher than normal concentrations of ligand TGF-beta, available for activation of the receptor for TGF-beta in tumor cells. Such surplus assets shall tion may cause and/or may be caused by malignant state of tumor cells. In some embodiments, the implementation of a malignant tumor subjected to a diagnostic or prognostic test for determining whether there is an amplification and/or overexpression of the receptor for TGF-beta, which leads to this increased activation of the receptor for TGF-beta. Alternative or additionally, the tumor can be subjected to a diagnostic or prognostic test for determining whether there is an amplification and/or overexpression of the receptor for TGF-beta in cancer, which is associated with excessive activation of the receptor. In the subgroup of these tumors excess aktivacija receptor may result from autocrine stimulation path.
"Autocrine" stimulating way is autostimulation due to the fact that the tumor cell produces the ligand TGF-beta and its cognate receptor TGF-beta. For example, the tumor may Express or sverkhekspressiya receptor TGF-beta and also to Express or sverkhekspressiya ligand TGF-beta (e.g., TGF-beta).
The tumor, which "sverkhekspressiya receptor TGF-beta, is one which has significantly higher concentrations of receptor TGF-beta on the cell surface compared to non-cancerous cell of the same tissue type. Such overexpression may be due to ample the qualifications of a gene or increased transcription or translation. The overexpression of the receptor for TGF-beta can be defined diagnostic or prognostic test in the evaluation of higher concentrations of the protein TGF-beta on the cell surface (e.g., by immunohistochemical analysis; IHC). Alternative or additionally you can define the levels encodes a TGF-beta nucleic acid in the cell, for example, fluorescent in situ hybridization (FISH; see WO 1998/45479, published in October 1998), southern-blotting, or polymerase chain reaction (PCR), such as quantitative PCR in real-time (RT-PCR). You can also study the overexpression of the receptor for TGF-beta in the definition of "exfoliating" antigen (e.g., the extracellular domain of the TGF-beta) in a biological fluid such as serum (for example, see U.S. patent No. 4933294 June 12, 1990; WO 1991/05264, published April 18, 1991; U.S. patent No. 5401638 dated March 28, 1995; and Sias et al., J. Immunol. Methods, 132: 73-80 (1990)). In addition to the above tests, the specialists in this field there are various in vivo tests. For example, you can expose cells in the body of the patient the effects of antibodies, which marked optional detectable label, e.g. a radioactive isotope, and it is possible to evaluate the binding of an antibody to cells in a patient, such as an external scan of radioactivity or analysis of biopsy material taken from the patient is and to the effects of antibodies.
In contrast to the tumor, which is not characterized by overexpression of the receptor for TGF-beta", is such that in the diagnostic test does not expresses a higher than normal concentration of the receptor for TGF-beta in comparison with non-cancerous cell of the same tissue type.
The tumor, which "sverkhekspressiya" ligand TGF-beta is one, which produces a much higher concentration of the ligand compared to non-cancerous cells of the tissue type. Such overexpression may be caused by gene amplification or increased transcription or translation. The overexpression of the ligand TGF-beta can be defined diagnostically when assessing concentrations of ligand (or nucleic acid encoding it) in a patient, e.g. biopsy material from tumors or various diagnostic tests, such as IHC, FISH, southern blotting, PCR or tests in vivo, described above.
"Hormone-independent tumor is such that proliferation is not dependent on the presence of the hormone that binds to a receptor expressed by cells in the tumor. Such tumors do not undergo clinical regression with the introduction of pharmacological substances or surgical interventions, which lead to a decrease in the concentration of hormone in the nearly tumors. Examples of hormone-independent tumors include androgen-independent prostate cancer, estrogen-independent tumor of the breast, a tumor of the endometrium and the ovarian tumor. Such tumors can start to develop as a hormone-dependent tumors and progress from the hormone-sensitive stage to hormone-resistant tumors after conducting antihormone therapy.
The term "cytotoxic agent", in the sense in which it is used here, refers to a compound that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211I131I125, Y90That Re186That Re188Sm153Bi212, P32and radioactive isotopes of Lu), and toxins such as toxins with a small molecule or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants.
"Chemotherapeutic agent" is a chemical compound suitable for the treatment of tumors. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; the ethyl is imini and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; nitrogen mustards such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, oxide hydrochloride mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, oralloy mustard; microsociety, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, calicheamicin, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, mycophenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, volumizing, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such occulusion, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; drugs are antagonists of adrenal hormones, such as aminoglutetimid, mitotane, trilostane; wspanialy folic acid, such as palenikova acid; Eagleton; aldophosphamide glycoside; aminolevulinic acid; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; the acetate slipline; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitrogen; pentostatin; penomet; pirarubicin; podovinnikova acid; 2-acylhydrazides; procarbazine; PSK®christening; razoxane; sizofiran; spirogermanium; tinoisamoa acid; triaziquone; 2,2',2"-trihlortrietilamin; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes (or taxoid), for example, paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NY) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DFO); retinoic acid; espiramicina; capecitabine and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Also in this definition are included antihormone tools that function for the regulation or inhibition of hormone action on tumors such as antiestrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutetimid, megestrol acetate MEGASE®, exemestane AROMASIN®, formestane, fadrozole, vorozole RIVTSOR®, letrozole, FEMARA® and anastrozole ARIMIDEX®; and anti-androgens such as flutamide, nilutamide, bikalutamid, leuprolide and goserelin; as well as troxacitabine (similar to 1,3-DIOXOLANYL cytosine); antisense oligonucleotides, particularly inhibiting the expression of genes in signaling pathways involved in cell proliferation, such as, for example, RKS-alpha, Ralf and H-Ras; vaccines such as vaccines based on gene therapy, for example, the vaccine ALLOVECTIN®vaccine, LEUVECTIN® vaccine VAXID®, PROLEUKIN® rIL-2; topoisomerase inhibitor 1 LURTOTECAN®; ABARELIX® rmRH; and pharmaceutical is Ki acceptable salt, acids and derivatives of any of the above.
In the sense in which it is used here, "drug aimed at EGFR" refers to a therapeutic agent that binds to EGFR and, optionally inhibits the activation of EGFR. Examples of such tools include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. patent No. 4943533, Mendelsohn et al.) and their variants, such as hyperserotonemia antibody 225 (S) and reconstructed human antibody 225 (N) (see WO 1996/40210, Imclone Systems Inc.); antibodies that bind to mutant EGFR type II (U.S. patent No. 5212290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. patent No. 5891996; and human antibodies that bind to EGFR (see WO 1998/50433, Abgenix). Anti-EGFR-antibody can be konjugierte with a cytotoxic drug, and thus, immunoconjugate (for example, see EP 659439, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD 1839 (Astra Zeneca), CP-358774 (OSI/Pfizer) and AG1478 effect.
"Therapeutic agent other than humanitariannet antibody" refers to any tool that is effective in the treatment of disorders associated with TGF-beta, other than these antibodies, and includes the t types, below.
"Antiangiogenic agent" refers to a compound that blocks or to some extent the negative impact on the development of blood vessels. Antiangiogenic factor may represent, for example, a small molecule or antibody that bind to a growth factor or growth factor receptor involved in angiogenesis. Examples include antagonists of vascular endothelial growth factor (VEGF), such as antibodies that specifically bind to VEGF, for example, AVASTIN®. "Antibody that binds to VEGF include chimeric, human and humanized antibodies as well as fragments, and also include antibodies that block or neutralize VEGF or block the binding of VEGF with one or more VEGF receptor, preferably both receptors.
The expression "the regulators of immune function in a mammal" refers to cytokines and growth factors that regulate the immune function of a mammal, including interleukins, tumor necrosis factor, lymphotoxin, epidermal growth factor, platelet-derived growth factor, TGF-α, the factor inhibiting the migration of macrophage activating factor, macrophage, fibroblast growth factor, macrophage activating factors, interferons and colony stimulating factors. These regulators can get the C natural sources, to produce using leukocytes to synthesize chemical methods, if it is appropriate, or to obtain recombinant methods. Preferred are IL-1, IL-2, IL-6 and IFN-β.
The term "cytokine" is a generic term for proteins secreted by a single population of cells that act on other cells as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and normal polypeptide hormones. In the cytokines included growth hormone such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogenic; factor-α and-β tumor necrosis; Mullerian-inhibiting substance; mouse associated with gonadotropin peptide; inhibin; activin; vascular endothelial factor growth; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; insulin-like factor-I and-II growth; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and-γ; colony stimulating factors (CSF), is such as macrophage CSF (M-CSF); granulocyte-macrophage-CSF(GM-CSF); and granulocyte CSF(G-CSF); interleukins (IL)such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit-ligand (KL). In the sense in which it is used here, the term cytokine includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalent variants of cytokines with the natural sequence.
The term "prodrug", in the sense in which it is used in this application refers to a precursor or derivative pharmaceutically active compound that has a lower cytotoxicity compared to the original drug and are capable of enzyme to be activated or to become more active in its original form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615thMeeting 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 of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphoramide prodrugs, sulfadimidine prodrugs, peptideatlas prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactosidase about what carstvo, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fertilizin and other prodrugs based on 5-ptoluidine that can turn into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be converted into a prodrug for use in this invention include, but are not limited to, chemotherapeutic agents described above.
"Liposome" is a small vesicles composed of various types of lipids, phospholipids and/or surfactant that is suitable for delivery of medication (such as anti-TGF-beta antibodies disclosed here and do not necessarily chemotherapeutic agent) to a mammal. Components of liposomes are typically arranged in a bilayer, similar to the arrangement of lipids in biological membranes.
The term "to the package insert" is used in relation to instructions, usually included in commercial packages of therapeutic products, that contain information about the indications, uses, dosage, introduction, contraindications and/or warnings concerning the use of such therapeutic products.
"Cardioprotector" is a compound or composition that prevents or reduces the indicate dysfunction of the myocardium (i.e. cardiomyopathy and/or congestive heart failure), associated with the introduction of a drug, such as an anthracycline antibiotic number and/or anti-TGF-beta antibody, to a patient. For example, cardioprotector can block or reduce mediated by free radicals cardiotoxic effect and/or to prevent or reduce damage caused by oxidative stress. Examples of cardioprotection included in this definition include dexrazoxane, chelate compound with iron (ICRF-187) (Seifert et al., The Annals of Pharmacotherapy, 28: 1063-1072) (1994)); a means for reducing the level of lipid and/or an antioxidant, such as probucol (Singal et al., J. Mol. Cell Cardiol., 27: 1055-1063 (1995)); amifostine (aminothiol 2-[(3-aminopropyl)amino]etention acidic phosphate ester, also known as WR-2721, and his dephosphorylation form for absorption by cells called WR-1065) and S-3-(3-methylaminopropyl)propylphosphonate acid (WR-151327), see Green et al., Cancer Research, 54: 738-741 (1994); digoxin (Bristow, M.R. In: Bristow M.R. ed. Drug-Induced Heart Disease (New York: Elsevier 191-215 (1980)); beta blockers such as metoprolol (Hjalmarson et al., Drugs, 47: Suppl 4: 31-9 (1994); and Shaddy et al., Am. Heart J., 129: 197-199 (1995)); vitamin E; ascorbic acid (vitamin C); extinguishers free radicals, such as olaoluwa acid, ursolic acid and N-acetylcysteine (NAC); the spin-exciting compounds such as alpha-phenyl-tribotechnical (PBN) (Paracchini et al., Anticancer Res., 13: 1607-1612 (1993)); selenoorganicheskikh compounds such as R (Elbesen) and the like.
"Isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid that encodes the antibody. The selected nucleic acid molecule is other than in the form or setting in which it is found in nature. Therefore, the selected nucleic acid molecules differ from the nucleic acid molecules in the form in which it is in natural cells. However, the selected molecule nucleic acid molecule includes nucleic acid found in cells that normally Express the antibody, where, for example, the nucleic acid molecule is in the position of chromosomes that is different from natural cells.
The expression "regulatory sequences" refers to DNA sequences necessary for the expression of the operatively linked coding sequence in a particular organism, the host. Regulatory sequences that are suitable for prokaryotes, for example, include a promoter, an optional operator sequence and a binding site with ribose the AMI. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is functionally linked"when it is placed in a functional relationship with another nukleinovokisly sequence. For example, DNA for proposedvalue or secretory leader is functionally linked to DNA for a polypeptide if it is expressed in the form of preprotein that participates in the secretion of the polypeptide; a promoter or enhancer functionally linked to the coding sequence if it affects the transcription of the sequence; binding site with ribosomes functionally linked to the coding sequence if it is located in such a way as to facilitate translation. As a rule, "functionally linked" means that linked DNA sequences are contiguous and in the case of a secretory leader are adjacent and are in the phase of reading. However, the enhancers are not necessarily adjacent. The binding occurs by legirovaniem in the relevant restriction sites. If such sites are not available, use synthetic adapters or linkers according to usual practice.
In the sense in which it uses these expressions, "cell", "cell line" and "cell culture" are used interchangeably, and in the e such definitions include the offspring. So, the words "transformants" and "transformed cells" include the primary data cells derived from their culture without taking into account the number of transpency. Also it should be understood that all such progeny may not be precisely identical in DNA content due to intentional or accidental mutations. Mutant offspring that has the same function or biological activity in the screening characteristic of the original transformed cells, is also included. In cases where there are certain signs that it will be clear from the context.
In the sense in which it uses the term "sample from an organism" refers to a fluid or biological sample which contains or may contain TGF-beta to determine. The sample includes liquids such as body fluids of humans or animals, for example, blood, serum, urine, amniotic fluid, extracts of tissues, cerebral spinal fluid and the like. For samples may require special processing, such as extraction prior to analysis, depending on the ability of the contained components to decomposition, aggregation or absorption in the container for storage.
II. Obtaining humanized anti-TGF-beta antibodies
In this area described methods for obtaining humanized antibodies, not the relative is relevant to human antibodies. Preferably humanitariannet antibody contains one or more amino acid residues inserted into it from a source that isn't human. Data non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can mostly be held according to the method of Winter and co-authors (Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing sequences of the hypervariable region on the corresponding sequences of a human antibody. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), which mainly less than an intact human variable domain is substituted by the corresponding sequence from species that are not relevant to humans. In practice, typically, humanized antibodies are human antibodies in which some hypervariable residues region and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.
Another method of obtaining humanized antibodies is described in published U.S. patent 2003/0017534, published on 23 January 2003, in which humanized antibodies and antibody preparations get from transgenic animals,non-human. Animals, non-human, receive genetic engineering to enable one or more loci gumanitarnogo immunoglobulin, which are able to undergo the rearrangement of genes and turning genes in transgenic animals, non-human, to produce different humanized immunoglobulins.
The choice of human variable domains, both light and heavy chains for use in obtaining humanized antibodies is very important to reduce antigenicity. According to the so-called method of "best fit" the sequence of the variable domain of the antibody rodents subjected to screening against a library of known sequences of human variable domains. The human sequence which is closest to that in rodents, taking on the human frame plot (FR) for gumanitarnogo antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chorthia et al., J. Mol. Biol., 196: 901 (1987)). In another method uses a particular frame plot derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same frame area can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).
Also the important to antibodies were humanitarian with retention of high affinity for the antigen and other useful biological properties. To achieve this goal, according to the preferred method, humanized antibodies produced by way of analysis of the source sequences and various speculative humanized products using three-dimensional models of the source and humanized antibodies. There usually are three-dimensional models of immunoglobulins, and they are known to specialists in this field. There are computer programs to illustrate and locations of possible three-dimensional conformational structures of selected sequences of immunoglobulins candidates. The study of these displays allows you to analyze the possible role of the residues in the functioning of the sequences of immunoglobulins candidates, i.e. to analyze the remains, which have an impact on the ability of the immunoglobulin-candidate for the presidential position to contact with the antigen. This way, FR residues can be selected and combined from the recipient and paste sequence to achieve the desired properties of the antibodies, such as increased affinity against the target antigen. Basically the remnants of the hypervariable region directly and primarily affect the binding to the antigen.
Example 2 below describes the production example of the s humanized anti-TGF-beta antibodies which are associated with TGF-beta. Humanitariannet antibody contains the remains of inhuman hypervariable region included in the human variable domain of the heavy chain and further comprises a replacement frame plot (FR) in a position selected from the group consisting of 48, 49, 67, 69, 71, 73 and 78 using the numbering system variable domains, represented by Kabat et al., above. In one embodiment, humanitariannet antibody contains replacement FR in two or more positions 48, 49, 67, 69, 71, 73 and 78; and in other embodiments, the implementation in three or more of such provisions. In preferred embodiments, the implementation of the antibody contains replacement FR at positions 49, 67 and 71, or regulations 48, 49 and 71, or the provisions of 49, 69 and 71, or the provisions of 49, 69, 71 and 73, or the provisions of 49, 71 and 73, or the provisions of 49, 71 and 78. It is preferable that had as little as possible replacement frame section to minimize immunogenicity, but also of great importance should be given efficiency. In fact, the substituted amino acids are those which preferably are conservative, so as not to cause changes in immunogenicity or efficacy. At position 48 is preferred substitution of valine for isoleucine, at position 49 is preferred substitution of alanine for glycine at position 67 is preferred I have is the replacement of phenylalanine to alanine, in the 69 position, it is preferable to replacement of phenylalanine to alanine, at position 71 is preferred replacement of arginine to alanine, at position 73 is preferred replacement of asparagine for lysine and at position 78 is preferred replacement of leucine to alanine.
Approximate humanitariannet interested antibody contains a complementarity determining residues variable domain of the heavy chain GYAFTNYLIE (sequence SEQ ID NO: 41), VNNPGSGGSNYNEKFKG (sequence SEQ ID NO: 42) or VINPGSGGSNYNEKFKG (sequence SEQ ID NO: 43); and/or SGGFYFDY (sequence SEQ ID NO: 44), optionally comprising amino acid modification data CDR residues, e.g. where the modifications mainly maintain or improve affinity of the antibody. For example, interesting variant antibodies can contain from about one to about seven or approximately five substitutions of amino acids in the above variable sequences of the heavy chain CDR. Such antibodies can be obtained by affinity maturation, for example, as described below. Preferably remains are two or more of GYAFTNYLIE (sequence SEQ ID NO: 41), VNNPGSGGSNYNEKFKG (sequence SEQ ID NO: 42) or VINPGSGGSNYNEKFKG (sequence SEQ ID NO: 43); most preferably all three; and/or SGGFYFDY (sequence SEQ ID NO: 44). The most preferred humanitariannet and tetelo contains the amino acid sequence of the variable domain of the heavy chain of sequence SEQ ID NO: 4, or one with GYAFTNYLIE (sequence SEQ ID NO: 41), VINPGSGGSNYNEKFKG (sequence SEQ ID NO: 43); and SGGFYFDY (sequence SEQ ID NO: 44).
Humanitariannet antibody can include defining complementarity remains of the variable domain of the light chain RASQSVLYSSNQKNYLA (sequence SEQ ID NO: 36) or RASQGISSYLA (sequence SEQ ID NO: 37); WASTRES (sequence SEQ ID NO: 38) or YASSLQS (sequence SEQ ID NO: 39); and/or HQYLSSDT (sequence SEQ ID NO: 40), for example, in addition to the remnants of the CDRs of the variable domain of the heavy chain, presented in the previous paragraph. Such humanized antibodies do not necessarily contain amino acid modifications of the above CDR residues, e.g. where the modifications mainly maintain or improve affinity of the antibody. For example, interesting variant antibodies can contain from about one to about seven or approximately five substitutions of amino acids in the above variable sequences of the light chain CDR. Such antibodies can be obtained by affinity maturation, for example, as described below. Preferably remains are two or more of RASQSVLYSSNQKNYLA (sequence SEQ ID NO: 36); WASTRES (sequence SEQ ID NO: 38); and/or HQYLSSDT (sequence SEQ ID NO: 40), most preferably all three. The most preferred humanitariannet antibody contains amino acid sequence of the variable domain is easily the chain in the sequence of SEQ ID NO: 3.
The present application also includes antibodies with Mature affinato that are associated with TGF-beta. The original antibody can be a human antibody or humanitariannet antibody, for example, contains a variable sequence of light and/or heavy chains, respectively, SEQ ID nos: 3 and 4 (i.e. option 5). Antibody with Mature affinato preferably binds to TGF-beta with affinato, greater than that for murine antibody 2G7 or option 5 (for example, increased from about 2 or about 4 times to about 100, or about 1000 times affinato, for example, when assessing TGF-beta extracellular domain (ECD) using ELISA).
Assumed various forms gumanitarnogo antibodies or antibodies with a Mature affinato. For example, humanitariannet antibody or antibody with Mature affinato may represent a fragment of the antibody, such as Fab, which is optional anywhereman with one or more cytotoxic agent(s) to obtain immunoconjugate. Alternative humanitariannet antibody or antibody with Mature affinato may represent intact antibody, such as an intact IgG1 antibody.
Have been developed various techniques to obtain fragments of antibodies, humanized antibodies. Traditionally, these fragments were obtained by proteolytic digestion of intact antic is l (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). Currently, however, these fragments can be obtained directly using recombinant host cells. For example, antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternative fragments, Fab'-SH can be directly obtained from E. coli and chemically bind with the formation of fragments F(ab')2(Carter et al., Bio/Technology, 10: 163-167 (1992)). According to another approach, the fragments F(ab')2you can directly select from the culture of the recombinant host cells. Specialists in this area other known methods of obtaining fragments of antibodies. In other embodiments, implementation of the antibody of choice is a single-chain Fv fragment (scFv). See WO 1993/16185; U.S. patent No. 5571894 and U.S. patent No. 5587458. The antibody fragment may also be a "linear antibody", for example, described in U.S. patent No. 5641870. Such fragments, linear antibodies can be a monospecific or bespecifically.
Bespecifically antibodies are antibodies that have binding specificity for at least two different epitopes. Approximate bespecifically antibodies can bind to two different epitopes of the protein TGF-beta. Other such antibodies may combine a binding site with TGF-beta with the binding site(s) for HER-2, EGFR, ErbB, ErbB3 and/or ErbB4. Alternative anti-TGF-beta-shoulder can be combined with an arm that binds to a triggering molecule on a leukocyte such as a molecule receptor T cells (e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus the protective cellular mechanisms for expressing TGF-beta cell. Bespecifically antibodies can also be used for the location of cytotoxic agents to cells which Express TGF-beta. These antibodies have binding TGF-beta shoulder and shoulder, which is associated with the cytotoxic agent (e.g., caporino, anti-interferon-α, Vinca alkaloid, a chain of ricin A, methotrexate or radioactive isotope hapten). Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically antibody F(ab')2).
In WO 1996/16673 describes bespecifically anti-TGF-beta/anti-FcγRIII-antibody and in U.S. patent No. 5837234 describes bespecifically anti-TGF-beta/anti-FcγRI-antibody. Bespecifically anti-TGF-beta/anti-Fcα-antibody described in WO 1998/02463. In U.S. patent No. 5821337 described bespecifically anti-TGF-beta/anti-CD3-antibody.
Methods of obtaining bespecifically antibodies known in the field. Traditional getting a full size of bispace the practical antibodies is based on the co-expression of two pairs of heavy chain-light chain immunoglobulin, where the two chains have different specificity (Millstein et al., Nature, 305: 537-539 (1983)). In the result of the arbitrary choice of immunoglobulin heavy and light chains of these hybridoma (quadroma) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which is usually carried out stages affinity chromatography, is quite cumbersome, and the product yield is low. Similar methods are disclosed in WO 1993/08829 and Traunecker et al., EMBO J., 10: 3655-3659 (1991).
According to another approach, the variable domains of the antibodies with the desired binding specificity (sites of Association of the antibody-antigen) hybridized sequences of the constant domain of immunoglobulin. Heriditary product is preferably constant domain of the heavy chain immunoglobulin comprising at least the hinge region, CH2 and CH3. It is preferable to have the first constant region of the heavy chain (SN) with the site required for binding to the light chain is present, at least one, hybridization product. DNA encoding fused heavy chain immunoglobulin and, if desirable, the light chain immunoglobulin, insert into individual expressing vectors and cotransfected in a suitable organism, the host. This provides the highest layer is the ability to adjust reciprocal plots of the three polypeptide fragments in the variants of implementation, when using unequal ratios of the three polypeptide chains in the design to ensure optimum output. However, it is possible to insert the coding sequences for two or all three polypeptide chains in one expressing vector when the expression of at least two polypeptide chains in equal ratios results in high output or when the ratios are of no particular value.
In the preferred embodiment, this approach bespecifically antibodies are composed of a hybrid heavy chain immunoglobulin with a first binding specificity in one arm, and a hybrid pair of heavy chain-light chain immunoglobulin (providing a second binding specificity) in the other shoulder. It was found that this asymmetric structure facilitates the separation of the desired especifismo connections from unwanted combinations of immunoglobulin chains, because the presence of the light chain of immunoglobulin in only one half of bespecifically molecules provides an easy way branch. This approach is disclosed in WO 1994/04690. For example, additional details of getting bespecifically antibodies see Suresh et al., Methods in Enzymology, 121: 210 (1986).
According to another approach described in U.S. patent No. 5731168, the boundary surface between a pair of antibody molecules can be constructed DL is receiving the maximum percentage of heterodimers, which are separated from the culture of recombinant cells. The preferred surface of the partition contains at least part of the domainN3 constant domain of the antibody. In this method, one or more small amino acid side chains from the interface of the first antibody molecules replaced with larger side chains (e.g. tyrosine or tryptophan). Create compensatory "cavities" of identical or similar size to the large side chain(s) on the boundary surface of the second molecule antibodies when replacing large amino acid side chains into smaller (e.g., alanine or threonine). This provides a mechanism to enhance the yield of heterodimer compared with unwanted end-products such as homodimers.
Bespecifically antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate can be combined with Avidya, and the other with Biotin. Such antibodies, for example, have been proposed for targeted impact on immune cells in contrast to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 1991/00360, WO 1992/200373 and EP 03089). Heteroconjugate antibodies can be obtained by any means cross-linkage. Suitable agents for cross-linkage are well known in this field and they described the, for example, in U.S. patent No. 4676980, along with a number of methods of cross-linkage.
The literature also describes methods of obtaining bespecifically antibodies, fragments of antibodies. For example, bespecifically antibodies can be obtained using chemical bonds. Brennan et al., Science, 229: 81 (1985) describe a method in which the intact antibody proteoliticeski split with obtaining fragments F(ab')2. These fragments regenerate in the presence of the dithiol-komleksoobrazuyuschee agent sodium arsenite to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide bonds. Then the resulting fragments Fab' is transformed into derivatives of dinitrobenzoate (TNB). Then one of the derivatives of Fab'-TNB again turned into Fab'-thiol by restoring mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB education especifismo antibodies. Received bespecifically antibodies can be used as a means for the selective immobilization of enzymes.
Recent achievements contribute to the direct allocation of fragments Fab'-SH from E. coli, which can be combined chemically with the formation of bespecifically antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe obtaining molecules completely gumanitarnogo especifismo antibody F(ab')2. Each is a fragment Fab' separately secreted from E. coli and subjected to the direct chemical combination with the formation of especifismo antibodies in vitro. Thus obtained bespecifically antibody capable of contacting cells, sverkhekspressiya receptor TGF-beta, and normal human T-cells, as well as "run" lytic activity of human cytotoxic lymphocytes to human target cells of a tumor of the breast.
Also describes the various methods of acquisition and allocation of fragments especifismo antibodies directly from a culture of recombinant cells. For example, were obtained bespecifically antibodies using latinovich "zippers". Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Peptides latinboy "zipper" of proteins Fos and Jun are associated with portions of the Fab' of two different antibodies by fusion of 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. The technology of obtaining "datel"described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), provides an alternative mechanism for obtaining fragments bespecifically antibodies. The fragments include the variable domain of the heavy chain (VH)connected to the variable domain light chain(V L) using a linker that is too short, so that the connection of the two domains on the same chain. Therefore, the domains of the VHand VLone fragment are forced to connect with complementary domains VHand VLanother fragment, thus forming a two antigenspecific site. There are reports of other strategies of obtaining fragments bespecifically antibodies when using dimmers single-chain Fv (sFv). See Gruber et al., J. Immunol., 152: 5368 (1994).
Provides antibodies with more than two valencies. For example, you can get thespecification antibodies. Tutt et al., J. Immunol., 147: 60 (1991).
It is possible to evaluate the growth inhibitory effect of antibodies when determining, for example, its ability to neutralize, to exert an antagonistic effect or inhibit the activity of TGF-beta, for example, to substantially prevent at least one of unwanted growth inhibitory, immunosuppressive, forming stroma (i.e. elements stroma, inflammatory cells, endothelial cells and fibroblasts) or anchor-independent growth-enhancing activity of the Mature TGF-beta, as defined in the literature. Thus, the antibody is able to block the activity of endogenous TGF-beta produced by suppressor tumor and lymphoid cells (T-cells). Od is they way to determine this achievement is to assess the inhibition of the absorption of 3H-thymidine cell line lung fibroblasts mink, for example, Mv-3D9, when increasing the concentration of the antibody, the activity of TGF-beta is steadily decreasing, linear or nonlinear. Line lung cells mink is very sensitive to the growth inhibitory action of TGF-beta, and this test is relatively simple to play with. Basically the test is carried out by incubation of the cells with a mixture of TGF-beta and antibodies in minimum essential medium containing 2 mm glutamine and 5% fetal bovine serum for 18-24 h at 37°C in an atmosphere of 5% CO2and then introduction 1 mccoury3H-thymidine in 20 µl and the collection after 4 h at 37°C. Preferably the antibody is able to inhibit the proliferation of cells under the action of TGF-beta to a greater extent compared with the monoclonal antibody 2G7. Another method of assessing growth inhibition action of antibodies is to determine the extent to which it inhibits TGF-beta in the test proliferation mesangial cells presented in the examples below. Antibodies are also suitable for production test binding receptor or radioreceptor analysis in the usual way when the incubation mixture of radioisotope-labeled TGF-beta (e.g., labeled with radioactive iodine rhTP-beta) and antibodies with cells containing the receptor of TGF-beta (e.g., pulmonary Phi is robustly mink, such as cell line Mv1Lu, which is available from ATS under ATSS No. CCL-64) and determining how the antibody blocks the binding of labeled TGF-beta-receptor.
To select antibodies that induce cell death, loss of membrane integrity, what the data indicates, for example, by the absorption of PI, Trypanosoma blue or 7AAD, can be evaluated by comparison with the control. The preferred test is the test for absorbance of PI using cells WT. According to this method, cells WT (which can be obtained from the American type culture collection (Manassas, VA)) were cultured in the medium Needle, modified by way of Dulbecco (D-MEM): Ham's F-12 (50:50) supplemented with 10% heat inactivated processing FBS (Hyclone) and 2 mm L-glutamine. (Thus, the test is carried out in the absence of complement and immune effector cells). Cells WT seeded with a density of 3×106the Cup in cups the size of 100×20 mm and cultured overnight. Then, the medium removed and replaced with the same fresh medium or medium containing 10 μg/ml of the appropriate monoclonal antibodies. Cells are incubated for 3 days. After each treatment, the monolayers are washed with phosphate buffered saline (PBS) and divide by treatment with trypsin. The cells are then centrifuged at 1200 rpm for 5 min at 4°C and the precipitate after price is ripperology resuspended in 3 ml of chilled on ice CA 2+buffer for binding (10 mm Hepes, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2and aliquot portions transferred into test tubes size 12×75 35-mm sieve-cover (1 ml per tube, 3 tubes per treatment) to remove cell clumps. Then in tubes make a PI (10 μg/ml). Samples can be analyzed using the flow cytometer FACSCANTMand software FACSCONVERTTMCellQuest (Becton Dickinson). Those antibodies that induce statistically significant levels of cell death according to the absorption of PI, you can select as inducing cell death antibody.
For selection of antibodies that induce apoptosis, there is a test binding of annexin using cells WT. Cells WT cultivate and sow the Cup, as mentioned in the preceding paragraph above. Then, the medium removed and replaced with the same fresh medium or medium containing 10 μg/ml of monoclonal antibodies. After incubation for 3 days, the monolayers are washed with PBS and the cells divide by treatment with trypsin. The cells are then centrifuged, resuspended in CA2+-buffer for binding and aliquot portions transferred into test tubes as described above for the production of the test determine cell death. Then in tubes contribute labeled annexin (e.g., annexin V-FTIC) (1 μg/ml). Samples can be analyzed using the flow cytometer FACSCANTMand so the software FACSCONVERT TMCellQuest (Becton Dickinson). Those antibodies that induce statistically significant levels of binding of annexin compared with the control, you can select as inducing apoptosis of cells antibodies.
In addition to inexensive test there is a test staining DNA with the use of cells WT. For setting the test cells WT treated with antibody of interest as described in the two paragraphs above, incubated with 9 μg/ml fluorescent probe HOECHST 33342TMfor 2 h at 37°C, then analyzed on a flow cytometer EPICS ELITETM(Counter Corporation) with the use of the software MODFIT LTTM(Verity Software House). Antibodies that induce a change in the percentage of apoptotic cells in 2 times and above (and preferably 3 times higher) compared to untreated cells (100% apoptotic cells), can be selected as causing apoptosis antibodies using this test.
For screening of antibodies that bind to an epitope in TGF-beta associated with the selected antibody can be an ordinary cross-blocking test, such as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternative or additionally you can spend epitope mapping methods known in this field.
The invention also relates to immunoconjugates, the content is relevant antibody, conjugated with a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., a toxin with a small molecule or enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants), or a radioactive isotope (i.e radioconjugates).
Chemotherapeutic agents suitable for such immunoconjugates described above. Conjugates of the antibody and one or more toxins with a small molecule, such as calicheamicin, maytansine (U.S. patent No. 5208020), trichoton and antitumor agent SS, are also included here.
In one preferred embodiment of the invention the antibody is conjugated with one or more molecules of maytansine (for example, from about 1 to about 10 molecules of maytansine molecule antibodies). For example, maytansine you can turn to May-SS-Me which can be restored to May-SH3 and subjected to interaction with modified antibody (Chari et al., Cancer Research 52: 127-131 (1992)) to obtain immunoconjugate maytansinoid-antibody.
Another interesting immunoconjugate includes anti-TGF-beta antibody conjugated with one or more molecules calicheamicin. Family of antibiotics calicheamicin can result in breakage of double-stranded DNA in subpicomolar concentrations. P is chornye analogues calicheamicin, which can be used include, but are not limited to, γ1I, α2I, α3IN-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research 53: 3336-3342 (1993) and Lode et al., Cancer Research 58: 2925-2928 (1998)). See also U.S. patent No. 5714586; 5712374; 5264586 and 5773001 included here for information.
Enzymatically active toxins and fragments thereof that can be used include a chain toxin of the pathogen diphtheria, neisvaziuosiu active fragments of the toxin of the pathogen diphtheria a chain of endotoxin And (from Pseudomonas aeruginosa), a chain of ricin a chain abrina And chain modeccin And alpha sarcin, proteins Aleurites fordii proteins of diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor of MOMORDICA CHARANTIA, Curtin, krotin, inhibitor saponaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. For example, see WO 1993/21232, published October 28, 1993.
The present invention further includes immunoconjugate formed by the antibody and the connection with nucleotidase activity (e.g., a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; Ncasa).
To obtain radioconjugates anti-TGF-beta antibodies have different radioactive isotopes. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, P32and radioactive isotopes of Lu.
Conjugates of the antibody and ototoksicescoe tools can be accessed using a variety of bifunctional agents for coupling proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and derivatives of bis-active fluorine (such as 1,5-debtor-2,4-dinitrobenzene). For example, you can get the immunotoxin ricin, as described in Vitetta et al., Science, 238: 1098 (1987). Labelled with carbon141-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide with the antibody. See WO 1994/11026. The linker can be "otdalennym linker"facilitating release of the cytotoxic drug in the cell. For example, you can use labile under the action of acid linker sensitive peptidase linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992)).
Alternatively, you can get a hybrid protein comprising an anti-TGF-beta antibody and cytotoxic agent, for example, recombinant techniques or peptide synthesis of the M.
In yet another embodiment, the antibody can be konjugierte with the "receptor" (such as streptavidin) for directions to the tumor, where the conjugate of the antibody-receptor is administered to the patient, followed by removal of unbound conjugate from the blood using a cleaning agent and then the introduction of a "ligand" (e.g., avidin), which anywhereman with a cytotoxic agent (e.g., radionucleotide).
Antibodies of the present invention can also be used in ADEPT by conjugation of the antibody with the prodrug activating enzyme which converts a prodrug (e.g., peptidase chemotherapeutic agent, see WO 1981/01145) in the active antitumor drug. See, for example, WO 1988/07378 and U.S. patent No. 4975278.
The enzyme component immunoconjugate suitable for ADEPT, includes any enzyme capable of influencing the prodrug in such a way as to transform it into the more active cytotoxic form.
Enzymes suitable for use in the method according to the invention, include, but are not limited to, alkaline phosphatase, suitable for converting phosphate-containing prodrugs into free drugs; arylsulfatase suitable for converting sulfate-containing prodrugs into free drugs; citizendiumissue suitable for converting non-toxic 5-forcit the Zina in anticancer drug, 5-fluorouracil; proteases, such as Serratia-protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which is suitable for converting peptide-containing prodrugs into free drugs; D-alanismorissette suitable for converting prodrugs that contain the substituents D-amino acid; a carbohydrate-splitting enzymes, such as β-galactosidase and neuraminidase, are suitable for converting glycosylated prodrugs into free drugs; β-lactamase, suitable for making drugs, derivatizing β-lactams, into free drugs; and penicillinases, such as penicillin-V-amidase or penicillin-G-amidase suitable for transformation of drugs, derivatizing respectively on their nitrogen of an amino group phenoxyacetyl or phenylacetylene groups, into free drugs. Alternative antibodies with enzymatic activity, also known in this area as "abzyme", can be used to convert the prodrugs of the invention into free active drugs (see, for example, Massey, Nature 328: 457-458 (1987)). Conjugates of the antibody-Abim can be obtained as described herein for delivery of abzyme in the population of tumor cells.
Enzymes suitable for the present invention, can covalently bind with anti-TGF-beta antibodies using meth is dick, well known in this field, such as the use of heterobifunctional cross-linking reagents mentioned above. Alternative hybrid proteins containing at least antigennegative region of the antibodies according to the invention is associated, at least functionally active leg of a suitable enzyme, can be constructed using methods of recombinant DNA, are well known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)).
Provided other modifications of the antibody. For example, the antibody can be associated with one of the various non-protein polymers, e.g. polyethylene glycol, polypropyleneglycol, polyoxyalkylene or copolymers of polyethylene glycol and polypropylenglycol. The antibody may also be included in the prepared microcapsules, for example, methods koatservatsii or interfacial polymerization (for example, microcapsules, respectively from hydroxymethylcellulose or gelatin and microcapsules of poly(methyl methacrylate)) and in colloidal systems for drug delivery (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsion. Such methods are disclosed in Remington's Pharmaceutical Sciences, 16thedition, Oslo, A., Ed., (1980).
Anti-TGF-beta antibodies disclosed here can also be formulated in the form of what immunoliposome. Liposomes containing the antibody are prepared by methods known in this field, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); U.S. patent No. 4485045 and 4544545; and WO 1997/38731, published on 23 October 1997, In U.S. patent No. 5013556 disclosed liposomes with enhanced circulation time.
Particularly useful liposomes can be obtained obraniakowi by evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-PE). Liposomes are passed through filters with defined pore size to obtain liposomes with the desired diameter. Fragments Fab' antibody of the present invention can be konjugierte with liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) by reaction of disulfide interchange. Chemotherapeutic agent is not necessarily inside the liposomes. See Gabizon et al., J. National. Cancer Inst., 81(19): 1484 (1989).
III. The vectors, cells of the host and recombinant methods
The invention also provides a selected nucleic acid encoding humanitariannet anti-TGF-beta antibody, vectors and cells of the host containing the nucleic acid, and recombinant methods for producing antibodies.
For recombinant obtain antibodies nucleic acid encoding it is isolated and inserted into a replicable vector tlepolemus cloning (amplification of the DNA) or for expression. DNA encoding a monoclonal antibody, is easily detected, and sequeiros using conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chain antibodies). Many vectors. Components of vectors typically include, but are not limited to, one or more of the following: a signal sequence, the starting point of replication, one or more marker genes, an enhancer, a promoter and termination sequence-transcription.
(i) Component signal sequence
Anti-TGF-beta antibody according to this invention can be obtained by recombinant not only directly, but also in the form of a hybrid polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-end of the Mature protein or polypeptide. Selected heterologous signal sequence is preferably one that is recognized and processed (i.e. cleaved signal peptidases) the host-cell. For prokaryotic host cells that do not recognize and ProcessInput natural signal sequence of the anti-TGF-beta antibodies, signal p is the sequence replace the prokaryotic signal sequence, selected, for example, from the group of alkaline phosphatase, penitsillinazy, lpp or leaders resistant to heat enterotoxin II. For secretion in yeast natural signal sequence can be replaced, for example, the leader of the yeast invertase leader, α factor (including the leaders of the α-factor of Saccharomyces and Kluyveromyces), acid phosphatase leader, the leader of glucoamylase C. albicans or signal sequence described in WO 1990/13646. When expression in mammalian cells have a signal sequence mammals, as well as viral secretory leaders, for example, the signal gD of herpes virus simple.
DNA for this predecessor are ligated in reading frame to DNA encoding the anti-TGF-beta antibody.
ii) a Component of the replication origin
Expressing and cloning vectors contain nukleinovokisly sequence that contributes to the vector to replicate in one or more cells of the host. Generally, in cloning vectors this sequence is one that promotes the vector to replicate independently of the chromosomal DNA of the host, and includes the starting point of replication or autonomously can replicate the sequence. Such sequences are well known for various bacteria, yeast and viruses. The replication origin of the plasmid pBR322 is the tsya suitable for most gram-negative bacteria, plasmid replication origin 2 MK suitable for yeast, and various viral starting point of replication (SV40, polyoma, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells. Usually a component of the replication origin is not necessary for expressing vectors mammals (you can use the replication origin SV40 only because it contains the early promoter).
iii) Component gene selection
Expressing and cloning vectors may contain a gene selection, also known breeding marker. Normal genes selection encode proteins that (a) give resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies or (C) supply critical nutrients that are missing in a complex environment, for example, the gene encoding racemase D-alanine for Bacilli.
In one example, the schema selection of used drug to stop the growth of the host cell. Data cells that are successfully transformed heterologous gene produce a protein which imparts resistance to the drug and, thus, survive in the mode selection. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
Another example coming up is their breeding markers for mammalian cells are those that facilitating the identification of cells competent to receive a nucleic acid encoding an anti-TGF-beta antibody, such as DHFR, thymidine kinase, metallothionein-I and-II, preferably genes metallothionein primates, adelaideans, ornithindecarboxilase etc.
For example, cells transformed gene DHFR selection, first identify the cultivation of all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. A suitable cell host, which used a wild-type DHFR is the cell line of Chinese hamster ovary (Cho) with a lack of DHFR activity.
Alternatively, you can select the cell owners (particularly wild-type hosts that contain endogenous DHFR)transformed or cotransformation DNA sequences encoding anti-TGF-beta antibody, protein DHFR wild-type and other breeding marker such as aminoglycoside-3'-phosphotransferase (ARN), by culturing cells in a medium containing selectivity of the agent for breeding marker such as aminoglycoside antibiotic series, for example, kanamycin, neomycin, or G418. See U.S. patent No. 4965199.
Suitable gene selection for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stichcomb et al., Nature, 282: 39 (1979)). Gene trp1 provide the t selection marker for a mutant strain of yeast with the lack of ability to grow in medium in the presence of tryptophan, for example, ATSC No. 44076 or RER-1. Jones, Genetics, 85: 12 (1977). The presence of the site trp1 in the genome of the yeast host cell then provides an effective environment for detecting transformation in culture in the absence of tryptophan. Similarly, the yeast strains c lack of Leu2 (ATSS 20622 or 38626) are complemented by known plasmids with genes Leu2.
In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of yeast Kluyveromyces. Alternative there are reports about expressing system for large-scale preparation of recombinant bovine chymosin for K. lactis. Van den Berg, Bio/Technology, 8: 135 (1990). Also disclosed are stable multicabinet expressing vectors for secretion of Mature human serum albumin industrial strains of Kluyveromyces. Fleer et al., Bio/Technology, 9: 968-975 (1991).
iv) promoter Component
Expressing and cloning vectors usually contain a promoter that is recognized by the body-master and functionally linked to a nucleic acid that encodes an anti-TGF-beta antibody. Suitable promoters for use with prokaryotic hosts include the phoA promoter, the promoter system β-lactamase and lactose, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac-promoter. However, suitable are other is known bacterial promoters. Promoters for use in bacterial systems will contain a sequence of Tyres Is Dalgarno (S.D.), functionally linked to DNA that encodes an anti-TGF-beta antibody.
Known promoter sequences for eukaryotes. In fact, all the genes of eukaryotes contain rich at the region located approximately 25 to 30 bases above the site where transcription. Another sequence found in 70-90 bases above the start of transcription of many genes, is the area CNCAAT (SEQ ID NO: 47), where N can be any nucleotide. At the 3'end of most eukaryotic genes is the sequence AATAAA (SEQ ID NO: 48), which may be the signal for addition of poly-a-tail to the 3'-end of the coding sequence. All sequence data respectively inserted into eukaryotic expressing vectors.
Examples of suitable promoter sequences for use with the owners-yeast include promoters for 3-phosphoglycerate or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphatedehydrogenase, glucokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, piruwatkinaza, triosephosphate, phosphoglucomutase and glucokinase.
Other yeast promoters, which are the Indus is chornye promoters, with the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, sociogram C, acid phosphatase, undergoing degradation enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use in yeast expression are additionally described in EP 73657. Yeast enhancers also are primarily used with yeast promoters.
Transcription of anti-TGF-beta antibodies from the vectors in the cells of the host mammal is controlled, for example, by promoters obtained from the genomes of viruses such as virus polyoma, avian pox, adenovirus (such as adenovirus 2), human papilloma virus bovine, avian sarcoma, cytomegalovirus, a retrovirus, hepatitis-b and most preferably simian virus 40 (SV40), heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter, heat shock promoter, provided such promoters are compatible with the system host cell.
Early and late promoters of SV40 virus respectively receive in the form of a fragment of the SV40 restriction, which also contains the replication origin in the Rus SV40. The immediate early promoter of human cytomegalovirus respectively obtained from restriction fragment HindIII E. System for the expression of DNA-hosts-mammals using human papilloma virus of cattle as a vector is disclosed in U.S. patent No. 4419446. Modification of this system is disclosed in U.S. patent No. 4601978. See also Reyes et al., Nature, 297: 598-601 (1982) on expression of cDNA for human β-interferon in mouse cells under the control of the promoter timedancing of the herpes virus simple. Alternative long terminal repeat of rous sarcoma virus can be used as a promoter.
v) Component enhancer
Transcription of DNA encoding anti-TGF-beta antibody according to this invention, in higher eukaryotes is often reinforce insert enhancer sequence into the vector. Currently, there are many enhancer sequences from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). As a rule, however, use an enhancer from a eukaryotic virus cells. Examples include the SV40 enhancer in the late side of the starting point of replication (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of polyoma on the late side of the starting point of replication and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18 (1982) enhancer e the cops for activation of eukaryotic promoters. The enhancer may be playserver in the vector at position 5' or 3' relative to the coding sequence of the anti-TGF-beta antibodies, but preferably it is located in the 5'-site from the promoter.
vi) The termination of transcription
Expressing the vectors used in eukaryotic cells-the hosts (for example, yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms)will also include sequences necessary for the termination of transcription and for stabilizing the mRNA. Typically, such sequences are 5'-end, in some cases, the 3'-end untranslated regions of eukaryotic or viral DNA, or cDNA. These areas contain nucleotide segments transcribed as polyadenylated fragments in the untranslated section of mRNA encoding anti-TGF-beta antibody. One suitable component of translation termination is the region polyadenylation bovine growth hormone. See WO 1994/11026 and expressing vectors disclosed there.
vii) Selection and transformation of host cells
Appropriate cell hosts for cloning or expression of the DNA in the vectors are the prokaryote, yeast, or higher eukaryote cells described here. Suitable prokaryotes for this whole shall include eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis R disclosed in DD 266710, published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred master of E. coli for cloning is E. coli 294 (ATCC 31 446), although suitable are other strains, such as E. coli B, E. coli X1776 (ATCC 31537), and E. coli W3110 (ATCC 27325). These examples are more illustrative than restrictive.
In addition to prokaryotes eukaryotic microbes such as filamentous fungi or yeast are suitable as hosts for cloning or expression coding for anti-TGF-beta antibody vectors. Saccharomyces cerevisiae, or common Baker's yeast are the most commonly used among lower eukaryotic microorganisms-hosts. However, there is usable a number of other genera, species and strains, such as Schizosaccharomyces pombe; Kluyveromyces, such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis, and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as A. nidulans and A. niger.
Appropriate cell hosts for the expression of glycosylated anti-TGF-beta antibodies derived from multicellular organisms. Examples of invertebrate cells include cells of plants and insects. Identified numerous baculovirus strains and the corresponding possible cell-hosts of insects, such owners, as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (Drosophila) and Bombyx mori. Widely known for a variety of viral strains for transfection, for example, a variant L-1 Autographa californica NPV and the strain Bm-5 Bombyx mori NPV, and such viruses can be used here as a virus of the present invention, particularly for transfection of cells Spodoptera frugiperda.
You can also use the culture of plant cells of cotton, corn, potatoes, soybeans, Petunia, tomatoes and tobacco as hosts.
However, the greatest interest was aroused by vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of suitable cell lines of mammalian hosts are the cell line of monkey kidney SV1 transformed by SV40 (COS-7, ATCC CRL 1651); line human embryonic kidney cells (293 or 293 cells, subcloned for cultivation in suspension culture, Graham et al., J. Gen. Virol., 36: 59 (1977)); cells SMOS is to young hamsters (KSS, ADS CCL 10); ovarian cells of Chinese hamsters/DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980), including DG44 (Urlaub et al., Som. Cell and Mol Gen., 12: 555-566 (1986)) and cell lines DP12); mouse Sertoli cells (TM4, 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); human cell carcinoma of the cervix (HELA, renowned for CCL2); cells of the kidneys of the dog MDCK, ATCC CCL 34); cells rat liver (BRL 3A, ATCC CRL 1442); cells of the human lung (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumor cells of the mammary gland of mice (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982)); the cells, MRC 5; FS4 cells and cell line human hepatoma (ner G2).
Cell host transformed using the above expressing or cloning vectors for the production of anti-TGF-beta antibodies and cultured in conventional nutrient medium, modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
viii) Culturing host cells
Cell owners used for the production of anti-TGF-beta antibodies according to this invention, can be grown in different environments. Suitable for culturing the host cells are different available industrial environment, such as environment Ham''s F10 (Sigma), minimalnayazhelaemy medium (MEM) (Sigma), RPMI-1640 (Sigma) and Wednesday Needle, modified by way of Dulbecco (DMEM) (Sigma). In addition, you can use any environment, described, for example, 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 1990/03430; WO 1987/00195 or in U.S. patent Re. 30985, as culture media for the host cells. In any of these environments can be added as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine or thymidine), antibiotics (such as GENTAMYCINTM), trace elements (usually defined as inorganic compounds present in the final concentrations in the micromolar level), and glucose or an equivalent energy source. You can also include any other necessary additives in suitable concentrations, which are known to experts in this field. Culturing conditions, such as temperature, pH and the like, are those previously used to host cells selected for expression, and they will be obvious to experts in this field.
ix) Purification of anti-TGF-beta antibodies
When using recombinant techniques, the antibody can be produced intracellularly or in perip osmoticheskoe space or directly to secrete into the environment. If the antibody is produced inside the cell, as a first stage, the debris of particles or cells of the host, or lysed fragments, is removed, for example, zentrifugenbau or ultrafiltration. Carter et al., Bio/Technology, 10: 163-167 (1992) describe a method for detecting antibodies that are secreted into periplasmatic space of E. coli. Briefly, the cell mass is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl (PMSF) for 30 min Cellular debris can be removed by centrifugation. When the antibody is secreted into the medium, supernatant of expressing such systems generally first concentrated using industrially available filter for concentrating the protein, for example, a block for ultrafiltrable AMICONTMor MILLIPORE PELLICONTM. You can include a protease inhibitor, such as phenylmethylsulfonyl (PMSF), on any of the above stages for the inhibition of proteolysis and you can add antibiotics to prevent the growth of random pollutants.
The composition of the antibodies obtained from cells can be purified using, for example, chromatography on hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography is the preferred method ocistring protein And ligand affinity depends on the species and isotype of any of the Fc domain of immunoglobulin, which is present in the antibody. Protein And can be used for purification of antibodies on the basis of human heavy chain γ1, γ2, or γ4 (Lindmark et al., J. Immunol. Meth., 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix is linked to the ligand affinity, most often represents the agarose, but there are other matrices. You can use mechanically stable matrices such as glass, controlled pore or poly(startdevice)benzene to provide faster flow and reduce processing time compared with agarose. In cases where the antibody contains a domain WithN3, it is suitable for cleaning resin is BAKERBOND ABXTM(J.T. Baker, Phillisburg, NY). There are also other techniques for protein purification such as fractionation on a column of ion exchange resin, precipitation with ethanol, obratsova HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM, chromatography on an anion - or cation-exchange resin (such as column poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate, depending on the antibodies that distinguish.
After any preliminary stage(s) of purification of a mixture containing the desired antibody and impurities, can be subjected to hydrophobic XP is matography in conditions of low pH using a buffer for elution at pH value in the range of about 2.5 to 4.5, previously performed at low salt concentrations (e.g., about 0-0,25 M salt).
IV. The pharmaceutical composition
Therapeutic compositions of the antibodies used in the present invention are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)) in the form liofilizovannyh compositions or aqueous solutions. Acceptable carriers, excipients 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 octadecyltrimethylammonium chloride; hexamethonium chloride; benzalconi chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, DISHA the IDA 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; metal complexes (e.g., complexes of Zn-protein); and/or nonionic surfactants such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG). Preferred liofilizovannye composition of anti-TGF-beta antibodies described in WO 1997/04801 included here for information.
The composition may also contain more than one active compound as necessary for specific indications, which are being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide antibodies that bind to HER-2, EGFR, TGF-beta (e.g., an antibody that binds to a different epitope on TGF-beta), ErbB3, ErbB4, or antigens vascular endothelial growth factor (VEGF) in a single composition. Alternative or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, antihormonal drug, a drug with a targeted effect on TGF-beta, an antiangiogenic drug and/or cardioprotector. Such m is likely respectively are in combination in amounts effective for the intended purpose.
The active ingredients can be included in microcapsules prepared, for example, methods koatservatsii or interfacial polymerization, for example, microcapsules from hydroxymethylcellulose or gelatin and microcapsules of poly(methyl methacrylate), respectively, in colloidal systems for the delivery of drugs (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980).
You can prepare medications with a slow release. Suitable examples of drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the antibody, where such matrices are in the form of shaped objects, for example, films or microcapsules. Examples of matrices with a slow release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol), polyactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, are not subject to the degradation of the ethylene vinyl acetate, degradiruete copolymers of lactic acid-glycolic acid such as LUPRONDEPOTTM(injectable microspheres composed of a copolymer of lactic acid-glycolic acid), and poly-D-(-)-3-guide is hydroxybutyric acid.
Compositions for use for administration in vivo must be sterile. This can easily be done by filtration through a membrane for sterile filtration.
V. Treatment with anti-TGF-beta antibodies
It is envisaged that the present invention anti-TGF-beta antibodies can be used to treat various diseases or disorders. Examples of conditions or disorders include benign or malignant tumors; leukemias and lymphoid malignancy; and other disorders such as neuronal, glial, astatically, hypothalamus, glandular, macrophagal, epithelial, stromal, blastocoele, inflammatory, angiogenic and immunologic disorders.
Basically infringement intended for treatment, is a violation associated with TGF-beta, most preferably a malignant disease. Examples of malignant tumors for treatment include, but are not limited to, carcinoma, blastoma and sarcoma, and certain types of leukemia or lymphoid malignancy. More specific examples of such malignant tumors include squamous cell malignant tumor (e.g., epithelial squamous cell tumor), malignant tumor of the lung, including small cell tumor of the lung, non-small cell lung evil is chestvennogo tumor of the lung, adenocarcinoma of the lung and squamous carcinoma of the lung, a tumor of the peritoneum, hepatocellular tumor, a stomach tumor, including tumors of the gastrointestinal tract, cancer of the pancreas, glioblastoma, cervical tumor of the uterus, ovarian tumor, liver tumor, malignant bladder cancer, hepatoma, malignant tumor of the breast, a tumor of the colon, tumor of the rectum, swelling of the colon, rectum, carcinoma of the endometrium or uterine carcinoma slyunnyh glands, malignant tumor, a prostate tumor, a tumor of the vulva, swelling of the thyroid gland, liver tumor, carcinoma of the anus, carcinoma of the penis and a tumor of the head and neck.
In one embodiment, a malignant tumor will be that which expresses (and can sverkhekspressiya) receptor TGF-beta. Examples of tumors that can Express/sverkhekspressiya receptor(s) TGF-beta include squamous cell tumor (e.g., epithelial squamous cell tumor), malignant tumor of the lung, including small cell tumor of the lung, non-small lung tumor, lung adenocarcinoma and squamous cell carcinoma of the lung tumor, a tumor of the peritoneum, hepatocellular tumor, a malignant tumor of the stomach, including the tumors of the gastrointestinal tract, pancreatic cancer, glioblastoma, cervical tumor of the uterus, ovarian tumor, liver tumor, bladder cancer, hepatoma, malignant tumor of the breast, a tumor of the colon, tumor of the rectum, swelling of the colon, rectum, carcinoma of the endometrium or uterine carcinoma of the salivary glands, kidney tumor, prostate tumor, a tumor of the vulva, swelling of the thyroid gland, liver tumor, carcinoma of the anus, carcinoma of the penis and swelling of the head and neck. However, the tumor to treatment with the antibody can be any tumor, not only those who simply Express/sverkhekspressiya receptor TGF-beta.
If the tumor for treatment may present one, which is characterized by intense activation of the receptor, TGF-beta, such intense activation may be a result of overexpression or increased production of the receptor for TGF-beta. In one embodiment, will be held diagnostic or prognostic test to determine the extent of tumor in a patient characterized by enhanced activation of the receptor for TGF-beta. For example, you can determine the amplification and/or overexpression of the receptor for TGF-beta in tumor. In this area there are different tests for determining such amplification and/or overexpression, for example, methods of immunohistochemistry (IHC); FISH, sa is used by blotting or PCR.
In addition, the overexpression or amplification of the receptor TGF-beta can be estimated using the diagnostic test in vivo, for example, the introduction of a molecule (such as an antibody)that is associated with the molecule for detection and labeled detectable label (e.g. a radioactive isotope) and external scanning the patient for localization of the label.
Tests to determine increases if humanitariannet antibody activity of TGF-beta in tumor suppression in vivo and in vitro, as described below.
A. Cytotoxic test
System L-929 is a useful test in vitro, which allows you to quickly determine the activity of the antibody, optionally in combination with a suitable regulator of immune function. The degree of correlation with the test tumor necrosis in vivo, Carswell et al., Proc. Natl. Acad. Sci. USA, 72: 3666 (1975) currently remains unknown; however, because it is specifically used murine tumor cells, the correlation should be high. In this test determines the activity of tumor necrosis factor (TNF-α) and lymphotoxin (TNF-beta). According to his principle of the test is the same as opened in U.S. patent No. 4457916 that uses mouse L-M cells and staining with methylene blue. However, it was shown that the test L-929 correlated (for TNF-α, obtained from cells HL-60) cytotoxicity for lines of human tumor cells.
In the system test L-929 cells L-929 receive during the night as monolayers in microtiter plates. The best sample was diluted two times the plate, irradiated with UV light and then applied on the resulting cell monolayers. Then in the culture medium in the wells make up to 1 μg/ml of actinomycin D. the Tablets incubated for 18 h at 37°C and tablets is assessed visually on a scale under a microscope. Each hole give 25, 50, 75 or 100%, expressing the degree of cell death in the hole. One unit of TNF activity is defined as the dilution at which there is a loss of 50% of the cells.
C. Tests in vivo
Drugs can also be tested for activity using the ability of anti-TGF-beta antibodies destroy or suppress the growth of tumors and to protect the animal with a tumor from death. Mice Balb/C vaccinated subcutaneously different types of tumor cells for the development of localized tumors. Lines of tumor cells include mouse MethA fibrosarcoma obtained in the form of a cell suspension of ascitic fluid, and MCF-7, carcinoma of the breast, which is injected in the form of 1 mm3bunch of cells.
For the production test, the mice female Balb/C (19-22 g) vaccinated subcutaneously with a needle rooms 26 suspension containing 5×105cells fibrosarcoma in 0.1 ml of medium or cells MCF-7. (Suspension cells fibrosarcoma preparing the 8-day ascitic fluid when counting the number of cells and the breeding environment, not containing serum). After 9-10 days, when the tumors become palpable, injected with 1 mg of TNF-α in the mouse, and the introduction of TNF-α I repeat, if it is desirable, in the following days. The results take into account when determining the tumor volume and survival of animals. The test is repeated using a separate serial injections of 1 μg TNF-α in mice and 10 mg/kg antibody A on the mouse. Test for the activity of antibodies compared with the data for these agents.
In those cases, when the tumor for treatment is hormone-independent tumor, it is possible to determine the expression of hormone (e.g., androgen and/or its cognate receptor in the tumor with any of the various available tests, such as described above. Alternative or additionally, the patient can be diagnose as having hormone-independent tumor, in the sense that the patient does not respond to antiandrogenna therapy.
In some embodiments, the implementation of the patient is given immunoconjugate containing anti-TGF-beta antibody conjugated with a cytotoxic agent. Preferably immunoconjugate and/or protein TGF-beta, with which it is associated, internalized cell, resulting in increased therapeutic efficacy immunoconjugate in relation to the death of tumor cells with which it is associated. In a preferred variant of the implementation of the cytotoxic agent acts intentionally, or has a negative effect on the nucleic acid in the tumor cell. Examples of such cytotoxic agents include maytansinoid, calicheamicin, ribonuclease and endonuclease DNA.
Anti-TGF-beta antibodies or immunoconjugate administered to the patient is a man known methods, such as intravenous administration, e.g., bolus or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes. Intravenous, intraperitoneal or subcutaneous administration of the antibody is preferred, despite the fact that subcutaneous or intraperitoneal routes are particularly preferred. The preferred scheme introduction is the introduction 2-3 times a week depending on the specific mammal that is being treated, the type of antibodies and other factors well known in the art. However, "work" other schemes.
Other therapeutic schemes can be combined with the introduction of anti-TGF-beta antibodies. The combined introduction includes co-administration, using separate compositions or in a single pharmaceutical composition, and the consequent introduction in any order, preferably where there is a time period while both (or all) active funds at the same time show their biological whom such activity.
In one preferred embodiment, the patient is treated with two different anti-TGF-beta antibodies.
Also, it may be desirable to combine the introduction of anti-TGF-beta antibodies or antibodies with the introduction of antibodies directed against another associated with tumor antigen. In this case, another antibody may, for example, be contacted with the antigen such as HER2, EGFR, ErbB3, ErbB4, vascular endothelial growth factor (VEGF) or b-cell surface marker or antigen (antigen expressed on the surface of b cells, which can be sent together with the antagonist, related), such as, for example, leukocyte surface markers CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 (for description see The Leukocyte Antigen Facts Book, 2ndEdition 1997, ed. Barclay et al., Academic Press, Harcourt Brace & Co., New York). Other b-cell surface markers include RP105, FcRH2, b-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, at btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA and 239287. Especially interesting b-cell surface marker preferably is expressed on b cells compared to other b-cell tissues of a mammal and may be expressed on precursor b cells and Mature b cells. Preferred b-cell surface marker is CD20 and CD22. In yet another and is too TGF-beta antibody can be combined with antiangiogenic agent, that inhibits angiogenesis. An example is a VEGF agonist, such as an antibody, for example, AVASTINTM.
In one embodiment, the treatment according to the present invention includes the combined introduction of anti-TGF-beta antibody (or antibodies) and one or more regulators of immune function in a mammal, such as cytokines and chemotherapeutic agents or inhibiting the growth of funds, including mutual introduction mixtures of different chemotherapeutic agents. Preferred chemotherapeutic agents include toxaphene (such as paclitaxel and docetaxel) and/or anthracycline antibiotics number. In relation to cooking and schemes for the introduction of such chemotherapeutic agents, they can be used according to the manufacturer's instructions, or as empirically determine a specialist in this field. Preparation and schemes for such chemotherapy are described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).
The antibody can be combined with antihormonal drug, for example, anti-estrogenic substance, such as tamoxifen, or an aromatase inhibitor such as anastrozole; antiprogesterone, such as onapristone (see, EP 616812), or antiandrogenna substance, such as flutamide, in dosages known for such molecules. In cases where treatment is subjected to hormone-n is dependent tumors, the first patient you can spend antihormone therapy, after which the tumor becomes independent of hormones, then you can enter anti-TGF-beta antibody (and optionally other means described herein).
In some cases it may be useful for co-administration to the patient cardioprotector (to prevent or reduce myocardial dysfunction associated with therapy) or one or more cytokines. You can also collaborate to introduce a cytotoxic drug. In addition to the above therapeutic schemes of the patient can be subjected to surgical removal of tumor cells and/or radiation therapy.
These anti-TGF-beta antibodies can be combined with the product, which is directed against the EGFR, such as discussed above in the section definitions with additional and potentially synergistic therapeutic effect.
Suitable dosages for any of the above simultaneously introduced funds are those that are currently used and can be reduced through the combined (synergistic) effect means and anti-TGF-beta antibodies.
For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease being treated, as defined above, the severity and course of the disease, the injected antibody for preventive or therapeutic purposes, previous therapy, the clinical history of the patient and response to the introduction of antibodies, and the decision of the attending physician. Antibody respectively administered to the patient once or treatment. Depending on the type and severity of the disease, the dose from about 1 μg/kg to 15 mg/kg (for example, 0.1 to 20 mg/kg) of antibody is an initial possible dose for administration to a patient, for example, one or more separate injections or continuous infusion. A typical daily dose may be in the range of from about 1 μg/kg to 100 mg/kg or higher, depending on the factors mentioned above. For re-introductions for several days or more, depending on the condition, treatment is continued until a desired suppression of existing symptoms.
The preferred dosage of the antibody will be in the range of from about 0.05 mg/kg to about 10 mg/kg So that the patient can enter one or more doses of approximately 0.5 mg/mg, 2.0 mg/mg, 4.0 mg/mg, or 10 mg/kg (or any combination of them). Such doses can be entered with an interval, for example every week or every three weeks (e.g. such that the patient received from about two to about twenty, for example, six doses of anti-TGF-beta antibodies). You can enter a higher initial dose, followed by the introduction of one or more lower doses. The approximate scheme dozorova the Oia includes the introduction of the initial loading dose, approximately 4 mg/kg, with subsequent introduction of a weekly maintenance dose of approximately 2 mg/kg anti-TGF-beta antibodies. However, you can use other dosing schedules. The effectiveness of this therapy is easily traced using conventional methods and tests.
An alternative antibody respectively administered serially or in combination with radiation treatment, irradiation or administration of radioactive substances, such as indicated in the UICC (Ed.), Klinische Oncologie, Springer-Verlag (1982).
In addition to the introduction of the patient protein antibodies present application includes the introduction of antibodies gene therapy. This introduction of nucleic acid that encodes the antibody included in the expression "the introduction of a therapeutically effective amount of antibodies". See, for example, WO 1996/07321, published on 14 March 1996 concerning the application of gene therapy to generate intracellular antibodies.
There are two basic approaches to the introduction of the nucleic acid (optionally in the vector) in cells of a patient in vivo and ex vivo. In terms of delivery in vivo nucleic acid is injected injected directly into the patient, usually in the place where you want antibody. For the treatment of ex vivo cells of the patient are removed, the nucleic acid is introduced into the data of the selected cells and the modified cells are administered to the patient either directly or, for example, and is encapsulated in porous membranes, who implanted the patient (see, for example, U.S. patent No. 4892538 and 5283187). There are various available methods of introducing nucleic acids into living cells. Methods vary depending on the transferred nucleic acid in cultured cells in vitro or transferred to the cells of the intended host in vivo. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the precipitation of calcium phosphate, etc. Commonly used vector for gene delivery ex vivo is a retrovirus.
The currently favored methods of transfer of nucleic acids in vivo include transfection with viral vectors (such as adenovirus, herpes virus simple I or adenopathy virus) and systems based on lipids (suitable lipids for mediated lipid transfer of the gene are, for example, DOTMA, DOPE and DC-Chol). In some situations it is desirable to provide a source of nucleic acid agent, which aims to target cells, such as specific antibodies against cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. When applied liposomes, proteins associated with the cell surface membrane protein associated with endocytosis, can be used for targeted and/or to facilitate uptake, e.g. capsid proteins or fragments thereof, have trapnest for a particular cell type, antibodies for proteins which undergo internalization in the blood, and proteins, which are aimed at intracellular localization and increased half-life. The technique of receptor-mediated endocytosis is described, for example, Wu et al., J. Biol. Chem., 262: 4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990). Review existing protocols marking of known genes and gene therapy, see Anderson et al., Science, 256: 808-813 (1992). See also WO 1993/25673 and cited there sources.
In one specific embodiment, the tumor, such as a malignant tumor of the lung, melanoma, cancer of the breast, malignant tumor, a tumor of the colon, rectal, pancreatic cancer, treated at a mammal, preferably a human, by administration to a mammal an effective amount of TGF-beta antibodies and antibody that binds to VEGF, optionally together with any other suitable means, are presented here. Preferably TGF-beta antibody is a monoclonal antibody, more preferably, which is associated with any one or more of the following: TGF-beta, TGF-beta2 and TGF-beta, and even more preferably of light is ivalsa, at least with TGF-beta or both TGF-beta and TGF-beta2, and most preferably is humanitariannet antibody presented here. In another embodiment, the antibody that binds to VEGF, is a monoclonal antibody, and more preferably it blocks or neutralizes VEGF and/or blocks binding of VEGF with one or more of its receptors.
In another embodiment, the invention provides a product containing substances suitable for the treatment of the disorders described above. The product includes a container and a label or an insert in the packaging for or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. of the Container can be made of various materials such as glass or plastic. The container contains a composition that is effective for treating the condition and may have a sterile access hole (for example, the container may be a bag or a vial for intravenous administration, with the locking device with the injection needle for subcutaneous injection). At least one active substance in the composition is humanitariannet anti-TGF-beta antibody. On the label or an insert in the packaging indicates that the composition is used for treating the condition of choice, t is someone like a malignant tumor. In one embodiment, the label or the liner in the packaging indicates that the composition comprising the antibody can be used for the treatment of disorders mediated TGF-beta, for example, for the treatment of malignant tumors, which expresses the receptor for TGF-beta. In addition, the label or the liner in the package can be stated that a patient who undergoes treatment, is the one with the tumor, which is characterized by increased activation of the receptor for TGF-beta. On the label or the liner in the package can also be stated that the composition can be used to treat the tumor, which is not characterized by overexpression of the receptor for TGF-beta. In other embodiments, the implementation on an insert in the packaging can be shown that the antibody or composition can be used for the treatment of malignant tumors of the breast (e.g., tumors of the breast with metastases); hormone-independent tumor; malignant neoplasm of prostate (e.g., androgen-independent prostate tumors); malignant tumor of the lung (e.g. small cell lung tumors); malignant tumor of colon, rectal or colorectal cancer; or any other diseases or disorders disclosed here.
In addition, the product may contain (a) a first container with a composition, the finding is camping in it, where the composition contains humanitariannet antibody, and b) a second container with a composition contained in it, where the composition comprises a therapeutic agent other than humanitariannet antibody. The product manufacture in this embodiment of the invention may optionally include an insert in the packaging, which indicated that the first and second compositions can be used in combination for the treatment of mediated TGF-beta violations, such as a malignant tumor. Such therapeutic agent may be any of the additional preparations described in the previous section (e.g., chemotherapeutic agent, an antiangiogenic drug, antihormonal drug, cardioprotector and/or a regulator of immune function in a mammal, including a cytokine). Alternative or additionally, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose. It may further include other materials desirable from a commercial and consumer standpoint, including other buffers, diluents, filters, needles and syringes.
VII. Non-therapeutic use of anti-TGF-beta antibodies
Antibodies (the example humanized anti-TGF-beta antibodies) of the invention have additional non-therapeutic applications.
For example, antibodies can be used as agents for affinity purification. In this way antibodies immobilized on a solid phase, such as resin SEPHADEXTMor filter paper, using methods well known in the field. The immobilized antibody is subjected to interaction with the sample containing the protein TGF-beta (or its fragment) for cleaning, and then the substrate is washed with a suitable solvent that will remove substantially all of the substance in the sample, with the exception of the protein TGF-beta, which is bound to the immobilized antibody. Finally, the substrate is washed with another solvent, such as glycine buffer, pH 5.0, which will separate the protein TGF-beta antibodies from.
Anti-TGF-beta antibodies can also be used in the formulation of diagnostic tests for protein TGF-beta, for example, when determining its expression in specific cells, tissues, or serum.
For diagnostic applications, the antibody typically will be labeled with detectable group. There are numerous labels, which in General can be grouped into the following groups:
(a) radioisotopes, such as35S14C,125I3H and131I. Antibody can be marked R is dostopa using methods, described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Wiley-Interscience, New York, Pubs. (1991)for example and radioactivity can be determined using a scintillation counter;
b) there is a fluorescent label, such as chelates of rare alkaline-earth metals (chelation Europe) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and taheny red. Fluorescent labels can be konjugierte with the antibody using methods, for example, disclosed in Current Protocols in Immunology, above. Fluorescence can be determined using fluorimetry;
C) there are different labels based enzyme substrates, and in U.S. patent No. 4275149 provides an overview on some of them. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate, which can be determined using various techniques. For example, the enzyme may catalyze a color change of the substrate, which can be determined spectrophotometrically. An alternative enzyme can change the fluorescence or chemiluminescence substrate. Methods for the quantitative determination of fluorescence described above. Chemiluminescent substrate becomes electronically excited by chemical reaction and may then emit light which can be defined (for example, using chemiluminometer), or eradiate energy to the acceptor fluorescence. Examples of enzymatic labels include luciferase (e.g., of Firefly luciferase and bacterial luciferase; U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, SharedAccess (e.g., glucose oxidase, galactosidase and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides and the like. Methods of conjugating enzymes to antibodies are described in O'sullivan et al., "Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay," in Methods in Enzym. (Ed., J. Langone &H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981).
Examples of combinations of enzyme-substrate, for example, include:
i) horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate, where the hydrogen peroxidase oxidizes a precursor ink (for example, orthophenylphenol (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));
ii) alkaline phosphatase (AP) with para-nitrophenylphosphate as chromogenic substrate; and
iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) or flourophenyl substrate 4-methylumbelliferyl-β-D-galactosidase.
Specialists in this area known for numerous other combinations of enzyme-substrate. is the overall review of it, see U.S. patent No. 4275149 and 4318980.
In some cases, the label kongugiruut indirectly with the antibody. Specialists in this field there are various methods for doing this. For example, the antibody can be konjugierte with Biotin and any of the tags belonging to the three groups listed above, you can konjugierte with Avidya or the like. Biotin selectively binds with Avidya and, thus, the label can be konjugierte with the antibody data indirectly. Alternative to achieve indirect conjugation of the label with the antibody, antibody kongugiruut with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above, kongugiruut with anti-hapten-antibody (e.g., anti-digoxin-antibody). Thus, it is possible to conduct indirect conjugation of the label with the antibody.
In another embodiment, the invention does not require the introduction of a label in an anti-TGF-beta antibody and its presence can be detected using a labeled antibody which binds with anti-TGF-beta antibody.
Antibodies of the present invention can be used in any known test, such as tests of competitive binding, direct and indirect sandwich tests and tests thus. Zola, Monoclonal Antibodies: A Manual of Techniques, p. 147-158 (CRC Press, Inc. 1987).
For immunohistochemistry the tumour sample may b the th just selected or frozen or may be in paraffin and fixed with a preservative, such as formalin.
Antibodies can also be used for diagnostic tests in vivo. Typically, the antibody have been labelled with a radionuclide (such as111In99Tc14C,131I125I3H,32P or35S) so that, for example, the tumor can be localized using immunoscintigraphy.
For convenience, the antibody of the present invention can be provided in the set, i.e. the packaged combination of reagents in predetermined amounts with instructions for setting a diagnostic test. When the antibody is labeled with an enzyme, the kit will include substrates and cofactors required for the enzyme (e.g., the predecessor of the substrate, which provides the detected chromophore or fluorophore). You can also include other additives such as stabilizers, buffers (for example, the buffer block or buffer for lysis), and the like. The relative amounts of the various reagents may be varied widely to provide concentrations in solution of the reagents which substantially optimize the sensitivity of the test. In particular, the reagents can be provided as dry powders, usually liofilizovannyh, including fillers which, when dissolved, will ensure R is the target of the reagent with the appropriate concentration.
This antibody will also be suitable for obtaining images of the in vivo when the labeled antibody is administered to a host, preferably into the bloodstream, and determine the presence and the presence of labeled antibody in the host. This technique of visualization, respectively, used in the diagnosis and treatment of tumors. Antibody respectively mark any group that is detectable from the owner, including the detected non-radioactive indicators, for example, nuclear magnetic resonance, or other methods known in this field. However, it is preferable that the label was radioactive label, including iodine, for example,125I and131I, selenium, bifunctional chelates, copper, for example,67Cu, technetium, for example,99mTc and rhenium, for example,186Re and188Re. The radioactive isotope kongugiruut protein by any means, including metal-chelating compounds or lactoperoxidase, or methods based Imogene for iodination.
Mouse monoclonal antibody 2G7 was deposited in the American type culture collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (ATCC) as HB10240 in 9/28/89; and mouse monoclonal antibody A was deposited as ATCC HB10241 in 9/28/89.
Additional details of the invention presents further non-limiting examples. The disclosure of all of the quotes in the above and in compressed form is included here for information.
Fabrication and characterization of a monoclonal antibody 2G7 and A
I. Definition ELISA
96-well tablets made of polystyrene covered purified TGF-beta with a concentration of 1 μg/ml at the rate of 100 µl/well in carbonate buffer pH 9.6 V for 18 h at 4°C. Coated plates were blocked with 0.5% bovine serum albumin (BSA) in PBS (named BPBS) for 1 h at 22°C, washed surface-active agent 0.05% tween-20TMin PBS (named PBST) and incubated with 100 μl of the supernatant of hybridoma for 1 h at 22°C. the Tablets were washed in PBST and the bound antibodies were detected with goat artemisinin IgG, conjugated with horseradish peroxidase (Tago, Burlingame, CA). The tablets were washed in PBST and contributed substrate o-phenylenediamine hydrochloride at the rate of 100 µl/well. The reaction was stopped after 15 min and was determined by optical density at 492 nm on a plate reader for tablets UVMAXTM(Molecular Devices, Palo Alto, CA).
II. Iodination gtr-beta
Purified TGF-beta was madirovalo modified method using sodium salt of n-chloro-para-toluensulfonate CHLORAMINE TTM(Greenwood et al., Biochem. J., 89: 114 (1963)). Briefly, 10 µg of purified gtr-beta marked 1 mcure Na125on ice with three successive additions of 20 μl of 0.1 mg/ml of sodium salt of n-chloro-para-toluensulfonate CHLORAMINE TTMR is zelennymi incubation for 2 minutes The reaction was stopped by sequential additions of 20 μl of 50 mm N-acetyltyrosine, 1M potassium iodide, followed by adding 200 μl of 8M urea. Audirovannyj gtr-beta was separated from free Na125I HPLC using a C18 column and gradient triperoxonane acid/acetonitrile and the fractions containing the main peak were pooled and stored at -70°C (specific activity 112 mccoury/µg).
III. The radioimmunoassay with capture antigen
Microtiter strips IMMULONTM2 "REMOVAWELL"TM(Dynatech, Chantily, VA) were coated with goat artemisinin IgG concentration of 5 µg/ml (Boehringer Mannheim) in carbonate buffer pH 9.6 V for 18 h at 4°C. the Wells were washed in PBST, blocked by PBS containing 0.1% gelatin (called PBSG), washed with PBST, and incubated with the supernatant of hybridoma for 4 h at 22°C. the Wells were washed in PBST and contributed 75,000 pulses / min/hole125I gtfr-beta in 100 μl of 0.1% gelatin in PBST and incubated for 2 h at 22°C. the Tablets were washed in PBST and quantify the associated125I gtfr-beta on the counter GAMMAMASTERTM(LKB, Sweden).
Specificity monoclonal anti-TGF-beta antibodies were also assessed on their ability to immunoprecipitate125I gtfr-beta or porcine platelet125I gtfr-beta2 (R & D Systems, Minneapolis, MN; specific activity 103,4 mccur the/µg). Two μg of purified monoclonal antibodies were incubated with 5×104pulses per min125I gtfr-beta or125I gtfr-beta2 for 2 h at 22°C. the immune complexes were besieged by protein-A agarose A-SEPHAROSETM(Repligen, Cambridge, MA)coated with rabbit artemisinin IgG (Boehringer Mannheim Biochemicals, Indianapolis, IN), and then washed 3 × with PBST. Complexes dissociatively of protein-A agarose A-SEPHAROSETMregenerating buffer, subjected to electrophoresis in 12% SDS-polyacrylamide gel (SDS-PAGE) and were autoradiographically.
V. Determination of affinity monoclonal anti-TGF-beta antibodies
Solid-phase radioimmunoassay described by Mariani et al., J. Immunol. Methods 71: 43 (1984), was used to determine the affinity of a monoclonal specific TGF-beta antibodies. Briefly, purified monoclonal anti-TGF-beta antibodies were applied to microtiter strips IMMULONTM2 "REMOVAWELL"TMin carbonate buffer pH 9.6 V for 18 h at 4°C. the Wells were washed and blocked as described above. 40,000 pulses per minute/hole125I gtfr-beta or pork125I gtfr-beta2 (R & D Systems) in 50 µl of the PBSG in 2-fold serial dilutions of unlabeled gtr-beta or pork gtr-beta2 calculated from 2500 to 9.7 ng/well in 50 μl of PBSG. The resulting mixture was incubated for 18 h at 4°C. the Wells were washed and radioactivity was determined as described above, and calculated affinity constants is Ecodom Scatchard (Munson and Pollard, Anal. Biochem., 107: 220 (1980)), which gives the same results as the analysis of nonlinear regression Antoni and Mariani, J. Immunol. Meth., 83: 61 (1985).
VI. Selection of monoclonal antibodies from ascitic fluid
The original hybridoma culture secreting the antibody, which was positive in the above tests, cloned by limiting dilution and cultured in ascitic fluid of mice Balb/C (Potter et al., JNCI, 49: 305 (1972)), premirovany primer PRISTANETM. Monoclonal antibodies were isolated from ascitic fluid using protein-A agarose A-SEPHAROSETMand suirable 0,1M acetic acid, 0.5m NaCl, pH 2,4, using accepted methods (Goding, J. Immunol. Methods 20: 241 (1978) and stored in sterile PBS at 4°C.
VII. Neutralizing monoclonal antibodies in vitro specific activity of TGF-beta
Used the test with TGF-beta in vitro on cell lines of lung fibroblasts mink, Mv-3D9 (subcloned from Mv1Lu obtained from the American type culture collection, Manassas, VA, under the number ATCC No CCL-64). Briefly, purified monoclonal anti-TGF-beta antibodies and control were incubated with gtr-beta, natural porcine TGF-beta2 (R & D Systems) or gtr-beta (Derynck et al., Nature, above) at a final concentration of 1000-2000 PCG/ml for 18 h at 4°C. 50 μl of these mixtures were made in 96-well microtiter tablets with subsequent addition of 1×10 cell Mv-3D9 50 μl of minimum essential medium containing 2 mm glutamine and 5% fetal bovine serum, and incubated for 18-24 h at 37°C in an atmosphere of 5% CO2. The wells were made 1 mccoury3H-thymidine in 20 μl and were collected after 4 h at 37°C and the radioactivity was determined by scintillation counter. The percentage inhibition of the absorption of3H-thymidine for each dilution of the standard TGF-beta used to calculate the activity of TGF-beta in PCG/ml in samples treated with negative control monoclonal antibody specific to TGF-beta monoclonal antibody.
VIII. Ittipiboon monoclonal antibodies
Ittipiboon TGF-beta-reactive monoclonal antibodies was performed using a device for fluorescent technology screening PANDEXTM. Polystyrene granules coated with rat antimachine-IgG-anticorodal, used for binding monoclonal antibodies from the culture supernatant, spilled in 96-well tablets PANDEXTM. The tablets were washed and brought conjugated with FITC rat monoclonal to antimelanoma the isotype specific reagents (Becton Dickinson Monoclonal Center). Quantify the associated fluorescence device for fluorescent technology screening PANDEXTM.
IX. Analysis of epitope
Ocimene the monoclonal anti-htfr-beta antibodies conjugatively with peroxidase from horseradish (HRP) according to the method of Nakane and Kawaoi, J. Histochem. Cytochem., 22: 1084 (1974). Covered gtr-beta the plates were incubated with 50 μg/ml of purified anti-htfr-beta-antibody or negative control in PBS for 2 h at 22°C. Then, a predetermined dilution of conjugate monoclonal anti-htfr-beta-antibody-HRP were made into tablets, and incubated for 1 h at 22°C. the Tablets were washed and added to the substrate and quantify the reactivity, as described above. The percentage of blocking heterologous monoclonal anti-htfr-beta antibodies was compared with self-reactive positive control.
X. Analysis of the Western blot turns
1 µg/lane gtr-beta were subjected to electrophoresis in 12% SDS-PAGE using a non buffer to determine the different reactivity of monoclonal antibodies with dimeric forms gtr-beta. Peptides were translationally on nitrocellulose paper and hybridized with the appropriate monoclonal antibody conjugated with HRP. Bound antibody was visualized using insoluble substrate 4-chloro-1-naphthol (Kirkegaard and Perry, Gathersburg, MD). The reaction was stopped after 15 min by thorough washing with distilled water and immunoblot dried and photographed.
Century Products anti-TGF-beta - and anti-TGF-beta2-specific monoclonal antibodies
In the original protocols immunization of mice Balb/C immune is zerouali gtr-beta (received and cleared, as described Derynck et al., Nature, above) in subcutaneous and intraperitoneal administration using different immunogenic preparations, doses and schemes and using complete and incomplete adjuvant's adjuvant. Immunization was performed before 11 weeks. In some mice the response was expressed in defined, but low titre anti-htfr-beta antibodies, and two of these mice were killed and spleen were used for hybridization. From 1152 original cultures only 84 supernatant were positive for anti-TGF-beta. 10 from these hybridomas were cloned and received monoclonal antibodies with low affinity, which could not be used to develop a test or treatment.
As an alternative strategy group of 10 female mice of Balb/C (Charles River Breeding Laboratories, Wilmington, MA) were injected with 5 μg/dose of purified TGF-beta in 100 μl of adjuvant DETOXTM(RIBI ImmunoChem Res. Inc., Hamilton MT) in the pads of the hind limbs at 0, 3, 7, 10 and 14 days. On the 17th day the animals were killed, removed, inguinal and popliteal lymph nodes and lymphocytes were separated from stromal nodes using stainless steel mesh. Suspensions of lymphocytes from all 10 mice were combined and hybridisable with murine myeloma lines H-Ag8.653 (Kearney et al., J. Immunol., 123: 1548 (1979)) using 50% polyethylene glycol 4000 adopted methods (Oi and Herzenberg, in Selected Methods in Cellular Immunology, B. Mishel and S. Schiigi, eds. (W. J. Freeman Co., San Fancisco, CA, 1980), p. 351. Hybrid cells were sown in General 1334 96-well tablets with a titer of 2×105cells/well, followed by selection of NAT (Littlefield, J. W., Science, 145: 709 (1964)) 1 day after hybridization.
1190 holes were reactive with the immobilized recombinant TGF-beta when ELISA. 18 of these cultures remained stable during the reproduction and cell lines were frozen. Data source culture was isotopically and analyzed for their ability to capture125I gtfr-beta and neutralize in vitro the activity of TGF-beta. Of the 18 original cultures, which were analyzed for neutralizing gtr-beta and then isotopically, two represented the isotype IgG1 Kappa; others represented the isotype IgG2b Kappa. It was found that only monoclonal antibodies specific to the subclass IgG1, showed inhibitory (neutralizing) activity against gtr-beta in vitro. Took three stable hybridoma that secretively high-affinity monoclonal antibodies against TGF-beta. The characteristics of these antibodies are presented in detail below.
C. Immunoprecipitate labeled with radioactive iodine TGF-beta
The experiments thus conducted to determine the ability of three monoclonal antibodies to recognize and precipitate TGF-beta in solution. The autoradiograph showed that m is noonline antibodies against TGF-beta 2G7, 4A11 and N was immunoprecipitated an equivalent amount125I gtfr-beta, while the control monoclonal antibody 6G12 gave a negative result. Immunoprecipitation band had an apparent molecular weight of about 14.5 KD. A competitive inhibition test was used to determine the affinity of interaction between TGF-beta and each of the monoclonal antibodies. Monoclonal antibody 2G7 and 4A11 had approximately equal, the higher the affinity, which was 1.2×108l/mol.
The experiments thus conducted to determine the ability of selected monoclonal antibodies to recognize and precipitate TGF-beta2 in the solution. The autoradiograph showed that in contrast gtr-beta only antibody 2G7 was immunoprecipitated125I-TGF-beta2 to a defined extent. When comparing antibody 4A11 and N with a negative control antibody was detected minor specific precipitation. These results were surprising in that in a cross-blocking has been shown that antibodies 4A11 and 2G7 were able to inhibit the binding of each other human gtr-beta. See table 1.
|vyzyvayuschee monoclonal antibody||The percentage of cross-blocking Mabs c TGF-beta|
Blocking monoclonal antibodies
|*Mab 456 represents a control antibody that vzaimodeistvie with CD4|
Collectively, the data indicate that the epitopes recognized by two monoclonal antibodies are different, but are either in close proximity or in some other way affect the binding of another at a distance. According to thus or cross-block was that N is another epitope, despite some blocking. This conclusion is also supported by data on not what tralization, below.
D. Analysis by Western blot turns with gtr-beta
Because the active form of TGF-beta is glycosilated, we conducted Western blot turns to determining whether the monoclonal antibodies recognize this form. All antibody 2G7, 4A11 and N were subjected to interaction mediated by the Western blot turns with the dimeric form of TGF-beta (unrestored). Antibody 2G7 gave a stronger band compared to 4A11 or N. As experience thus control antibody 6G12 gave a negative result. This type of reactivity was also observed in direct Western-blotting with HRP-conjugates data monoclonal antibodies.
In conclusion, the performed Protocol using immunization pad extremities merge lymph nodes after many unsuccessful attempts to eliminate tolerance gtr-beta using different immunization schemes and dosing in mice Balb/C and SN with complete and incomplete adjuvant's adjuvant. Basically, this procedure was deemed suitable for quick response with very high affinity to these weak antigens in contrast to the results of experience Dasch et al., above that received neutralizing TGF-beta and TGF-beta2 monoclonal antibodies using purified bovine derived from bone TGF-beta2 in adjuvant who's adjuvant as an immunogen in mice Balb/C.
All three monoclonal antibodies were associated with gtr-beta in Western blot turns, ELISA, cross-blocking and thus. Two of the anti-htfr-beta antibodies neutralized the activity gtr-beta in vitro, while only one of the two antibodies neutralized the activity of TGF-beta2 and TGF-beta in test cell lung fibroblasts mink. Neutralizing TGF-beta antibodies also blocked the binding of labeled radioactive iodine gtr-beta in radioreceptor test, indicating that neutralization activity gtr-beta in vitro may be due to blocking of the receptor.
Humanized antibody 2G7
The variable domains of the murine monoclonal antibody 2G7 first cloned into a vector that allows you to produce a chimeric mouse/human Fab fragment. Allocated total fraction of RNA from hybridoma cells using a kit for the extraction of RNA STRAGENETMfollowing the protocols of the manufacturer. The variable domains of the amplified RT-PCR, gel purified and inserted into a derivative of plasmid pUCl19 containing human constant domain Kappa and human domainn1, as described previously (Carter et al., Proc. Natl. Acad. Sci. (USA), 89: 4285 (1992) and U.S. patent No. 5821337). The resulting plasmids were transformed into strain IS E. coli for expression of the Fab fragment. Cultivation ku is tour, induction of protein expression and purification of Fab fragment was performed as described previously (Werther et al., J. Immunol., 157: 4986-4995 (1996); Presta et al., Cancer Research, 57: 4593-4599 (1997)).
DNA sequencing of chimeric clones allowed us to identify residues of CDRs (Kabat et al., above). When using oligonucleotide site-directed mutagenesis all six of these areas CDR inserted into complete human skeleton schastok (VLKappa subgroup I and VHsubgroup III)in the plasmid VX4, as described previously (Presta et al., Cancer Research, 57: 4593-4599 (1997)). Protein obtained from "CDR-exchange" expressed and purified as described above. Conducted experiments on linking to compare the two options. In short, the tablet NUNC MAXISORPTMcovered 1 μg per ml of the extracellular domain of TGF-beta (ECD; obtained as described in WO 1990/14357) in 50 mm carbonate buffer, pH of 9.6, overnight at 4°C and then blocked with diluent for ELISA (0,5% BSA, 0.05% of nonionic surfactant POLYSORBATETM, PBS) at room temperature for 1 h Serial dilution in diluent for ELISA were incubated in tablets within 2 hours After washing were detected bound to the Fab fragment with biotinylated mouse anti-human Kappa antibody (ICN 634771), then with streptavidin, conjugated to peroxidase from horseradish (Sigma) and using 3,3',5,5'-tetramethylbenzidine (Kirkegaard ∓ Perry Laboratories, MD) as substrate. Was determined by the absorbance at 450 nm. Linking "CDR-exchange" Fab was significantly lower compared with the binding of chimeric Fab fragment.
To restore binding gumanitarnogo Fab designed mutants using DNA from "CDR-sharing" as a matrix. With the use of the computer program it was found that these mutations lead to changes in the balance of the human frame portion at their mouse opposites in positions where change could affect the conformation of the CDR and the surface of the separation of the antibody-antigen. The mutants are presented in table 2 (note: the numbering of amino acids is expressed as in Kabat et al., above). Sequence see figure 1-4.
|Designation of mutations FR gumanitarnogo antibody 2G7|
|Mutant No.||Replacement frame plot (FR) in comparison with human anti-TGF-beta consensus sequence (SEQ ID NO: 6)|
|Option 4||ArgH71Ala, AlaH49Gly,|
|VA is iant 5||ArgH71Ala, AlaH49Gly, PheH67Ala|
|Option 6||ArgH71Ala, AlaH49Gly, LeuH78Ala|
|Option 709||ArgH71Ala, AlaH49Gly, ValH48Ile|
|Option 710||ArgH71Ala, AlaH49Gly, IleH69Leu|
|Option 11||ArgH71Ala, AlaH49Gly, AsnH73Lys|
|Option 712||ArgH71Ala, AlaH49Gly, IleH69Leu, AsnH73Lys|
Options 3 and 4 used as an intermediate for obtaining humanized Fab variants with higher numbers. Option 5, with changes AlaH49Gly, PheH67Ala and ArgH71Ala, had restored binding to that of the original chimeric Fab fragment antibody 2G7 as options 709 and 11 (figure 5). I believe that the options 710 and 712 have the same binding chimeric fragment, but the option 712 has an additional mutation frame section, which may be undesirable because of increased immunogenicity. Additional remains of FR or CDR, such as L3, L24, L54 and/or N can be modified (for example, replace as follows: GlnL3Met, ArgL24Lys, ArgL54Leu, GluH35Ser). Replacement, which could be desirable to improve the stability, represent a substitution of leucine or isoleucine for methionine to reduce oxidation, or is the Amen asparagine in CDR on other residues to reduce the possibility of deliciouse. Alternative or additional humanitariannet antibody can have a Mature affinity (see above) to further improve or improve its affinati and/or biological activity.
Plasmids for the expression of moderaterna IgG was designed by sublimemovies domains VL and VH chimeric fragment antibody 2G7, and humanized variants of Fab 5, 709 and 11 in the previously described pRK vectors for expression in mammalian cells (Gorman et al., DNA Prot. Eng. Tech., 2: 3-10 (1990)). Briefly, each design was Fab EcoRV digested and BlpI to cut the VL fragment, which was cloned in the EcoRV sites/BlpI plasmid pDR1 (see Fig.6) for the expression of the complete light chain domains (VL-CL). Additionally, each design Fab uncoupled PvuII and ApaI for clipping VH fragment, which was cloned into the PvuII sites/ApaI plasmid pDR2 (see Fig.7) for the full expression of heavy chain domains (VH-CH1-CH2-CH3).
For each variant IgG was performed transient transfection with cotranslational expressing the light chain plasmid and expressing the heavy chain plasmids in a line of human embryonic kidney cells transformed with adenovirus, 293 (Graham et al., J. Gen. Virol., 36: 59-74 (1977)). Briefly, 293 cells were split the day before transfection and were sown in a medium containing serum. The next day I prepared the precipitate using the calcium phosphate from dahapute the Neu DNA light and heavy chains together with a DNA vector pADV ANTAGE TM(Promega, Madison, WI) and was added dropwise into tablets. Cells were incubated over night at 37°C, then washed with PBS and cultured in medium without serum for 4 days, when collected, air-conditioned environment. Antibodies were isolated from the culture supernatant using protein-A agarose A-SEPHAROSE CL-4BTM, then was replaced by a buffer of 10 mm sodium succinate, 140 mm NaCl, pH 6.0, and concentrated using microconcentrators METHOD to 10TM(Amicon). The protein concentration was determined by analysis of the absorption at 280 nm or by quantitative analysis of amino acids.
Additional modifications option 5 IgG hu2G7 conducted to determine which CDR contributes to binding, some CDR you can revertively in sequence loci Kappa human germ line without loss of activity or for the stabilization of antibodies. They are listed in table 3 and represented by amino acid differences between version 5 and data options.
|Designation of mutations CDR gumanitarnogo antibody 2G7|
|Mutant No.||Replace the CDR in comparison with human anti-TGF-beta antibody option 5|
|Varian is 5 (V5H.V5L)|
|H2N1.V5L||Same as alternative 5, except that Asn51 replaced by Ile in CDR H2|
|V5H.g1L2||Same as alternative 5, except that the CDR L2 revertive in the sequence of the Kappa locus of the human germ line L8/L9/L14/L15:YASSLQS (SEQ ID NO: 39|
|V5H.g1L1glL2||Same as alternative 5, except that the CDR L1 revertive in the sequence of the Kappa locus of the human germ line L8/L9:RASQGISSYLA (SEQ ID NO: 37), and CDR L2 revertive in the sequence of the Kappa locus of the human germ line L8/L9/L14/L15:YASSLQS (SEQ ID NO:39)|
|H2NI.g1L1glL2||Same as alternative 5, except that the CDR L1 revertive in the sequence of the Kappa locus of the human germ line L8/L9:RASQGISSYLA (SEQ ID NO: 37) and CDR L2 revertive in the sequence of the Kappa locus of the human germ line L8/L9/L14/L15:YASSLQS (SEQ ID NO: 39) and Asn51 replaced by Ile in CDR H2|
The name for the sequence germline used for CDR-L1, is L8/L9, as shown in figure 4 in Cox et al., Eur. J. Immunol., 24: 827-836 (1994) and five at Schable and Zachau, Biol. Chem. Hoppe-Seyler, 374: 1001-1002 (1993). For CDRL2 sequence of the germline of the named L8/L9/L14/L15 (see Cox et al., is use and Schable and Zachau, above).
Spent a reversal in the sequence of the Kappa locus of human germ-line (gl) in all CDR, but only revertant the germ line, above, showed binding (see Fig). From the data presented on this figure, it follows that V5H.g1L2 with CDR L2, revertively in the sequence of the Kappa locus of the human germ line, is still associated with TGF-beta, as V5H.V5L. Two options V5H.g1L1g1L2 and H2NI.g1L1g1L2 and H2NI.V5L not contacted as chimeric antibody.
Test proliferation of murine mesangial cells used for testing control antibodies and several humanized antibodies (V5H.V5L, V5H.g1L2, H2NI.V5L, V5H.g1L1g1L2 and H2NI.g1L1g1L2). The research Protocol is the following:
1 day: mouse mesangial cells were planted in 96-well plates in medium (a mixture eagle medium modification of Dulbecco and environment Hams F12 with 95% fetal bovine serum with 5% of 14 mm HEPES buffer) and were cultured overnight;
2 day: TGF-beta in three different concentrations (100 ng, 10 ng and 1 ng) and five different types of humanized TGF-antibodies (20 μg/ml) were diluted with medium without serum was added to the cells. Mouse TGF-antibody was used as control (2G7);
on the 4th day: after 48 h incubation, 20 μl of a buffer for reaction (CELLTITER 96 AQUEOUS ONE SOLUTION REAGENTTM(PROMEGA INC. the number rolled is GU G3580)) were introduced into each well of the tablet, and incubated for 2 hours Absorbance (OD) was determined at 490 nm.
H2NI.V5L (20 μg/ml) completely blocked the inhibition of cells induced by TGF-beta in a concentration of 1 ng/ml, which is the same result obtained using chimeric mouse control antibody (see Fig.9). Option 5 V5H.V5L also blocked the inhibition of cells similar to control.
Various humanized antibodies were tested for their activity to neutralize various TGF-beta in comparison with the antibody 2G7 using cell line T of fibroblasts destroyed embryos Swiss mice, stimulated one of the three TGF-beta in vitro, and then was defined as the activity of their proliferation. The results are presented on figure 10-14. These figures indicate that humanitariannet antibody H2NI.V5L surpassed in its blocking activity control antibody 2G7. Other humanized tested antibodies H2NI.g1L2 (CDR L2, revertively in the sequence of the Kappa locus of the human germ line) and V5H.g1L2 (CDR L2, revertively in the sequence of the Kappa locus of the human germ line) showed comparable inhibitory activity, despite the fact that V5H.g1L2 were the least effective for all TGF-beta-3.
In conclusion, humanized antibodies V5H.V5L, V5H.g1L2, H2NI.V5L, H2NI.g1L2 and options 709, 710 and 711 are the most predpochtitel is passed humanitarianism options because they are comparable in extent associated with TGF-beta, as chimeric antibody (chimH.chimL; fragment Fab 2G7) and/or neutralize TGF-beta or block the inhibition of cells induced by TGF-beta in vitro, and have minimal changes wireframe plot of all tested humanized antibodies, which will minimize the risk of the manifestation of the immune response in patients. In addition, H2NI.V5L is particularly preferred antibody, because it has higher neutralizing activity of all three isoforms of TGF-beta (TGF-beta 1, 2, 3) and may have increased stability due to changes in CDR H2. In addition, it can be expected that these humanized antibodies that have shown an increased ability to block the activity of all three ligands TGF-beta in vitro compared with mouse monoclonal antibody 2G7, will function better in comparison with 2G7 in various indications, presented below, due to their higher ability to inhibit all induced by TGF-beta effects, as shown in the test fibroblasts induced by TGF-beta (figure 10-14).
Therapy of recurrent or refractory malignant tumors of the prostate
This antibody is a full-sized, humanitariannet monoclonal the th antibody (produced by cells SNO) against TGF-beta. It is shown as the only means for the treatment of hormone-refractory (androgen-independent) malignant neoplasm of prostate patients. The primary endpoint for evaluating the effectiveness include overall survival compared with best drugs (mitoxantrone/prednisone), when used as one tool, and portability. Secondary endpoints efficiency include the time to disease progression, speed of response, quality of life, pain and/or duration of response. The antibody is administered intravenous (IV) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
The antibody is also shown in combination with chemotherapy for the treatment of hormone-refractory (androgen-independent) prostate tumors in patients. The primary endpoint for evaluating the effectiveness include overall survival compared with chemotherapy and portability. Secondary endpoints efficiency include the time to disease progression, speed of response, quality of life, pain and/or duration of response. The antibody is administered intravenous (IV) used when the on or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
Examples of drugs that can be combined with the humanized anti-TGF-beta antibody for the treatment of prostate tumors (e.g., androgen-independent prostate tumors)include inhibitor farnesyltransferase; antiangiogenic drug (for example, anti-VEGF antibody); a drug with a targeted action against EGFR (e.g., S or ZD1839); anti-HER-2 antibody HERCEPTIN® or anti-ErbB-antibody which induces apoptosis such as 7C2 or 7F3, including humanized variants or variants with Mature affinity; S or humanitariannet antibody S; other anti-TGF-beta antibody (e.g. monoclonal TGF-beta antibody); a cytokine (such as IL-2, IL-12, G-CSF or GM-CSF); antiandrogen, such as flutamide or cyproterone acetate); leuprolide; suramin; chemotherapeutic agent such as vinblastine, estramustine, mitoxantrone, liarozole (means, blocking the metabolism of retinoic acid), cyclophosphamide, anthracycline antibiotics number, such as doxorubicin, taxon (e.g., paclitaxel or docetaxel) or methotrexate, or any combination of the above drugs, as vinblastine/estramustine or cyclophosphamide/doxorubicin/methotrexate; prednisone; hydrocortisone is whether their combination. You can enter the standard dose data for different products, for example, 40 mg/m2/week for docetaxel (TAXOTERETM®); 6 (AUC)carboplatin and 200 mg/m2for paclitaxel (TAXOL®).
Since TGF-β is also involved in the development of prostate tumors (Shah et al., Cancer Research, 62: 7135-7138 (2002)), the antibody can be tested in models of prostate tumors (for example, on the TRAMP transgenic mice and transplanted cells (PC-3) to forecast the effectiveness of the treatment.
Therapy of malignant mammary gland tumors
This antibody is shown as the only means for the treatment of patients with malignant tumor of the breast, in particular, but not limited to patients with metastases. The primary endpoint evaluation of effectiveness include speed of response and tolerability. Secondary endpoints efficacy include overall survival, time to disease progression, quality of life, and/or duration of response. The antibody is administered intravenous (IV) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
This humanizer the bath antibody is also shown in combination with chemotherapy for the treatment of patients with tumor of the breast. The primary endpoint evaluation of effectiveness include overall survival compared with the same chemotherapy and portability. Secondary endpoints efficiency include the time to disease progression, speed of response, quality of life and/or duration of response. Humanitariannet antibody injected (in/in) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
Examples of drugs that can be combined with the humanized anti-TGF-beta antibody for the treatment of breast cancer (e.g., tumors of the breast with metastasis, which is not characterized by overexpression of TGF-beta), include chemotherapeutic agents such as anthracycline antibiotics (eg, doxorubicin), cyclophosphamide, taxon (e.g., paclitaxel or docetaxel), navelbine, xeloda, mitomycin C, compounds of platinum, oxaliplatin, gemcitabine, or combinations of two or more of these drugs, such as doxorubicin/cyclophosphamide; anti-HER-2 antibody HERCEPTIN® or anti-ErbB-antibody which induces apoptosis such as 7C2 or 7F3, including humanized variants of Il the options with Mature affinity; S or humanitariannet antibody S; other anti-TGF-beta antibody (e.g. monoclonal TGF-beta antibody), antiestrogen (e.g., tamoxifen), aromatase inhibitor (e.g., anastrozole); ihibitor farnesyltransferase, antiangiogenic drug (for example, anti-VEGF antibody); a drug with a targeted action against EGFR (e.g., S or ZD1839); a cytokine (such as IL-2, IL-12, G-CSF or GM-CSF), or a combination of the above drugs. You can use the standard dose for such additional products.
The present humanitariannet antibody is shown in combination with anti-HER-2 antibody HERCEPTIN® or rhuMAb 2C4 for the treatment of patients with tumors of the breast, especially with metastasis. The primary endpoint for evaluating the effectiveness include speed of response and tolerability. Secondary endpoints efficiency include the time to disease progression, overall survival compared with some anti-HER-2 antibody HERCEPTIN® or rhuMAb 2C4, quality of life and/or duration of response. RhuMAb 2C4 injected (in/in) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. Antibody rhuMAb 2C4 put in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration). Anti-HR-2-antibody HERCEPTIN® is injected in/with an initial loading dose, equal to 4 mg/kg, with subsequent introduction of a weekly maintenance dose of 2 mg/kg of Anti-HER-2 antibody HERCEPTIN® supplied in the form of liofilizirovannogo powder. Each ampoule anti-HER2 antibody HERCEPTIN® contains 440 mg of the anti-HER2 antibody HERCEPTIN®, to 9.9 mg L-histidine HCl, 6.4 mg L-histidine, 400 mg α, α-trehalose dihydrate and 1.8 mg surfactant POLYSORBATE 20TM. Recovery 20 ml of bacteriostatic water for injection (BWFI), containing 1.1% benzyl alcohol as preservative, gives 21 ml of a solution containing many doses containing 21 mg/ml anti-HER2 antibody HERCEPTIN® with a pH of approximately 6,0.
Using mouse antibody 2G7 in the epithelial cells T of spontaneous mouse mammary tumor cells (1,5x105) was instilled into fat breast mouse (0 day). In this model, palpable primary tumor appear after 1 week; secondary metastases appear in the lungs after 2 weeks in the liver after 3 weeks and in the bones after 4-5 weeks. Tissue was collected in week 5. Antibody 2G7 and two control IgG antibody was administered intraperitoneally to mice at a dose of 25 mg/kg 3x/week and was determined in serum products TGF-beta cells T in harm tissue culture (or blood in the experiments in vivo) using industrially available set for staging ELISA production R&D systems.
On figa shows the production of TGF-beta cells T and the norm is ranked on murine epithelial cells C as control using ELISA in vitro. On FIGU presents the influence of antibody 2G7 on the production of TGF-beta cells T in vivo in mice with tumors (+anti-TGF-beta) compared with mice without tumors (control), treated (control) antibodies (IgG isotype, which is antidilutive antibody) and compared to mice with tumors (+control), treated with control antibodies. These results indicate that epithelial tumor cells T has produced more TGF-beta compared with the control epithelial cells C in vitro (figa) and that the antibody 2G7 reduced the amount of free TGF-beta in the blood compared with mice treated with control antibody (pigv).
The concentrations of free TGF-beta in mice treated with anti-TGF-beta antibody 2G7, coincide with the previously obtained results, showing that anti-TGF-beta antibodies may alter the availability of TGF-beta in vivo (Wojtowicz-Praga et al., Immunother. Emphasis Tumor Immunol., 19(3): 169-75 (1996). Erratum in: J. Immunother. Emphasis Tumor Immunol., 19(5): 386 (1996), Verma UM (corrected Verma UN)). In addition, when used herein, the antibody (2G7) does not prevent the carrying out ELISA, because adding 2G7 in the control plasma did not affect the readings ELISA at a dilution of 1:10, 1:100 and 1:1000 in comparison with the solvent. Ziyadeh et al., Proc. Natl. Acad. Sci. USA, 97: 8015-20 (2000).
On figa presents the results of histol the strategic research points and figv presents the values of the tissue mass for secondary lung tumors obtained on the model of the tumor cells used in Fig, with the control antepartum IgG and anti-TGF-beta 2G7, injected into mice as described for Fig, indicating that anti-TGF-beta antibodies lead to the decrease of the degree, the number of affected shares, tissue mass in g and the weight of the lungs in the form of a percentage of body weight compared with control. Secondary tumors of the lung detected by computer tomography ex vivo.
On Fig presents data on the quantification of lung tumors on the above model in mice using uCT, indicating the volume and number of tumors, despite the fact that anti-TGF-beta 2G7 reduces the volume of the tumors compared with the control IgG, with the introduction of as 2G7 and control mice as described for Fig (i.e. 25 mg/kg 3×/week intraperitoneally).
On figa presents data on the volume of tumors on the above model in mice as a function of time in days after injection of cells using the scheme of 25 mg/kg 3×/week intraperitoneally control IgG with saline solution or with Taxol and 25 mg/kg 3×/week intraperitoneally anti-TGF-beta 2G7 with Taxol, indicating that the latter scheme was the most effective in reducing the volume of the tumors. On FIGU shown that anti-TGF-beta 2G7 in combination with chemotherapy is designed for people who do a lot of tissues - lungs, spleen and tumors compared with control IgG/saline and IgG/chemotherapy.
In addition, the antibody 2G7 reduced blood concentrations of vascular endothelial growth factor (VEGF) compared to the control IgG on this model in mice. On Fig presents the levels of VEGF in plasma (PCG/ml) in mice without tumors (normal) or in mice with breast tumors T treated with control IgG (control) or anti-TGF-beta 2G7 (TGF-beta) (25 mg/kg 3×/week intraperitoneally for control and anti-TGF-beta). Each point represents an individual mouse. The horizontal line indicates the average value for the group.
In conclusion, on the model of mammary gland tumors from epithelial cells derived from a spontaneous mammary tumors in mice Balb/C (T), which was injected into the fat pad of the breast syngeneic mice, it was found that the antibody 2G7 leads to a decrease in the level of free TGF-beta in the blood and the concentration of VEGF in the blood, and they are characterized by small, but reliable temporary ability to reduce primary tumor growth compared with control. Treatment with antibody 2G7 at an early stage reduced secondary tumors of the lung.
In addition, early treatment with antibody 2G7 significantly weakened induced by a tumor of the breast bone destruction, and euwww place on this model, that was installed using the same scan that and for lungs. See table 4. In this table, trabecular number refers to the number of trabeculae (small spicules of bone, which take place in the bone marrow and trabecular thickness refers to the average thickness of these trabeculae. Both these parameters specify the amount of bone and determine the micro-computer tomography and algorithm for the quantitative determination of various bone parameters.
|The definition of regeneration of bones|
|The number of trabeculae||The thickness of trabeculae||Amount of bone volume (BV)/total||The surface of the bones/BV||Mineral density|
|Antibody 2G7 without primary tumor||-2,8%||Not assessed||-4,8%||Not assessed||Not assessed|
|With the primary tumor||-7,2%||-22,5%||-28,3%+28,6%||-15,9%|
|Antibody 2G7 with the primary tumor||+6,5%||+7,2%||+14,3%||-6,6%||+6,3%|
It should be noted that the percentages refer to:
1) compared to normal mice (i.e. without tumors) for samples "2G7 without primary tumor" and "primary tumor";
2) relative to mice with tumors treated with control IgG antibodies to sample 2G7 with the primary tumor."
The antibody 2G7 tested on models of breast cancer (PymT)described Maglione et al., Transgenic Polyoma middle-T mice model premalignant mammary disease, Cancer Res., 61(22): 8298-305 (2001) and Lin et al., Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases, Am. J. Pathol., 163(5): 2113-26 (2003). Both antibody (control IgG (antidilutive antibody) and anti-TGF-beta 2G7) was administered in the same manner: 25 mg/kg 3×/week intraperitoneally. On figa shows the effect of anti-TGF-beta 2G7 compared to control IgG on tumor volume as a function of the growth of tumors in the day for PymT model, indicating that the antibody 2G7 leads to a reduction of tumor volume over time compared to control IgG. On FIGU shown that the tumor weight was also decreased under the action of TGF-beta antibody 2G7 compared with the control IgG. It was also the mouth is attached, the concentration of VEGF was decreased in PymT tumors compared with tumors Her2.
Based on these data we can assume that the humanized variants of the antibody 2G7 will operate similarly 2G7 in terms of their impact on the growth and metastasis of tumors.
In contrast to epithelial cells T epithelial cells Her+ not synthesized TGF-beta in high concentrations in vitro, their growth is inhibited under the action of TGF-beta in vitro (Siegel et al., Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis. Proc. Natl. acad. Sci. USA, 100(14): 8430-5 (2003)), and their growth was not suppressed in the processing of TGF-beta in vivo. In addition, the concentration of VEGF was increased in this model with the introduction of anti-TGF-beta antibody 2G7 compared with the control IgG.
Staining of the three models of breast cancer (T, PymT and Her2) on the basement membrane (collagen IV), endothelial cells (CD31) or support vessels cells called pericyte (SMA or NG2), revealed differences in the data models for these three components. These components are not limited to any theory, can serve to predict the sensitivity of tumors to treatment with anti-TGF-beta antibodies.
The proposed bifunctional nature of the role of TGF-beta in the development of tumors using diagnosticum to determine how/if those who HT will respond, can be used in the application of TGF-beta inhibitory strategy for the treatment of tumors. For example, it may be important to determining whether you will or will not tumor cells in the patient to maintain sensitivity to the growth inhibitory action of TGF-beta.
Not limited to any one theory, presented below is a list of potential diagnostic markers for selection of patients/tumors, the most responsive to treatment with TGF-beta, where this list is not limiting:
1) the expression of one or more of the three isoforms of TGF-beta, TGF-beta, -2 and/or -3, particularly at higher concentrations, with special attention to TGF-beta:
a) this will include a number of different types of tumors, including, but not limited to malignant tumors of the breast, pancreas, prostate, kidney, lung and skin (melanoma). It was found that there is a significant overexpression of TGF-beta in these types of tumors compared with normal tissues of the same type;
b) Her2-negative breast tumor growth in contrast to Her2-positive breast tumors, as the former may respond better to treatment with antibodies, as indicated by higher expression of TGF-beta. In this regard, it was found that in these types of tumors occurred reliable, surgex Russia TGF-beta compared with normal tissues of the same type;
2) confirmation #1 products of tumor cells, irrespective of TGF-beta, also occurs in the stroma/environment;
3) mutation and decrease in the expression of one or more receptors TGF-beta, especially, but not limited to, TGF-beta RI or TGF-RII (type-IIR);
4) mutations or changes in the concentration or localization of molecules in TGF-beta signaling pathway, including, but not limited to: SMAD/SMAD, c-myc, CDC25A, p15INK4B, p21WAFl/CiP1 and p27K1P1;
5) changes in other signaling pathways known that they affect the activity of TGF-beta, including, but not limited to: FoxG1, Jagged/Notch, CDK2 and CDK4, and especially Her2/neu, the concentration of the estrogen receptor, the activity of Ras activity phosphatidylinositol-3-kinase (PI3K), akt and MARK, as well as the status of p53. In addition, the above diagnostic markers for determining which tumors can be treated, you can use multiple markers for assessment (in the tumor and/or on the periphery) of biological activity of anti-TGF-beta antibodies in patients before and after treatment, including, but not limited to:
1) the concentration of TGF-beta in tumor tissue and in the blood (as shown on FIGU, as well as data from immunohistochemistry (IHC), showing the expression of the protein TGF-beta in stained tissue sections of tumor xenografts Colo205 and tumors Calu6, tumors HPAC and human tumor adenocarcinoma duct breast the drive);
2) the concentration of VEGF in the tumor or in the blood (as shown on Fig, as well as data showing that levels of VEGF are increased in Her2-positive model, when provided with the antibody 2G7 compared with control);
3) the concentration of molecules, such as SMAD/SMAD in TGF-beta signaling pathway in the tumor and/or in peripheral cells, such as mononuclear cells (BMC);
4) the indicator function of immune cells, especially the activity of NK, T cells and macrophages.
Therapy of malignant tumors of the lung
The present humanitariannet antibody is shown as the only means for the treatment of stage IIIb or IV non-small cell lung tumors (NSCLC). The primary endpoint evaluation of effectiveness include speed of response and tolerability. Secondary endpoints efficacy include overall survival, time to disease progression, quality of life and/or duration of response. Humanitariannet antibody injected (in/in) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
Humanitariannet antibody is also shown in combination with chemioterapia treatment of patients with non-small cell lung tumor lung metastases. The primary endpoint evaluation of effectiveness include overall survival compared with conventional therapy and tolerability. Secondary endpoints efficiency include the time to disease progression, speed of response, quality of life and/or duration of response. The antibody is administered intravenous (IV) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
Examples of additional drugs that can be combined with this antibody for the treatment of tumors of the lung include chemotherapeutic agents such as carboplatin, taxon (e.g., paclitaxel or docetaxel), gemcitabine, navelbine, cisplatin, oxaliplatin, or a combination of any of these drugs, such as carboplatin/docetaxel; anti-Her-2 antibody HERCEPTIN® or anti-ErbB-antibody which induces apoptosis such as S or 7F3, including humanized antibodies or their variants with Mature affinity; S or humanitariannet antibody S; other anti-TGF-beta-antibodies (e.g. monoclonal TGF-beta antibody); an inhibitor farnesyltransferase; antiangiogenic drug (for example, anti-VEGF antibody); drug steinebrunner action against EGFR (e.g., C225 or ZD1839); a cytokine (such as IL-2, IL-12, G-CSF or GM-CSF), or a combination of the above drugs.
Therapy of malignant tumors of the colon and rectum
The present humanitariannet antibody is shown as the only means for the treatment of malignant tumors of the colon and rectum with metastases. The primary endpoint evaluation of effectiveness include speed of response and tolerability. Secondary endpoints efficacy include overall survival, time to disease progression, quality of life and/or duration of response. Humanitariannet antibody injected (in/in) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
Humanitariannet antibody is also shown in combination with chemotherapy for the treatment of patients with tumors of the colon and rectum with metastases. The primary endpoint evaluation of effectiveness include overall survival compared with conventional therapy and tolerability. Secondary endpoints efficiency include the time to disease progression, speed of response, quality of life and and/or duration of response. Humanitariannet antibody injected (in/in) weekly or every three weeks at a dose of 2 or 4 mg/kg, respectively, until disease progression. The antibody is supplied in the form of a liquid composition containing a lot of doses (20 ml tank with a concentration of 20 mg/ml or higher concentration).
Examples of chemotherapeutic agents used to treat cancer of the colon and rectum, which can be combined with the humanized antibody that binds to TGF-beta, include 5-fluorouracil (5-FU), leucovorin (LV), CPT-11, levamisole, or a combination of any two or more of them, for example, 5-FU/LV/CPT-11. You can enter the standard dose for such chemotherapeutic agents. Other drugs that can be combined with anti-TGF-beta antibody for the treatment of tumors of the colon and rectum include inhibitor farnesyltransferase; antiangiogenic drug (anti-VEGF antibody); a drug with a targeted action against EGFR (e.g., C225 or ZD1839); a cytokine (such as IL-2, IL-12, G-CSF or GM-CSF); anti-HER-2 antibody HERCEPTIN® or anti-ErbB-antibody which induces apoptosis such as S or 7F3, including humanized antibodies or their variants with Mature affinity; S or humanitariannet antibody S; other anti-TGF-beta antibodies (e.g. monoclonal TGF-beta antibody) or a combination of the above PR the preparations.
Taking into account the possible role of TGF-beta in the development of malignant tumors of the colon antibody can be tested in models of tumors of the colon (for example, NT and NST) and it is assumed that they are "working".
Therapy of melanoma
Based on the results of monoclonal antibody 2G7 in this example, we can assume that the humanized antibodies are suitable for the treatment of malignant melanoma. Mouse anti-TGF-beta antibody 2G7 tested on models of melanoma in animals. Specifically, in syngeneic mice SEM, which were subcutaneously grafted cells murine melanoma (B16F10 or VV), treatment with anti-TGF-beta antibody 2G7) scheme 25 mg/kg 3×/week intraperitoneally resulted in reduction of the size of the primary tumor compared with the processing of IgG isotype control (antidilutive antibody) (25 mg/kg 3×/week intraperitoneally) (Fig, 24 and 25). In this model, B16 treatment with anti-TGF-beta antibody 2G7 also reduced the percentage of mice with lung tumors (i.e., the incidence of lung tumors) (figa) and the number of lung tumors (pigv) compared with the control, when using each method of quantitative determination of tumors, i.e. determination of the surface ("surface"), histology ("pathology"), the quantitative determination of all visible tumors after the svetlenija tissues ("bleached") or data micro-computer tomography ("CT"). See also Fig, 26.
It was found that tumor cells Calu-6 (human non-small cell lung carcinoma) (American type culture collection (ATSS), Manassas, VA) produce TGF-beta in vitro. Cells Calu-6 induced the expression of VEGF and SMA in fibroblasts in vitro, where this action is inhibited by treatment of a murine anti-TGF-beta antibody 2G7. Based on these data, we can assume, without being limited to any one theory that TGF-beta may participate in the activation of stromal cells in the environment of the tumor. Conducted xenotransplantation data cells “Nude” mice (see, for example, Gourdeau et al., Mol. Cancer Ther., 3: 1375-1384 (2004)) and determined the volume of tumors after treatment control IgG2b antibody used in the experiment on B16 described above (25 mg/kg 3×/week intraperitoneally) along with the anti-VEGF antibody (A) (5 mg/kg 3×/week intraperitoneally), mouse anti-TGF-beta antibody 2G7 (25 mg/kg 3×/week intraperitoneally) and a combination 2G7 and A (doses presented above). The results of determination of the volume of tumors and masses of tumors presented respectively on Fig and 28, and they indicate that the combination of antibody 2G7 and anti-VEGF antibodies was the most effective treatment, followed by antibody 2G7. The results indicate that the two antibodies (anti-TGF-beta and anti-VEGF) are additive and/or synerg is critical for each other.
Based on these data, it can be assumed that these humanized antibodies will also reduce primary and secondary tumors present in malignant melanoma. Specifically, humanitariannet antibody H2NI.V5L can be tested on two models, which have been shown to be effective mouse antibody 2G7:
1) cells, mouse melanoma (B16) in syngeneic mice and
2) tumor cells Calu-6 (human NSCLC) xenografts in “Nude” mice.
On the model of B16 cells implanted into mice subcutaneously. On both models the processing of anti-TGF-beta antibodies, including H2NI.V5L (25 mg/kg 3×/week) or control antibodies (25 mg/kg 3×/week) start when there is a palpable tumor. The tumor determine 2-3 times a week.
Before testing any of the antibodies assess the ability H2NI.V5L inhibition induced by TGF-beta growth of fibroblasts (NIH3T3).
If the Fc fragment H2NI.V5L is not necessary for the activity of the mice, then we can assume that H2NI.V5L will have the same activity, or higher activity than the original murine antibody 2G7 mice. If the Fc fragment is required for the activity of the mice, then we can assume that H2NI.V5L will not possess the same activity than the original murine antibody 2G7 mice. You can, however, feels the ü, what H2NI.V5L (and other humanized antibodies, as claimed here) will keep efficiency in humans, because the human Fc will be active.
The pharmacokinetics of the antibody 2G7 in normal mice and mice with tumors
The purpose of this study was to determine the pharmacokinetics of parametrov (RK) mouse anti-TGF-beta antibody 2G7 in normal mice and mice with tumors.
The scheme of experiment:
Model animals were mice Balb/C with the induced cells T tumors of the breast. Four groups were administered a single dose of 43 mg/kg:
- group 1: mice without tumors/
- group 2: mice with tumors/
- group 3: mice with tumors in a/b
- group 4: mice with tumors p/
N=3 mice in the group for the time point. Each mouse blood was collected 3 times.
Preparing the serum to determine the TGF-beta using ELISA after 5, 15, 30, 60 min, 3, 6, 24 h; 3, 7, 10, 14, 21 day.
The results show that the rate of elimination of murine anti-TGF-beta antibodies is higher in mice with tumors compared with normal mice. In addition, there is a higher 95% bioavailability of the antibody with the/b and p/C of the introduction. The half-life in mice with tumors was 2-3 days.
Thus, the pharmacokinetic profile of antibody 2G7 is acceptable. In normal mice, the half-life is ntitle amounted to 4 days, which is in the range observed for other antibodies and hybrid proteins in normal mice. In mice with tumors clearance antibodies was approximately 2 times higher, which is not a likely factor in this dose. Bioavailability was higher than 95%, indicating that in b/W and n/K route of administration are appropriate ways for the administration of drugs. The presence of the half-life of 2-3 days, justifies the scheme introduction 2-3 times a week.
1. Humanitariannet antibody that binds to TGF-beta containing the variable domain of the heavy chain (VH), which contains non-human residues hypervariable region GYAFTNYLIE (SEQ ID NO: 21), VINPGSGGSNYNEKFKG (SEQ ID NO: 43) or VNNPGSGGSNYNEKFKG (SEQ ID NO: 22) and SGGFYFDY (SEQ ID NO: 23), included in the human domain of the VH, where the specified variable domain comprises a frame plot (FR), containing the remains of the frame region SEQ ID NO: 6 and contains one or more substitutions in SEQ ID NO: 6 in a position selected from the group consisting of 48, 49, 68, 70, 72, 74 and 79.
2. Humanitariannet antibody according to claim 1, containing replacement FR at positions 49, 68 and 72.
3. Humanitariannet antibody according to claim 2, in which position 49 alanine substituted for glycine at position 68 phenylalanine replaced with alanine and at position 72 arginine is replaced by alanine.
4. Humanitariannet antibody according to claim 1, containing replacement FR in regulations 48, 49 and 72.
5. Humanitariannet antibody according to claim 4, in which position 48 valine is replaced by isoleucine, at position 49 alanine substituted for glycine in position 72 arginine replaced by Alan is N.
6. Humanitariannet antibody according to claim 1, containing replacement FR at positions 49, 70 and 72.
7. Humanitariannet antibody according to claim 6, in which position 49 alanine substituted for glycine at position 70 isoleucine replaced with leucine and at position 72 arginine is replaced by alanine.
8. Humanitariannet antibody according to claim 6, in which additional replacement FR is in position 74.
9. Humanitariannet antibody of claim 8, in which position 74 asparagine is replaced by lysine.
10. Humanitariannet antibody according to claim 1, containing replacement FR at positions 49, 72 and 74.
11. Humanitariannet antibody of claim 10, in which position 49 alanine substituted for glycine at position 72 arginine is replaced with alanine and at position 74 asparagine is replaced by lysine.
12. Humanitariannet antibody according to claim 1, containing replacement FR at positions 49, 72 and 79.
13. Humanitariannet antibody according to item 12, in which position 49 alanine substituted for glycine at position 72 arginine is replaced with alanine and at position 79 leucine is replaced by alanine.
14. Humanitariannet antibody according to any one of claims 1 to 13, containing the remains of determining the complementarity of the plot (CDR) of the variable domain of the light chain (VL) RASQSVLYSSNQKNYLA (SEQ ID NO: 18) or RASQGISSYLA (SEQ ID NO: 7); WASTRES (SEQ ID NO: 19) or YASSLQS (SEQ ID NO: 8) and HQYLSSDT (SEQ ID NO: 20).
15. Humanitariannet antibody according to any one of claims 1 to 13, containing the remains of determining the complementarity of the plot (CDR) vari is belingo domain light chain (V L) RASQSVLYSSNQKNYLA (SEQ ID NO: 18); WASTRES (SEQ ID NO: 19) and HQYLSSDT (SEQ ID NO: 20).
16. Humanitariannet antibody according to any one of claims 1 to 13, containing the amino acid sequence of domain VLin SEQ ID NO: 3.
17. Humanitariannet antibody according to any one of claims 1 to 13, containing the remains of determining the complementarity of the plot (CDR) of the VH domain GYAFTNYLIE (SEQ ID NO: 21); VNNPGSGGSNYNEKFKG (SEQ ID NO: 22) and SGGFYFDY (SEQ ID NO: 23).
18. Humanitariannet antibody according to any one of claims 1 to 13, containing the remains of determining the complementarity of the plot (CDR) of the VH domain GYAFTNYLIE (SEQ ID NO: 21); VINPGSGGSNYNEKFKG (SEQ ID NO: 43) and SGGFYFDY (SEQ ID NO: 23).
19. Humanitariannet antibody according to any one of claims 1 to 3, containing the amino acid sequence of the VH domain, is presented in SEQ ID NO: 4.
20. Humanitariannet antibody according to any one of claims 1 to 13, which represents an intact IgG1 antibody.
21. Humanitariannet antibody according to any one of claims 1 to 13, which is an antibody fragment.
22. Humanitariannet antibody according to item 21, which is a Fab fragment.
23. Humanitariannet antibody according to any one of claims 1 to 13, which is not conjugated with a cytotoxic agent.
24. Humanitariannet antibody according to any one of claims 1 to 23, which is conjugated with a cytotoxic agent.
25. Composition for treating disorders mediated TGF-beta containing humanitariannet antibody according to any one of claims 1 to 24, and the media.
26. Vydeleny what I nucleic acid, coding humanitariannet antibody according to any one of claims 1 to 24.
27. A vector containing a nucleic acid according p.
28. A host cell containing the nucleic acid according p.
29. The method of obtaining gumanitarnogo antibodies, providing for the cultivation of the host cell by p so that the expressed nucleic acid and is produced by an antibody.
30. The method according to clause 29, additionally providing for the selection of antibodies from the culture of the host cell.
31. The method according to item 30, in which the antibody is isolated from the culture medium of the host cell.
32. The method according to any of p-31, in which the cultivation of the cell host cotransfected with a vector containing a nucleic acid encoding a variable domain of the heavy chain, and with a vector containing a nucleic acid encoding a variable domain of the light chain.
33. A method of treating disorders mediated TGF-beta in a mammal, involving the administration to a mammal an effective amount of gumanitarnogo antibody according to any one of claims 1 to 24.
34. The method according to p, in which the mammal is a Primate.
35. The method according to p or 34, in which the mammal is a human.
36. The method according to any of PP and 34 in which the violation is fibrosis, damage to the arteries, infection, rheumatoid arthritis or malignant tumors of the ü.
37. The method according to p in which a malignant tumor is a malignant tumor of the colon, cancer of the colon and rectum, cancer of the rectum, cancer of the lung, cancer of the breast, malignant ovarian tumor or a malignant melanoma.
38. The method according to any of PP-34, additionally providing for the administration to a mammal an effective amount of a therapeutic agent, other than humanitariannet antibody.
39. The method according to § 38, in which therapeutic agent is a chemotherapeutic agent, a cytotoxic drug, a cytokine, a means of inhibiting the growth, angiogenic drug or antibody.
40. The method according to § 39, in which therapeutic agent is an antiangiogenic drug.
41. The method according to § 38, in which therapeutic agent is an antibody.
42. The method according to paragraph 41, in which the antibody binds to vascular endothelial growth factor.
43. The method according to paragraph 41, in which the antibody binds to the antigen Her-2.
44. The method according to any of PP-43, in which the antibody is an intact antibody.
45. The method according to any of PP-43, in which the antibody is an antibody fragment.
46. The method according to item 45, in which the fragment of the antibody, not only is em a Fab fragment.
47. The method according to any of PP-43, in which the antibody is conjugated with a cytotoxic agent.
48. The method according to any of PP-43, in which the antibody is not conjugated with a cytotoxic agent.
49. Method of detecting TGF-beta in a sample from an organism, which involves contacting gumanitarnogo antibody according to any one of claims 1 to 24 with a sample from an organism and determining whether the binding of an antibody to TGF-beta.
50. The product containing container, including humanitariannet antibody according to any one of claims 1 to 24, and instructions for the consumer with an indication for treatment mediated TGF-beta disorders in a mammal antibody in an effective amount.
51. The product according to item 50, optionally containing container, comprising a therapeutic agent other than humanitariannet antibody, where the instructions tell the user how to treat a violation of antibody in combination with a tool in effective amounts.
52. The product according to item 50 or 51, where the mammal is a human.
53. A method of treating a malignant tumor in a mammal, involving the administration to a mammal an effective amount of antibodies against TGF-beta and the antibody according to any one of claims 1 to 24, which binds to vascular endothelial growth factor.
54. The method according to item 53, in which the mammal is a human.
55. the manual on item 53 or 54, in which the antibody against TGF-beta associated with any one or more of the following: TGF-beta, TGF-beta2 and TGF-beta.
56. The method according to any of PP and 54, in which the antibody binds to TGF-beta.
57. The method according to any of PP and 54, in which the antibody binds to TGF-beta and GF-beta2.
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
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
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.
SUBSTANCE: strain 5A10 of hybridomal line of cells of mouse Mus. museums, producing monoclonal antibodies to immunoglobulin IgG of cattle (C) is permanent line of cells and is suitable for biotechnology in elaboration of preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 71. Antibody titers in native culture liquid constitute 1:32-1:64, in ascitic liquid 1:640-1:5120 in immuno-enzymatic analysys. Monoclonal antibodiesproduced by strain are specific to immunoglobulin IgG of cattle and do not react with immunoglobulins of sheep. Peroxydase-marked monoclonal antibodies ensure high sensitivity and specificity of IEA for detection of antibodies to C leucosis virus in biological material. Strain 5A10 - producent of monoclonal antibodies to immunoglobulin IgG of cattle can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis.
EFFECT: application of said test-system will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.
SUBSTANCE: obtained is strain 1H8 of hybridomal line of cells of mouse Mus. musculus - producent of monoclonal antibodies to IgG of sheep, suitable for biotechnology in elaboration of diagnostic preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 73. When determined by method of immuno-enzymatic analysys (IEA) antibodies titers in native culture liquid constituted in IEA 1:32-1:64, in ascitic liquid 1:640-1:5120. Monoclonal antibodies are specific to immunoglobulin IgG of sheep and do not react with immunoglobulin of cattle. When used for fixation on solid phase of glycoproteidal antigen of cattle (C) leucosis virus (in composition of complex glycoproteidal antigen- monoclonal antibodies of sheep to glycoproteidal antigen) in IEA, they ensure strength of fixation and optimal availability of antigen for antibodies in testes samples.
EFFECT: strain 1H8 can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.
1 tbl, 4 ex
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
SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.
EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.
10 cl, 26 dwg, 2 tbl, 22 ex
SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.
EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.
7 cl, 13 dwg, 4 tbl, 7 ex
SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.
EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.
16 cl, 34 dwg, 7 tbl, 20 ex
SUBSTANCE: strain A-4A7 is prepared by fusion of mouse myeloma cells of line SP2/0.Agl4 with mouse lymphocytes of line Balb/c immunised by introduction in pads of a purified preparation AMGF (alpha2-microglobulin of fertility) separated from amniotic fluid, and deposited in the transplantable mammal cell culture collection of the Research Institute of Human morphology of the Russian Academy of Medical Science numbered 131/2002. Strain A-4A7 synthesises monoclonal antibodies (MCA) of IgGI class specifically reacting in solid-phase immune-enzyme analysis (IEA) with AMGF isoforms of endometrial, follicular and sperm nature. Activity of the strain: cultural supernatant contains MCA 3-5 mkg/ml, while ascetic fluid contains MCA 2-5 mg/ml. The antibody titre in cultural fluid is 1:500-1:1000, in ascetic fluid up to 1:1×107. A-4A7 bonds various AMGF protein glycoforms produced in male and female reproductive organs. Application of MCA A-4A7 as an immunodiagnosis test systems allows for high-specific and high-sensitive (1 ng/ml) quantitative analysis of various AMGF/glykodeline isoforms in biological liquids.
EFFECT: new compounds are characterised with valuable biological properties.
SUBSTANCE: genetic makers of siSTRIKE-neo vector producing interfering RNA (siRNA), are inhibitors of reproduction of human immunodeficiency virus type 1. Invention allows producing effective anti-HIV preparations of siRNA produced in cells by the administered genetic makers containing palindrome intended for formation of siRNA production and selected with using non-virus and virus models.
EFFECT: invention can be used in medicine and researches.
12 cl, 3 dwg, 3 tbl, 3 ex
SUBSTANCE: invention concerns biotechnology and represents a new alpha galactosidase, a DNA molecule coding it. Besides the invention concerns an expression vector containing such DNA molecule, and also a cell transformed by the vector. The invention also concerns the method for making alpha-galaktobiose disaccharides with using new alpha galactosidase.
EFFECT: invention allows for high-efficient alpha-galaktobiose disaccharides.
15 cl, 5 dwg, 1 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.
EFFECT: invention allows improving effectiveness of cancer prevention.
28 cl, 7 dwg, 2 tbl, 1 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex
SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.
EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.
20 cl, 62 dwg, 20 ex
SUBSTANCE: invention is related to the field of biotechnology, specifically, to separation and identification of new genes of spiramycins biosynthesis track and to new polypeptides, which participate in this biosynthesis, and may be used to produce acyltransferase, which is responsible for modification of platenolid in position 3. Polynucleotide coding acyltransferase, which is responsible for modification of platenolid in position 3, cells of bacterium Streptomyces type are transformed, and strain-producer of end polypeptide is made.
EFFECT: increased extent of production and purity of produced spiramycin.
26 cl, 41 dwg, 44 tbl, 31 ex
SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.
EFFECT: creation of model cat for research of retroviral infection.
24 cl, 10 dwg, 6 tbl, 8 ex
SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.
EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.
10 cl, 26 dwg, 2 tbl, 22 ex
SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.
EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.
35 cl, 13 dwg, 36 tbl, 14 ex
SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.
EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.
16 cl, 3 tbl, 7 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex