Ox40l antibodies and methods for using

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

 

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority 35 U.S.C. § 119 to provisional application U.S. No. 60/751377, registered on December 16, 2005, the full contents of which are hereby incorporated by reference.

The SCOPE of the INVENTION

The present invention mainly relates to the fields of molecular biology. More specifically, the invention relates to antibodies against OX40L and their applications.

BACKGROUND of the INVENTION

OX40 ligand person (OX40L), membrane-bound representative of the superfamily of tumor necrosis factor predominantly expressed on activated antigen presenting cells such as dendritic cells, and to a lesser extent B-cells. See Sugamura, K et al., Nature Reviews Immunology 4:420 (2004); Weinberg AD, Trends Immunol. 23(2):102 (2002). OX40L, also known as receptor ACT-4, TNF4 human GP34 and CD134L, binds to the OX40 receptor (OX40), which is also known as CD134, ACT-4, the antigen CD134, ACT35 antigen and TNR4 person. OX40 is a protein activated T-cells, which is followed by binding of the T-cell receptor (TCR). The binding of CD40 activated antigen presenting cells increases the expression of OX40L. The binding of OX40 by OX40L is for co-stimulating effector T-cells, and costimulate activates the production of cytokines by T cells-helper 1 and T-helper 2. The interaction of OX40-OX40L t is the train important for the formation of T-cell memory, they contribute to the survival of effector T-cells after premirovany antigen. Blocking OX40L in vivo reduced the severity of rheumatoid arthritis, experimental autoimmune disease (EAE), an acute disease graft-versus-host (GVHD), inflammatory bowel disease, experimental leishmaniasis and collagen-induced arthritis in animal models. See Sugamura, K et al., Nature Reviews Immunology 4:420 (2004); Weinberg AD, Trends Immunol. 23(2):102 (2002).

Anti-OX40L and antibodies that recognize OX40L mentioned, for example, in WO 95/12673; WO 95/21915; WO 99/15200; Baum, P.R., et al., EMBO J. 13 (1994) 3992-4001; Imura, A., et al., Blood 89 (1997) 2951-2958; Imura, A., et al., J. Exp. Med. 183 (1996) 2185-2195; Kjaergaard, J., et al., J. Immunol. 167 (2001) 6669-6677; Lane, P., J. Exp. Med. 191 (2000) 201-206; Mallett, S., and Barclay, A.N., Immunol. Today 12 (1991) 220-223; Mallett, S., et al., EMBO J. 9 (1990) 1063-1068; Ndhlovu, L.C., et al., J. Immunol. 167 (2001) 2991-2999; Oyhshima, Y., et al., J. Immunol. 159 (1997) 3838-3848; Rogers, P.R., et al., Immunity 15 (2001) 445-455; Stüber, E., and Strober, W., J. Exp. Med. 183 (1996) 979-989; Stüber, E., et al., Gastroenterology 115 (1998) 1205-1215; Takahashi, &., et al., J. Virol. 75 (2001) 6748-6757; Takasawa, N., et al., Jpn. J. Cancer Res. 92 (2001) 377-382; Taylor, L., and Schwarz, H., J. Immunol. Meth. 255 (2001) 67-72; Weinberg, A.D., et al., Nature Medicine 2 (1996) 183-189; Weinberg, A.D., et al., Semin. Immunol. 10 (1998) 471-480; Weinberg, A.D., Trends Immunol. 23 (202) 102-109; Wu, T., et al., Transplant. Proc. 33 (2001) 217-218; Higgins, L.M., et al., J.Immunol. 162 (1999) 486-493; Yoshioka, T., et al., Eur. J. Immunol. 30 (2000) 2815-2823; and Wang, Q., et al. Tissue Antigens 64:566-574 (2004). Mouse antibodies to human OX40L commercially available from MBL International Corp. (TAG-34) and R and D Corp (clones 159403 and 159408).

Clearly, Thu is still a need for tools who have clinical characteristics that are optimal for the development of these devices as therapeutic agents. The described invention meets this need and provides additional advantages.

All cited here references, including patent applications and publications cited as references in full.

The INVENTION

The invention is based in part on the identification of many compounds bind to OX40L (such as antibodies and fragments thereof). OX40L is an important and preferred target for therapy, and the invention relates to compositions and methods on the basis of binding OX40L. The substances according to the invention bind to OX40L, as described herein, are important therapeutic and diagnostic tools for use when exposed to pathological States associated with expression and/or activity of the ways involving OX40L-OX40 receptor. Thus, the invention relates to methods, compositions, kits and articles related to the binding OX40L.

In one aspect the invention relates to the selected antibody to OX40L, where a full-size IgG form of the antibody specifically binds to OX40L man with a binding affinity of about 10 nm or better. As is common in this area, the binding affinity of the ligand with the its receptor can be determined using any of a variety of tests and expressed in terms of sets of quantitative values. Thus, in one embodiment, the implementation of the affinity of binding is expressed as Kd values and reflects its own binding affinity of (for example, with minimized effects of avidity). Typically and preferably, the binding affinity of measure in vitro or in a cell-free, or associated with cells of the system. Any number of assays known in this field, including the assays described herein can be used to obtain measurements of the affinity of binding, including, for example, Biacore, radioimmune assay (RIA) and ELISA.

In one aspect the invention relates to the selected antibody, which binds to the region OX40L binding to OX40 receptor. In some embodiments, implementation of the selected antibody binds to the polypeptide containing the extracellular domain of human OX40L or consisting of him.

In one aspect the invention relates to the selected antibody to OX40L, which competes with the OX40 receptor for binding to OX40L.

In one aspect the invention relates to the selected antibody to OX40L, which inhibits, reduces and/or blocks the activity of OX40L.

In one aspect the invention relates to the antibody to OX40L, which inhibits, reduces and/or blocks the proliferation of T-cells.

In one aspect the invention relates to the antibody to OX40L, that inhibits, reduces, and/is whether blocking the survival of activated T-cells.

In one aspect the invention relates to the antibody to OX40L, which inhibits, reduces and/or blocks the secretion of IL-2 T-cell memory.

In one aspect the invention relates to the antibody to OX40L, containing at least one, two, three, four, five and/or six sequences of the hypervariable region (HVR)selected from the group consisting of: RSSQSIVHGNGNTYLE (SEQ ID NO:1), RSSQSPVHSNGNTYLH (SEQ ID NO:2), RVSNRFS (SEQ ID NO:3), KVSNRFS (SEQ ID NO:4), FQGSHVPYT (SEQ ID NO:5), SQSTHIPWT (SEQ ID NO 6), SYWLN (SEQ ID NO:7), SYWMH (SEQ ID NO:8), MIDPSDSETHYNQVFKD (SEQ ID NO:9), EIDPSNGRTNYNEKFKS (SEQ ID NO:10), GRGNFYGGSHAMEY (SEQ ID NO:11) and ERSPRYFDV (SEQ ID NO:12).

In one aspect the invention relates to the antibody to OX40L, containing at least one, two, three, four, five and/or six sequences of the hypervariable region (HVR)selected from the group consisting of: (a) HVR-L1 containing the sequence RSSQSIVHGNGNTYLE (SEQ ID NO:1) or RSSQSPVHSNGNTYLH (SEQ ID NO:2); (b) HVR-L2, containing the sequence RVSNRFS (SEQ ID NO:3) or KVSNRFS (SEQ ID NO:4); (c) HVR-L3, containing the sequence FQGSHVPYT (SEQ ID NO:5) or SQSTHIPWT (SEQ ID NO:6); (d) HVR-H1 containing the sequence SYWLN (SEQ ID NO:7) or SYWMH (SEQ ID NO:8); (e) HVR-H2 containing the sequence MIDPSDSETHYNQVFKD (SEQ ID NO:9) or EIDPSNGRTNYNEKFKS (SEQ ID NO:10); and (f) HVR-H3 containing the sequence GRGNFYGGSHAMEY (SEQ ID NO:11) or ERSPRYFDV (SEQ ID NO:12).

In one of the embodiments the antibody according to the invention contains a variable domain light chain having posledovatel the ability:

and contains a variable domain of a heavy chain having the sequence:

In one aspect the invention relates to the antibody to OX40L, containing at least one, two, three, four, five and/or six sequences of the hypervariable region (HVR)selected from the group consisting of: (a) HVR-L1 containing the sequence RSSQSIVHGNGNTYLE (SEQ ID NO:1)or HVR-L1 containing the sequence RSSQSPVHSNGNTYLH (SEQ ID NO:2); (b) HVR-L2, containing the sequence RVSNRFS (SEQ ID NO:3)or HVR-L2 containing a sequence KVSNRFS (SEQ ID NO:4); (c) HVR-L3, containing the sequence FQGSHVPYT (SEQ ID NO:5), or HVR-L3, containing the sequence SQSTHIPWT (SEQ ID NO:6); (d) HVR-H1 containing the sequence SYWLN (SEQ ID NO:7), or HVR-H1 containing the sequence SYWMH (SEQ ID NO:8); (e) HVR-H2 containing the sequence MIDPSDSETHYNQVFKD (SEQ ID NO:9), or HVR-H2 containing the sequence EIDPSNGRTNYNEKFKS (SEQ ID NO:10); and (f) HVR-H3 containing the sequence GRGNFYGGSHAMEY (SEQ ID NO:11), or HVR-H3 containing the sequence ERSPRYFDV (SEQ ID NO:12).

In one aspect the invention relates to the antibody to OX40L, containing at least one, two, three, four, five and/or six sequences of the hypervariable region (HVR)selected from the group consisting of: (a) HVR-L1 containing the sequence RSSQSIVHGNGNTYLE (SEQ ID NO1); (b) HVR-L2, containing the sequence RVSNRFS (SEQ ID NO:3); (c) HVR-L3, containing the sequence FQGSHVPYT (SEQ ID NO:5); (d) HVR-H1 containing the sequence SYWLN (SEQ ID NO:7); (e) HVR-H2 containing the sequence MIDPSDSETHYNQVFKD (SEQ ID NO:9); and (f) HVR-H3 containing the sequence GRGNFYGGSHAMEY (SEQ ID NO:11).

In one of the embodiments the antibody according to the invention contains a light chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of RSSQSIVHGNGNTYLE (SEQ ID NO:1), RVSNRFS (SEQ ID NO:3) and FQGSHVPYT (SEQ ID NO:5). In one of the embodiments of the antibody containing light chain HVR-L1 having the amino acid sequence RSSQSIVHGNGNTYLE (SEQ ID NO:1). In one of the embodiments, the antibody includes a light chain HVR-L2 having the amino acid sequence RVSNRFS (SEQ ID NO:3). In one of the embodiments, the antibody includes a light chain HVR-L3 having the amino acid sequence FQGSHVPYT (SEQ ID NO:5). In one of the embodiments the antibody according to the invention contains a heavy chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of SYWLN (SEQ ID NO:7), MIDPSDSETHYNQVFKD (SEQ ID NO:9) and GRGNFYGGSHAMEY (SEQ ID NO:11). In one of the embodiments, the antibody contains a heavy chain HVR-H1 having the amino acid sequence SYWLN (SEQ ID NO:7). One is from embodiments the antibody contains a heavy chain HVR-H2, having the amino acid sequence MIDPSDSETHYNQVFKD (SEQ ID NO:9). In one of the embodiments, the antibody contains a heavy chain HVR-H3 having the amino acid sequence GRGNFYGGSHAMEY (SEQ ID NO:11). In one of the embodiments the antibody according to the invention contains a heavy chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of SYWLN (SEQ ID NO:7), MIDPSDSETHYNQVFKD (SEQ ID NO:9) and GRGNFYGGSHAMEY (SEQ ID NO:11), and light chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of RSSQSIVHGNGNTYLE (SEQ ID NO:1), RVSNRFS (SEQ ID NO:3) and FQGSHVPYT (SEQ ID NO:5).

In one of the embodiments the antibody according to the invention contains a variable domain light chain having the sequence:

In one of the embodiments the antibody according to the invention contains a variable domain of a heavy chain having the sequence:

In one of the embodiments the antibody according to the invention contains a variable domain light chain having the sequence:

and contains a variable domain of a heavy chain having the sequence:

In one aspect of the invention regarding the seeking for the antibody to OX40L, containing at least one, two, three, four, five and/or six sequences of the hypervariable region (HVR)selected from the group consisting of: (a) HVR-L1 containing the sequence RSSQSPVHSNGNTYLH (SEQ ID NO:2); (b) HVR-L2, which contains a sequence of KVSNRFS (SEQ ID NO:4); (c) HVR-L3, containing the sequence SQSTHIPWT (SEQ ID NO:6); (d) HVR-H1 containing the sequence SYWMH (SEQ ID NO:8); (e) HVR-H2 containing the sequence EIDPSNGRTNYNEKFKS (SEQ ID NO:10); and (f) HVR-H3 containing the sequence ERSPRYFDV (SEQ ID NO:12).

In one of the embodiments the antibody according to the invention contains a light chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of RSSQSPVHSNGNTYLH (SEQ ID NO:2), KVSNRFS (SEQ ID NO:4) and SQSTHIPWT (SEQ ID NO:6). In one of the embodiments, the antibody includes a light chain HVR-L1 having the amino acid sequence RSSQSPVHSNGNTYLH (SEQ ID NO:2). In one of the embodiments, the antibody includes a light chain HVR-L2, having the amino acid sequence of KVSNRFS (SEQ ID NO:4). In one of the embodiments, the antibody includes a light chain HVR-L3 having the amino acid sequence SQSTHIPWT (SEQ ID NO:6). In one of the embodiments the antibody according to the invention contains a heavy chain containing at least one, at least two or all three of HVR sequence selected and the group, consisting of SYWMH (SEQ ID NO:8), EIDPSNGRTNYNEKFKS (SEQ ID NO:10) and ERSPRYFDV (SEQ ID NO:12). In one of the embodiments, the antibody contains a heavy chain HVR-H1 having the amino acid sequence SYWMH (SEQ ID NO:8). In one of the embodiments, the antibody contains a heavy chain HVR-H2 having the amino acid sequence EIDPSNGRTNYNEKFKS (SEQ ID NO:10). In one of the embodiments, the antibody contains a heavy chain HVR-H3 having the amino acid sequence ERSPRYFDV (SEQ ID NO:12). In one of the embodiments the antibody according to the invention contains a heavy chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of SYWMH (SEQ ID NO:8), EIDPSNGRTNYNEKFKS (SEQ ID NO:10) and ERSPRYFDV (SEQ ID NO:12), and light chain containing at least one, at least two or all three of HVR sequence selected from the group consisting of RSSQSPVHSNGNTYLH (SEQ ID NO:2), KVSNRFS (SEQ ID NO:4) and SQSTHIPWT (SEQ ID NO:6).

In one of the embodiments the antibody according to the invention contains a variable domain light chain having the sequence:

In one of the embodiments the antibody according to the invention contains a variable domain of a heavy chain having the sequence:

In one of the embodiments the antibody according to the invention contain the it variable domain light chain, having the sequence:

and contains a variable domain of a heavy chain having the sequence:

As is well known in this area and as described in more detail below, the position of the amino acids/defining the boundaries of the hypervariable region of the antibody may vary depending on the context, and in this area known different definitions (as described below). Some provisions within the variable domain can be viewed as a hybrid hypervariable position in the sense that these provisions can be considered to be located within the hypervariable region, based on one set of criteria, while they are considered outside the hypervariable region on the basis of a different set of criteria. One or more of these provisions can also be found in the extended hypervariable regions (as further defined below).

In some embodiments, the implementation of the antibody is a monoclonal antibody. In some embodiments, the implementation of the antibody is a polyclonal antibody. In some embodiments, the implementation of an antibody selected from the group consisting of chimeric antibodies, affinity-Mature antibodies, gumanitarnogo antibodies and human antibodies is and. In some embodiments, the implementation of the antibody is an antibody fragment. In some embodiments, the implementation of the antibody is a Fab, Fab', Fab'-SH, F(ab')2or scFv.

In one of the embodiments the antibody is a chimeric antibody, such as antibody containing antigennegative sequences from non-human donor grafted on heterologous not owned by the person, human or humanitarian sequence (for example, the sequence of frame and/or constant domain). In one embodiment, the implementation of the non-human donor is a mouse. In one of the embodiments antigennegative sequence is synthetic, for example, obtained by mutagenesis (e.g., by screening using the phage display technique, and so on). In one embodiment, the implementation of the chimeric antibody according to the invention has the murine V-regions and human C region. In one embodiment, the implementation of the murine V-region light chain fused to human light chain Kappa. In one embodiment, the implementation of the murine V-region heavy chain fused with a human C region of IgG1.

Humanized antibodies of the invention include antibodies that have amino acid substitutions in FR and variants affinity maturation with substitutions in the CDR grafted. Replacement of the n amino acids in the CDR or FR are not limited to amino acids, present in the antibodies of the donor or recipient. In other embodiments, implementation of the antibodies according to the invention additionally contain substitutions of amino acid residues in the Fc-region, resulting in improved effector functions, including strengthening the functions of the CDC and/or ADCC, and the killing of B-cells. Other antibodies of the invention include antibodies having specific substitutions that improve stability. In other embodiments, implementation of the antibodies according to the invention contain substitutions of amino acid residues in the Fc region, thereby weakening the effector functions, such as the weakening of the functions of the CDC and/or ADCC and/or weaken the killing of B-cells. In some embodiments, the implementation of the antibodies according to the invention are characterized by a decrease in binding (such as the absence of binding factor) of the human complement C1q and/or Fc-receptor of human natural killer (NK) cells. In some embodiments, the implementation of the antibodies according to the invention are characterized by a decrease in binding (such as the absence of binding to FcγRI, FcγRIIA and/or FcγRIIIA person. In some embodiments, the implementation of the antibodies according to the invention belong to the IgG class (e.g., IgG1 or IgG4) and contain at least one mutation in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and/or P329 (numbering according to EU index). In some embodiments, the implementation of the antibodies with the may contain the mutations L234A/L235A or D265A/N297A.

In one aspect the invention relates to polypeptides anti-OX40L containing any of the presented here antigenspecific sequences, where the polypeptides anti-OX40L specifically bind to OX40L.

In one aspect the invention relates to immunoconjugates (interchangeably referred to as "conjugate antibodies with drug" or "ADC")that contains any of these antibodies against OX40L, conjugated with a substance such as a drug.

Antibodies of the invention bind (in some embodiments, the implementation of specifically associated with OX40L and, in some embodiments, the exercise may modulate one or more aspects of the effects associated with OX40L, including as non-limiting examples of activation OX40L, molecular signal transmission in the direction from OX40L, activation of OX40 receptor, molecular signal transmission in the direction from the receptor OX40, impaired binding of OX40 receptor with OX40L, multimerization OX40L and/or disruption of any biologically important biological involving OX40L and/or OX40 receptor, and/or treatment and/or prevention of immune disorders (such as asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, a disease graft-versus-host (GVHD) and/or system red is alcance); and/or the treatment or prevention of disorders associated with expression and/or activity of OX40L (such as increased expression and/or activity of OX40L). In some embodiments, the implementation of the antibody of the invention specifically binds to OX40L. In some embodiments, the implementation of the antibody specifically binds to a region of binding OX40L receptor OX40. In some embodiments, implementation of the selected antibody binds to the polypeptide containing the extracellular domain of human OX40L, consisting of, or consisting of his being. In some embodiments, the implementation of the antibody specifically binds to OX40L with a Kd of approximately 1 nm or stronger. In some embodiments, the implementation of the antibody specifically binds to OX40L with a Kd of about 10 nm or stronger. In some embodiments, the implementation of the antibody according to the invention reduces, inhibits and/or blocks the activity of OX40L in vivo and/or in vitro. In some embodiments, the implementation of the antibody competes for binding to OX40L with the OX40 receptor (reduces and/or blocks binding of OX40 receptor with OX40L).

In one aspect the invention relates to the use of the antibodies according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders, such as immune disorder. In some embodiments, the implementation of rastra the STV is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus. In some embodiments, the implementation of the disorder is a disease caused by a virus, bacteria or other infectious agent, such as caused by the virus immunopathology, such as pathology caused by infection with influenza virus or RSV, or related viruses, for example, in the lung. See mainly US2005/0069548 A1. In some embodiments, the implementation of the disorder is an arthritic (acute and chronic rheumatoid arthritis, including juvenile rheumatoid arthritis and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, collagen-induced arthritis type II, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, of still's disease, vertebral arthritis, osteoarthritis, chronic progredient arthritis, arthritis deformans; primary chronic arthritis, reactive arthritis, menopausal arthritis, arthritis due to a decrease in production of estrogen and ankylosing spondylitis/rheumatoid spondylitis), autoimmune if theproliferation disease inflammatory hyperproliferative skin diseases, psoriasis such as blaskovic psoriasis, guttate psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and syndrome Jobe, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, rash, dermatitis herpetiformis, coins dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis and atopic dermatitis, x-linked Hyper-IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS), such as sinapticeski MS, primary progressive MS (PPMS), and relapsing remitirse MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis, atoxicity sclerosis, optic neuromyelitis (NMO), inflammatory bowel disease (IBD) (for example, bol is June Crown, mediated by the immune system diseases of the gastrointestinal tract, inflammation of the gastrointestinal tract, colitis such as ulcerative colitis,colitis ulcerosamicroscopic colitis, collagenosis colitis, polypous colitis, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), inflammation of the intestine, gangrenous pyoderma, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including respiratory distress syndrome, adult or acute (ARDS), meningitis, inflammation of all or part of the choroid of the eye, inflammation, iris, chorioidea, an autoimmune hematological disorder, disease, graft versus host, angioedema, such as hereditary angioedema, damage to the cranial nerve as in meningitis, herpes pregnant, pemphigoid pregnant, scrotal itching, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis and limbic encephalitis, and/or encephalitis of the brain stem, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, degranulated the EIT uveitis, valanchery uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with nephrotic syndrome and without it, such as acute or chronic glomerulonephritis such as primary GN, mediated by the immune system GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranosa-proliferative GN (MPGN), including type I and type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis, including limited ploskokletochnyi balanitis, balanoposthitis, annular erythema, persistent dichromates erythema, erythema multiforme, granuloma annulare, brilliant zoster, sclerotic and atrophic lichen, simple chronic zoster, spinelessly vitiligo, lichen planus, lamellar ichthyosis, epidermolizei hyperkeratosis, premalignant keratosis, gangrenous pyoderma, allergic conditions and responses, food Allergy, drug Allergy, Allergy to insect bites, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, disgestrotical eczema, and vesicular palmoplantar eczema, asthma such asasthma bronchiale, bronchial asthma and auto munna asthma, allergic asthma and children asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal blood group A-B-O in the process of pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, impaired leukocyte adhesion, lupus, including lupus nephritis, lupus cerebri, baby erythematosus, vnepochechny erythematosus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, lupus alopecia, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated red lupus, juvenile diabetes mellitus (type I), including child IDDM, diabetes mellitus in adults (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic large arteries, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, Wegener, including lymphomatoid Wegener, Wegener's granulomatosis, agranulocytosis, vasculitis, including vasculitis, vasculitis of large vessels, including rheumatic rheumatica and giant cell arteritis diagnostics ('s arteritis)), vascular lesions is lit vessels of medium caliber (including Kawasaki disease and polyarteritis polyarteritis/periarteritis nodosa), microscopic polyarteritis, immunovaccine, vasculitis in the Central nervous system, cutaneous vasculitis, vasculitis in patients with hypersensitivity, necrotizing vasculitis such as systemic necrotizing vasculitis associated with ANCA vasculitis, such as vasculitis or syndrome Cerca-Strauss (CSS) and associated with ANCA vasculitis vessels of small caliber, temporal arteritis diagnostics, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, anemia of Diamond-Blackfan, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison disease, true red cell anemia or aplasia (PRCA), a deficiency of factor VIII, hemophilia A, autoimmune(s) neutropenia(s), cytopenia, such as pancytopenia, leukopenia, diseases involving diabetes leukocytes, inflammatory disorders in the Central nervous system, Alzheimer's disease, Parkinson's disease, a syndrome of multiple organ failure, such as secondary syndromes septicemia, trauma or hemorrhage, disease, mediated by complexes of antigen-antibody, a disease associated with antibodies against the glomerular basement membrane, antiphospholipid syndrome, motor neuritis, allergic neuritis, disease/syndrome behceta, syndrome Castellana syndrome?, Raynaud's syndrome, with ngram Sjogren, the syndrome of Stevens-Johnson, pemphigoid, such as bullous pemphigoid and skin pemphigoid, pemphigus (including ordinary pemphigus, foliate pemphigus, pemphigoid mucosa and erythematous pemphigoid), autoimmune polyendocrinopathy, illness or Reiter syndrome, thermal injury due to an autoimmune condition, preeclampsia, a disorder caused by immune complexes, such as immune complex nephritis, mediated by antibodies jade, neirolepticalkie disorder, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenia (e.g., such as occurs in patients with myocardial infarction), including the number of thrombotic thrombocytopenic purple (TTP), post-transfusion purple (PTP), heparin-induced thrombocytopenia, and autoimmune or immunopositive thrombocytopenia, including, for example, idiopathic thrombocytopenic purple (ITP)including chronic or acute ITP, scleritis such as idiopathic keratoscleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic tireo is the CIO (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, a disease of graves, polyglandular syndromes such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as myasthenic syndrome Lambert-Eaton or the syndrome of Eaton-Lambert syndrome, stiff person" or "stiff subject, encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergicaand experimental allergic encephalomyelitis (EAE), myasthenia gravis, such as associated with thymoma myasthenia gravis, cerebellar degeneration, neuromyotonia, needs to be or needs to be-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), obliterative bronchiolitis (regardless of transplant) vs. NSIP, Guillain-Barre syndrome, a disease Berger (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, is otrokovicky pustular dermatosis, transient contritione dermatitis, cirrhosis, such as primary biliary cirrhosis and pneumocytes syndrome autoimmune enteropathy, celiac or coeliac disease, sprue-celiac disease (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, such as mixed cryoglobulinemia, amyotrophic lateral sclerosis (ALS; disease Lu Goering), disease coronary artery disease, autoimmune ear disease such as autoimmune disease of the inner ear (AIED), autoimmune hearing loss, polyhedra, such as refractory or recurrent or relapsing polyhedric, pulmonary alveolar proteins syndrome Kogan /recipricocity interstitial keratitis bell's palsy, a disease/sweet syndrome, autoimmune the eyes, pain associated with shingles, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and the monoclonal gammapathy unclear significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, kalapati, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, paroxysmal paralysis, and kalapati CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomer lockers (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune Hepatology disorder, fibromyalgia, multiple endocrine failure, syndrome Schmidt, inflammation of the adrenal gland, atrophy of the stomach, presenilny dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinizing polyneuropathy, Dressler syndrome, focal alopecia, CREST syndrome (calcinosis, Raynaud's syndrome, dysmotility of the esophagus, sclerodactyly and telangiectasia), male and female autoimmune infertility, e.g., due to the presence of antibodies to sperm, mixed disease of connective tissue, Chagas disease, rheumatic fever, habitual miscarriage, alveolitis among agricultural workers, polymorphic erythema, postcardiotomy syndrome, Cushing's syndrome, pulmonary Allergy birders, allergic granulomatous of anyit, benign lymphocytic anghit syndrome Alport, alveolitis such as allergic and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, cyanosis, schistosomiasis, ascariasis, aspergillosis, syndrome Sumpter syndrome Kaplan, dengue, endocarditis, endomyocardial fibrosis, trim the significant interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, fibrosing mediastinal, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, elevated persistent erythema, hemolytic disease of the newborn, eosinophilic fasciitis, syndrome, Shulman syndrome, still's, filarias, cycle, such as chronic cycle, heterochronies cycle, iridocyclitis (acute or chronic) or cycle Fuchs, purple's disease-Seleina, infection with the human immunodeficiency virus (HIV), SCID, acquired immune deficiency syndrome (AIDS), infection by echoviruses, sepsis (system inflammatory response syndrome (SIRS)), groove toxins, pancreatitis, hyperthyroidism, infection with parvovirus, a virus infection rubella, post-vaccination syndromes, congenital infection with rubella, the virus infection Epstein-Barr, infectious parotitis, Evans syndrome, autoimmune gonadal failure, horey of Sydenham, poststreptococcal jade, thromboangiitis obliterans, thyrotoxicosis, the " dryness " of the spinal cord, chorionic, the giant cell rheumatica, chronic pneumonitis with hypersensitivity, conjunctivitis, such as spring Qatar, keratoconjunctivitis sicca and epidemic keratoconjunctivitis, idiopathic nephritic syndrome, nephropathy with minimal changes, benign family damage and damage during ischemia-rap is hushi, the reperfusion of organs in transplantation, autoimmune reaction in the retina, joint inflammation, bronchitis, chronic obstructive airway disease/lung, silicosis, atty, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular disease), such as arteriosclerotic encephalopathy and arteriosclerotic retinopathy, aspermatogenesis, autoimmune hemolysis, Beck disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylaxis endophthalmitis, allergic enteritis, knotty leprosy erythema, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, a disease of Hammana-rich, sensorineural hearing loss, the paroxysmal hemoglobinuria, hypogonadism, regional REIT, leukopenia, infectious mononucleosis, transverse myelitis, primary the idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous pyoderma, quervain's thyroiditis, acquired atrophy of the spleen, the non-malignant thymoma, lymphovascular limit, vitiligo, toxic shock syndrome, food poisoning, conditions involving infiltration of T-cells, the defect of leukocyte adhesion, immune responses associated with acute and delayed hypersensitivity mediated what takinami and T-lymphocytes, diseases involving diabetes leukocyte syndrome of multiple organ lesions mediated by complexes of antigen-antibody diseases, directed against the basement membrane of the glomeruli, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed disease of connective tissue, nephrotic syndrome, insult, polyendocrine failure, autoimmune polyglandular syndromes, including polyglandular syndrome type I, idiopathic adult hypoparathyroidism (AOIH), cardiomyopathy such as cardiomyopathy with dilation acquired bullous bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or negniy sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal bone, maxilla, or sphenoid bone, allergic sinusitis associated with eosinophils disorder such as eosinophilia, eosinophilia with infiltration into the lungs, symptoms of eosinophilia-myalgia syndrome Leffler, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma or containing granuloma eosinophils, anaphylaxis, spondyloarthropathies, seronegative, spondyloarthr is t, polyendocrine autoimmune disease, sclerosing cholangitis, chronic mucocutaneous candidiasis sclera and episclera syndrome Bruton, the transient gammaglobulinemia newborn syndrome Wiskott-Aldrich syndrome ataxia-telangiectasia, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, such as chronic arthroleptis, lymphadenitis, reduction of blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic disorders associated with hypersensitivity, glomerulonephritis, reperfusion injury, ischemic reperfusion disorder, reperfusion injury of the myocardium and other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other inflammatory disorders of the Central nervous system, ocular and orbital inflammatory disorders, syndromes, associated with the transfusion of granulocytes induced by cytokines toxicity, narcolepsy, acute severe inflammation, chronicalsometimes inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvula and endometriosis. Other examples, which in some cases cover the examples listed above, include as non-limiting examples of autoimmune rheumatologic disorders (such as rheumatoid arthritis, Sjogren syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid syndrome, and psoriatic arthritis), autoimmune gastrointestinal disorders and liver disorders (such as inflammatory bowel disease (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis and coeliac disease), vasculitis (such as, for example, associated with ANCA vasculitis, including vasculitis Cerca-Strauss, Wegener's granulomatosis and polyarteritis), autoimmune neurological disorders (such as multiple sclerosis, needs to be myoclonus syndrome, myasthenia gravis, optic neuromyelitis, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), renal disorders (such as, for example, glomerulonephritis syndrome? and illness Berger), autoimmune dermatologic disorders(such as, for example, psoriasis, urticaria, rash, common pemphigus, bullous pemphigoid and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune diseases of the ear (such as, for example, a disease of the inner ear and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplantation, GVHD and autoimmune endocrine disorders (such as, for example, diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison disease and autoimmune thyroid disease (e.g., graves ' disease and thyroiditis)).

In one aspect the invention relates to compositions containing one or more antibodies according to the invention and a carrier. In one of the embodiments the carrier is pharmaceutically acceptable.

In one aspect the invention relates to nucleic acids encoding antibodies against OX40L according to the invention.

In one aspect the invention relates to vectors containing the nucleic acid according to the invention.

In one aspect the invention relates to compositions containing one or more nucleic acids according to the invention and is of the eh. In one of the embodiments the carrier is pharmaceutically acceptable.

In one aspect the invention relates to a cell host containing a nucleic acid or vector according to the invention. The vector may refer to any type, such as a recombinant vector, such as expressing vector. You can use any of many host cells. In one of the embodiments a host cell is a prokaryotic cell, for example,E. coli. In one of the embodiments a host cell is a eukaryotic cell, for example cell of a mammal, such as a cell Chinese hamster ovary (CHO).

In one aspect the invention relates to methods of producing antibodies according to the invention. For example, the invention relates to methods of producing antibodies against OX40L (which, as defined herein, includes a full-sized antibody and its fragments), where the method includes the expression in a suitable cell host recombinant vector according to the invention, encoding the indicated antibody, and the allocation of the indicated antibodies.

In one aspect the invention relates to a product containing container, and a composition contained within the container, where the composition comprises one or more antibodies against OX40L according to the invention. In one embodiment, the implementation of the comp the position contains a nucleic acid according to the invention. In one of the embodiments a composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one of the embodiments of the product according to the invention further comprises instructions for introduction of the composition (e.g., antibody) to a subject (such as instructions for any of the methods described here).

In one aspect the invention relates to a kit containing a first container containing a composition comprising one or more antibodies against OX40L according to the invention and a second container containing a buffer. In one of the embodiments the buffer is pharmaceutically acceptable. In one of the embodiments a composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one of the embodiments set further comprises instructions for administration of the composition (e.g., antibody) to a subject.

In one aspect the invention relates to the use of antibodies against OX40L according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders, such as immune disorder. In some embodiments, the implementation of the disorder is autoimmune the second disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus.

In one aspect the invention relates to the use of nucleic acids according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders, such as immune disorder. In some embodiments, the implementation of the disorder is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus.

In one aspect the invention relates to the use of the expressing vector according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders, such as immune disorder. In some embodiments, the implementation of the disorder is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemno is lupus erythematosus.

In one aspect the invention relates to the use of host cell according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders, such as immune disorder. In some embodiments, the implementation of the disorder is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus.

In one aspect the invention relates to the use of the product according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders, such as immune disorder. In some embodiments, the implementation of the disorder is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus.

In one aspect the invention relates to the use of the kit according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of disorders such as immune disorders is. In some embodiments, the implementation of the disorder is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus.

The invention relates to methods and compositions suitable for modulating disease States associated with expression and/or activity of OX40L, such as increased expression and/or activity or undesired expression and/or activity.

The methods according to the invention can be applied to influence any suitable pathological condition. Typical disorders described in this specification and include immune disorders.

The methods according to the invention can optionally include additional stages of treatment. In one aspect the invention relates to a method comprising introducing an effective amount of antibodies against OX40L in combination with an effective amount of another therapeutic agent. For example, in one embodiments, the method further includes a step in which the targeted cell and/or tissue is subjected to treatment with steroids. Antibody to OX40L can be entered periodically or in combination with another therapeutic agent, which is a Ki is effective for those purposes, or in the same composition or in separate compositions. Introduction antibodies against OX40L and another therapeutic agent can be performed simultaneously, for example, in the form of a single composition or as two or more separate compositions using the same or different routes of administration. Alternative or additionally, the introduction can be performed sequentially, in any order. Alternative or additional stages can be implemented as a combination of both sequential and simultaneous, in any order. In specific embodiments, the implementation between the introduction of two or more compositions may be present intervals ranging from minutes to days, to weeks, to months. For example, the other therapeutic agent can be entered at the beginning, before antibodies against OX40L. However, it also assumes simultaneous introduction or the introduction of antibodies against OX40L in the beginning. In specific embodiments, the implementation between the introduction of two or more compositions may be present intervals ranging from minutes to days, to weeks, to months.

In another aspect the invention relates to methods of detection, OX40L, where the methods include the detection of complex OX40L-OX40L antibody to in the sample. The term "detection"as used in this description, includes qualitative and/or quantitative, detecti the (levels of measurement) compared with the control or without comparison with him.

In another aspect the invention relates to methods of diagnosis of a disorder associated with expression and/or activity of OX40L, where the methods include the detection of complex OX40L-OX40L antibody to" in a biological sample from the patient with a disorder or a suspect in possession of them. In some embodiments, the implementation of the expression of OX40L is increased expression or abnormal expression. In some embodiments, the implementation of the disorder is an immune disorder.

In another aspect the invention relates to any antibodies against OX40L, described here, where the antibody to OX40L contains detektiruya tag.

In another aspect the invention relates to a complex of any of antibodies against OX40L, described here, and OX40L. In some embodiments, the implementation of the complex is a complex in vivo or in vitro. In some embodiments, the implementation of the antibody to OX40L been labelled with a detectable label.

BRIEF DESCRIPTION of DRAWINGS

In Fig. 1A and 1B presents the amino acid sequence of the variable regions of murine monoclonal antibodies to human OX40L 8E12. A - variable region light chain 8E12 (SEQ ID NO:13). B - variable region of the heavy chain 8E12 (SEQ ID NO:15). Amino acids are numbered according to Kabat.

In Fig. 2A and 2B presents the amino acid sequence of the variable regions of murine mono is analnogo antibodies to human OX40L 13G5. A - variable region light chain 13G5 (SEQ ID NO:14). B - variable region of the heavy chain 13G5 (SEQ ID NO:16). Amino acids are numbered according to Kabat.

In Fig. 3 graphically shows that the introduction of monoclonal antibodies against OX40L 8E12 and 13G5 competitive inhibited the binding of OX40 receptor human with human OX40L.

In Fig. 4 graphically shows that the introduction of monoclonal antibodies against OX40L 8E12 and 13G5 inhibited the proliferation of T-cells in the analysis of cell-based.

In Fig. 5 graphically shows that the introduction of monoclonal antibodies against OX40L 8E12 and 13G5 inhibited the production of IL-2 T-cell memory.

In Fig. 6 graphically shows that the introduction of monoclonal antibodies against OX40L 8E12 and 13G5 inhibited the survival of activated T-cells.

DETAILED description of the INVENTION

The invention here relates to antibodies against OX40L, which is suitable, for example, for the treatment or prevention of disease States associated with expression and/or activity of OX40L, such as increased expression and/or activity or undesired expression and/or activity. In some embodiments, the implementation of the antibodies according to the invention is used to treat immune disorders (such as asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GVHD and/or systemic lupus erythematosus).

In another aspect, the anti-Christ. ate to OX40L according to the invention find utility as reagents for detection and/or selection OX40L, as, for example, the detection of OX40L in different types of tissues and cells.

The invention additionally relates to methods for producing antibodies against OX40L, polynucleotides coding for antibodies against OX40L and cells containing polynucleotides encoding antibodies against OX40L.

General procedures

Described or cited here methods and procedures, as a rule, specialists in this field are well understood and applied in the conventional practice, using conventional methods, such as, for example, are widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Definitions

"Selected" antibodies are antibodies that have been identified and isolated and/or extracted from a component of its natural environment. Contaminant components of its natural environment are materials that may impede the use of antibodies for diagnosis or treatment, and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, the implementation of antibodies purified (1) Bo is her than 95 wt.% antibodies as determined by the Lowry method, and most preferably more than 99 wt.%, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a sequencing machine with rotating cups, or (3) to homogeneity with SDS-PAGE in reducing or non conditions using Kumasi preferably blue or silver staining. Selected antibodies include antibodiesin situwithin recombinant cells, since in this case there is no at least one component of the natural environment antibodies. However, as a rule, the selected antibodies get through at least one stage of cleaning.

"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 antibodies. The selected nucleic acid molecule is in a different form or environmental conditions than in nature. The selected nucleic acid molecules therefore are distinguished from molecules of nucleic acid, which exists in natural cells. However, the selected nucleic acid molecule includes m is lukulu nucleic acid, in cells that normally Express the antibody, where, for example, the location of the nucleic acid molecule in the chromosome differs from location in natural cells.

The term "residue numbering variable domain as in Kabat" or "numbering provisions of amino acids as in Kabat", and its variations, refers to the numbering system used for the variable domains of the heavy chain or variable light chain domains of data collection for antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). When using this numbering system the actual linear amino acid sequence may contain fewer amino acids or more amino acids corresponding to the shortening FR or CDR variable domain or paste in them. For example, the variable domain of the heavy chain may include only the insertion of amino acids (residue 52a according to Kabat) after residue 52 H2 and inserted residues (e.g. residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 heavy chain FR. The residue numbering according to Kabat can be defined for these antibodies through alignment regions of homology sequence of the antibody with the "standard" are numbered according to Kabat sequence.

"The binding affinity of"generally refers to the force is not the total amount is valentich interactions between a single binding site of a molecule (for example, antibodies) and communicating with it by a partner (e.g., antigen). Unless specified otherwise, as used in the present description, "the affinity of binding" refers to the self-affinity of binding, which reflects the interaction of 1:1 between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X with respect to its partner Y can usually presented via dissociation constants (Kd). The affinity can be measured by using common methods known in this field, including the methods described here. Antibodies with low affinity, usually associated with antigen slowly and have a tendency to mild dissociation, whereas antibodies with high affinity, usually associated with antigen faster and have a tendency to stay longer linked. Many ways to measure the affinity of binding is known in this field, any of which can be used for the purposes of the present invention. Specific illustrative embodiments of described below.

In one embodiment, the implementation of the Kd" or "Kd" according to this invention is measured by analyzing the binding of radioactively labelled antigen (RIA)performed with the Fab version of interest antibodies and their antigen, as described by the following analysis, which ISM is accelerate the binding affinity of Fab in solution with the antigen by trim Fab minimum concentration ( 125I)-labeled antigen in the presence of a serial dilution of unlabeled antigen, with subsequent removal of the associated antigen on the tablet coated with an antibody to Fab (Chen, et al., (1999) J. Mol Biol 293:865-881). To determine the conditions of the analysis of tablets for micrometrology (Dynex) cover during the night of 5 μg/ml capture antibody anti-Fab (Cappel Labs) in 50 mm sodium carbonate (pH 9,6), and then score 2% (wt./about.) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In readsorbing tablet (Nunc No. 269620) 100 PM or 26 PM [125I]-antigen are mixed with serial dilutions of interest Fab (for example, in accordance with the assessment of antibodies anti-VEGF, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). Interest Fab then incubated over night; however, the incubation can be continued for a longer period (for example, 65 hours)to ensure achievement of equilibrium. After this mixture is transferred into a tablet for capture to incubate at room temperature (for example, within one hour). The solution is then removed and the plate washed eight times with 0.1% tween-20 in PBS. After the tablets are dried, add the scintillator (MicroScint-20; Packard) at the rate of 150 μl/well and take readings with tablets on the gamma counter Topcount (Packard) for ten minutes. Conc is tion of each Fab, who give less than 20% or equal to 20% of the maximum binding is chosen for use in the analysis of competitive binding. According to another variant implementation of the Kdor Kdmeasured using the methods of analysis based on surface plasmon resonance using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with CM5 chips with immobilized antigen at ~10 units resonance signal (RU). In brief biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) before injection with flow rate of 5 μl/minute to achieve approximately 10 units of the resonance signal (RU) associated protein. After injection of the antigen 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements twofold serial dilution of Fab (0.78 nm to 500 nm) are injected in PBS with 0.05% tween-20 (PBST) at 25°C with a flow rate of approximately 25 μl/min Rate of Association (konand the rate of dissociation (koff) is calculated using a simple model linking one-to-one Langmuir (software to calculate BIAcore, ver the Oia 3.2) by simultaneous approximation sensogram Association and dissociation. The equilibrium dissociation constant (Kd) is calculated as the ratio of koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol. Biol 293:865-881. If the velocity of the Association exceed 106M-1S-1in the above analysis based on surface plasmon resonance, the velocity of the Association can be determined using the method of quenching the fluorescence, which measures the increase or decrease in the intensity of the emitted fluorescence (excitation = 295 nm; emission = 340 nm, a bandwidth of 16 nm) at 25°C 20 nm of the antibody to the antigen (Fab form) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen measured in the spectrometer, such as a spectrophotometer, equipped with a stopped stream (Aviv Instruments)or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a stirred cuvette.

"Rate of Association" or "speed link", or "kon"according to this invention can also be defined in the same way, surface plasmon resonance as described above using a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with CM5 chips with immobilized antigen at ~10 units resonance signal (RU). In brief biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxycut what inimigos (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) before injection with flow rate of 5 μl/minute to achieve approximately 10 units of the resonance signal (RU) associated protein. After injection of the antigen 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements twofold serial dilution of Fab (0.78 nm to 500 nm) are injected in PBS with 0.05% tween-20 (PBST) at 25°C with a flow rate of approximately 25 μl/min Rate of Association (konand the rate of dissociation (koff) is calculated using a simple model linking one-to-one Langmuir (software to calculate BIAcore, version 3.2) by simultaneous approximation sensogram Association and dissociation. The equilibrium dissociation constant (Kd) is calculated as the ratio of koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol. Biol 293:865-881. If the velocity of the Association exceed 106M-1S-1in the above analysis based on surface plasmon resonance, the rate of Association is preferably determined using the method of quenching the fluorescence, which measures the increase or decrease in the intensity of the emitted fluorescence (excitation = 295 nm; emission = 340 nm, a bandwidth of 16 nm) at 25°C 20 nm of the antibody to the antigen (Fab form) in PBS, pH 7.2, the presence in trustusa concentrations of antigen measured in the spectrometer, such as a spectrophotometer, equipped with a stopped stream (Aviv Instruments)or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a stirred cuvette.

The term "vector"as used in the present description, is intended to refer to nucleic acid molecules that can carry another nucleic acid to which it was connected. One type of vector is a "plasmid", which refers to a circular double-stranded loop of DNA that can be ligitamate additional segments of DNA. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be ligitamate in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are administered (e.g., bacterial vectors having a bacterial replication origin and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the host, and thus they are replicated together with the genome of the host. Moreover, certain vectors are capable of driving the expression of genes with which they are functionally linked. Such vectors are denoted here as "expressing recombinant vectors" (or simply, "recombinant what's vectors"). As a rule, expressing the vectors used in the methods of recombinant DNA, are often in the form of plasmids. In the present description "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector in common practice.

"Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substance that can be embedded into a polymer using a DNA or RNA polymerase or by using synthetic reactions. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. In the presence of modified nucleotide can be performed prior to Assembly of the polymer or after it. The nucleotide sequence can be interrupted dinucleotide components. Polynucleotide can be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotide analogue, mezhnukleotidnyh modifications such as, for example, the R, modification uncharged bridges (for example, methylphosphonate, phosphotriesterase, potamididae, carbamates and the like) and charged bridges (for example, phosphorothioate, phosphorodithioate etc.), modification, containing suspended components, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine and the like), modification by intercalators (e.g., acridine, psoralen, etc.), modification, containing chelators (e.g., metals, radioactive metals, boron, metals, oxidizing agents, and the like), modification, containing alkylating agents, modification modified bridges (e.g., alpha anomeric nucleic acids, etc), as well as unmodified forms of polynucleotide(s). Further, any hydroxyl group, usually present in sugars, can be replaced, for example, phosphonate groups, phosphate groups, protected by conventional protective groups, or activated for more links with additional nucleotides, or they can be konjugierte with solid or semi-solid media. 5'- and 3'-terminal OH you can fosforilirovanii or replace amines or organic components of the blocking group of 1-20 carbon atoms. You can also get other derivatives of hydroxyl with conventional protective groups. Polynucleotide which can also contain analogous forms of sugars ribose or deoxyribose, which are widely known in this field, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, alpha-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose form of sugars, furanose form of sugars, sedoheptulose, acyclic analogs and basic nucleoside analogues, such as methylribose. One or more fosfolipidnyh bridges can be replaced by alternative linking groups. These alternative linking groups include as non-limiting examples of variants of implementation, where the phosphate is replaced by P(O)S("tiat"), P(S)S ("ditial"), "(O)NR2(amidate"), P(O)R, P(O)OR', CO or CH2(formatall"), in which each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. You do not want all the links in polynucleotide was identical. The preceding description applies to all mentioned here polynucleotides, including RNA and DNA.

"Oligonucleotide", as used in the present description generally refers to short, usually single-stranded, as a rule, synthetic polynucleotides that, typically, but not necessarily, consist of less than approximately 200 n is cleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides equally and fully applicable to oligonucleotides.

The term "OX40 ligand" (interchangeably referred to as "OX40L"), as used in the present description, refers, unless specifically or contextually indicated otherwise, to any natural or variant (or natural or synthetic) OX40L polypeptide. The term "natural sequence" specifically encompasses naturally occurring truncated or secreted forms (e.g., the sequence of the extracellular domain), naturally occurring variant forms (e.g., alternative splanirowannya forms) and naturally occurring allelic variants. The term "OX40L wild-type"generally refers to a polypeptide containing the amino acid sequence of a naturally occurring protein OX40L. The term "sequence OX40L wild-type"generally refers to the amino acid sequence represented in naturally occurring OX40L.

The term "receptor OX40" (interchangeably referred to as "OX40"), as used in the present description, refers, unless specifically or contextually indicated otherwise, to any natural or variant (or natural or synthetic) is peptide OX40. The term "natural sequence" specifically encompasses naturally occurring truncated or secreted forms (e.g., the sequence of the extracellular domain), naturally occurring variant forms (e.g., alternative splanirowannya forms) and naturally occurring allelic variants. The term "OX40 wild-type"generally refers to a polypeptide containing the amino acid sequence of a naturally occurring protein OX40. The term "sequence OX40 wild-type"generally refers to the amino acid sequence represented in naturally occurring OX40.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, polyspecific antibodies (for example, bespecifically antibodies as long as they possess the desired biological activity) and may also include certain antibody fragments (as described here in more detail). Antibodies can be human, humanitarianism and/or affinity-Mature.

The term "variable" refers to the fact that the sequence of certain portions of the variable domains differ significantly among the Titel and used in the binding and specificity of each particular antibody against the specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called areas of complementarity determining (CDR), or hypervariable regions in the variable domains, both light chain and heavy chain. More highly conserved part of the variable domains are called the framework (FR). Each of the variable domains of the natural heavy and light chain contains four FR-region, largely accepting the configuration of the β-layer, connected by three CDRs, which form loops connecting the β structure-layers and in some cases forming part thereof. CDRs in each chain are located together in close proximity to regions FR and together with other CDR circuits are involved in the formation antigennegative site of antibodies (see Kabat et al., Sequences of Protein of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding the antibody to the antigen, but they have different effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The papain cleavage of antibodies produces two identical antigenspecific fragment, called "Fab-fragments, each with a single antigennegative site, and a residual "Fc fragment", the name given to the which reflects its ability to easily crystallize. Treatment with pepsin yields F(ab')2-the fragment that has two antihistamine areas, and he is still capable of forming cross-linkage with the antigen.

"Fv" is the minimum antibody fragment which contains a complete antipersonnel and antigennegative plot. In double-stranded varieties Fv this area consists of a variable domain of the dimer of one heavy and one light chains with strong non-covalent bond. In the single-stranded species Fv variable domain of one heavy and one light chains can be covalently linked by a flexible peptide linker, so that the light and heavy chains can be connected in a "dimeric" structure analogous to the structure of double-stranded varieties Fv. In this configuration, the three CDRs of each variable domain interact with the formation antigennegative area on the surface of the dimer VH-VL. Together, the six CDR give antigennegative the specificity of the antibodies. However, even a single variable domain (or half of an Fv, containing only three CDRs specific antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

Fab-fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. ab'fragments differ from Fab fragments by the addition of a few residues at the C-end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of antibodies. Fab'-SH here designated as Fab'in which the residue(s) cysteine constant domains bear(ut) free thiol group. F(ab')2-fragments of the antibodies of the original was obtained as pairs of Fab'-fragments, which have a hinge cysteine each other. Also other known chemical cross-linking of fragments of antibodies.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be attributed to one of two clearly distinct types, called Kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be attributed to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1and IgA2. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins are called α, δ, ε, γ and µ, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.

"Antibody fragments" contain only a portion of an intact antibody, where the part is preferably retains at least one, before occhialino most or all of the functions, in the norm associated with this part when it is present in an intact antibody. Examples of fragments of antibodies include Fab, Fab'and F(ab')2and Fv fragments; dimeric antibodies; linear antibodies; single-chain molecule antibodies; and polyspecific antibodies formed from fragments of antibodies. In one of the embodiments the antibody fragment contains antigennegative the site of the intact antibody and thus retains the ability to bind to the antigen. In another embodiment, the fragment of the antibody, for example a fragment which contains the Fc-region, retains at least one biological functions normally associated with the Fc-region, when it is present in an intact antibody, such as FcRn binding, modulation of time half-life of antibodies, ADCC function and binding of complement. In one embodiment, the implementation of a fragment of the antibody is a monovalent antibody, which has in vivo by time-life is essentially similar to that of the intact antibody. For example, such an antibody fragment may contain antigennegative shoulder associated with the Fc sequence, capable of giving in vivo stability of the fragment.

The term "hypervariable region", "HVR" or "HV", when used in this description refers to the regions of the variable domain of antibodies, which are hypervirial lname on sequence and/or form a structured loops. Typically, antibodies contain six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Use a range of definitions hypervariable region, and they are considered in this description. By Kabat, region complementarity determining (CDR), determined on the basis of the variability of the sequence, and use them often (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Instead, Chothia refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). Hypervariable region AbM are a compromise between Kabat CDRs by and structural hinges on Chothia, and their use in software for modeling antibodies Oxford Molecular's AbM. "Contact" hypervariable region determined on the basis of analysis of available crystal structures of the complexes.

Hypervariable region may contain "extended hypervariable region"as follows: 24-36 (L1), 46-56 (L2) and 89-97 (L3) in the VL and 26-35 (H1), 47-66 or 49-66, or from 50 to 66 (H2), 93-101 or 93-102 (H3) in the VH. The remains of the variable domain are numbered according to Kabat et al., above for each of these definitions.

"Framework" or "FR" residues are those residues of the variable domain, which differ from the residues of the hypervariable region, as defined in this specification.

"Humanized" forms do not show the category of the human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from the immunoglobulin, which is not human. Basically, humanized antibodies are human immunoglobulins (recipient antibody)in which residues of the hypervariable region of the recipient to replace the remnants of the hypervariable region species, non-human (donor antibody)such as mouse, rat, rabbit or non-human Primate, which possess the desired specificity, affinity and activity. In some cases, remnants of the framework region (FR) of a human immunoglobulin replace the corresponding residues are not human. Furthermore, humanized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are carried out to further improve the properties of the antibodies. Typically, humanized antibodies contain essentially all, of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to loops immunoglobulin, non-human, and all or substantially all of the FR represent the sequence of FR of a human immunoglobulin. Also humanized EN is the body not necessarily contain at least part of a constant region (Fc) of an immunoglobulin, typically a constant region of 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). See also the following cited here, review articles and links: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

"Chimeric" antibodies (immunoglobulins) are part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a specific class or subclass of antibody, while the remainder of the chain(s) is(are) identical(s) or homologous(s) 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 possess the desired biological activity (U.S. patent No. 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). As used in the present description, humanized antibodies belong to the subclass of chimeric antibodies.

"Single-chain Fv" or "scFv-fragments of the antibodies contain the VH and VL domains of antibodies, where these domains are present in a single polypeptide chain. Typically, peptide scFv further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

"Antigen" is a predetermined antigen with which the antibody can selectively contact. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Preferably the antigen target is a polypeptide.

The term "dimeric antibody" refers to small fragments of antibodies with two antihistamine areas where the fragments contain the variable domain of the heavy chain (VH)connected to the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). When using a linker that is too short to provide the pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigenspecific plot. Dimeric antibodies are more fully described in, for example, in EP 404097; WO 93/11161 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

"Human antibodies" are antibodies that have an amino acid sequence that corresponds to the sequence of the antibody produced by the person, and/or which the s is obtained using any of the methods for producing human antibodies, as described in this description. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigennegative remains.

"Affine-Mature antibodies are antibodies with one or more changes in one or more of its CDR, which leads to an increase in the affinity of antibodies against the antigen, compared to a parent antibody which does not have these(and) change(s). Preferred affinity-Mature antibodies will have nanomolar or even picomolar values of affinity against the target antigen. Affine-Mature antibodies produced using methods known in this field. In Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by the rearrangement of VH and VL domains. Random mutagenesis of residues in the CDR and/or frame residues described in Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

"The effector functions of antibodies belong to such kinds of biological activity which are peculiar to the Fc-region (natural sequence Fc region or a variant amino acid sequence Fc region) of an antibody and vary depending on the isotype of the antibody. Examples of effector functions of antibodies including the up: the binding of C1q and complementability cytotoxicity; the binding of Fc-receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g. B-cell receptor and activation of B-cells.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig associated with Fc-receptors (FcR)present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages), cause specific to these cytotoxic effector cells to bind to the carrier cell antigen target and then destroy target cells by cytotoxins. Antibodies arm cytotoxic cells and are absolutely necessary for such destruction. The basic cell for the implementation of the ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the activity of interest molecules against ADCC can analyze ADCCin vitrosuch as analysis, described in U.S. patent No. 5500362 or 5821337, or in U.S. patent No. 6737056, Presta. Suitable for such analyses effector cells include mononuclear cells of peripheral blood and (PBMC) and natural killer (NK) cells. Alternative or additional activity of interest molecules against ADCC can be evaluatedin vivofor example, in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998).

"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 carry out ADCC include mononuclear cells of peripheral blood (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, thus are preferred PBMC and NK-cells. Effector cells can be isolated from a natural source, for example from the blood.

The terms "Fc receptor" or "FcR" describe a receptor that binds to the Fc region of antibodies. Preferred FcR FcR is the man with the natural sequence. Moreover, a preferred FcR FcR is associated with IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and alternatively splanirowannya forms of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid after what euteleostomi, which differ mainly by their cytoplasmic domains. Activating receptor FcγRIIA in its cytoplasmic domain contains immunoreceptor activating motif-based tyrosine (ITAM). Inhibiting receptor FcγRIIB in its cytoplasmic domain contains immunoreceptor inhibitory motif-based tyrosine (ITIM) (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). The term "FcR" in the present description include other FcR, including FcR, which will be identified in the future. 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)) and regulates the homeostasis of immunoglobulins. In WO00/42072 (Presta) described variants of the antibodies with increased or reduced binding to FcR. The contents of this patent publication, in particular, shown here as a reference. See also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

Known methods of measuring binding to FcRn (see, e.g., Ghetie 1997, Hinton 2004). You can analyze the binding to human FcRn in vivo and the half-life in serum polypeptides that bind with high affinity to human FcRn, for example, in transgenic mice or transfected with the human cell lines that Express the dominant human FcRn, or primates, which enter the Fc variants of the polypeptides.

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

Variants of polypeptides with altered amino acid sequences of the Fc-region and increased or decreased ability to bind C1q described in U.S. patent No. 6194551B1 and WO99/51642. The contents of these patent publications, in particular, shown here as a reference. See also, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

"Blocking" antibody or antibody antagonists are antibodies that inhibit or reduce the biological activity of the antigen with which they are associated. Preferred blocking antibodies or antibody antagonist essentially or completely inhibit the biological activity of the antigen.

"Disorder" or "disease" is any condition that is likely to have a beneficial effect treatment substance/molecule or method according to the invention. Nowlocal chronic and acute disorders or diseases, including such pathological conditions that make individuals mammal to the disorder. Non-limiting examples of disorders to be treated according to this description, include asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis and systemic lupus erythematosus.

"Autoimmune disease" herein is a disease or disorder arising in native tissues of the individual and directed against them, or an individual symptom or manifestation, or condition that occurs as its consequence. For many of these autoimmune and inflammatory disorders may exist a number of clinical and laboratory signs, including as non-limiting examples, hypergammaglobulinemia, high levels of autoantibodies, sediment complexes antigen-antibody in the tissues, the positive effect of treatment with corticosteroids or immunosuppressive drugs and clusters of lymphoid cells in the affected tissues.

As used in the present description, "treatment" refers to clinical intervention aimed at changing the natural course of the disease of the individual or cell being treated, and it can be done or for prophylaxis or during the course of clinical pathology. W the undesirable effects of treatment include preventing occurrence or recurrence of disease, mitigation of symptoms, reduction of any direct or indirect pathological consequences of the disease, decrease the speed of disease progression, improvement or temporary relief of the disease state, and remission or improved prognosis. In some embodiments, the implementation of the antibodies according to the invention are used to delay development of a disease or disorder.

"Individual" is a vertebrate animal, preferably a mammal, more preferably a human. Mammals include as non-limiting examples of farm animals (such as cows), animals used in sports, Pets (such as cats, dogs and horses), primates, mice and rats.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and animals from the zoo that is used in sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably the animal is a human.

"Effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

"Therapeutically effective amount" visesthakul according to the invention, agonist or antagonist may vary due to factors such as the disease state, age, sex and weight of the individual, and the ability of a substance/molecule, agonist or antagonist to cause the desired response in the individual. Therapeutically effective amount is the amount at which any toxic or detrimental effects of the substance/molecule, agonist or antagonist were outweighed by therapeutically beneficial effects. "Prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily as a prophylactic dose is used in subjects prior to disease or at its early stage, the prophylactically effective amount will be less than therapeutically effective amount.

The term "cytotoxic agent"as used in the present description, refers to a substance that inhibits the functioning of cells or stops it and/or causes destruction of cells. It is assumed that the term shall include radioactive isotopes (e.g., At211I131I125, Y90That Re186That Re188Sm153Bi212, P32and radioactive isotopes of Lu), chemotherapeutic drugs, such as methotrex is t, adriamicin, Vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating tools, enzymes and fragments thereof such as nucleotidase enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants, and the various antitumor or anticancer means described below. Other cytotoxic tools described below. Comorienne means causes destruction of tumor cells.

"Chemotherapeutic agent" is a chemical compound suitable for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide CYTOXAN®; alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; Betulinol acid; camptothecin (including the Isla synthetic analogue topotecan (COSMEGEN ®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcystein, scopoletin and 9-aminocamptothecin); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllin acid; teniposide; cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancreastatin; sarcodictyin; spongistatin; nitrogen mustard gas analogues, such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novemberin, finestein, prednimustine, trofosfamide, uramustine; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; antibiotics such as andinavia antibiotics (for example, calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, including dynemicin a, spiramycin; and the chromophore neocarzinostatin and similar chromophores chromoproteins indinavir antibiotics, aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, calcination, chromomycin, dactinomycin, daunorubicin, distrubition, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN®(misrepresented the number of morphosyntactical, cyanomethaemoglobin, 2-pyrrolidinecarbonyl and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; analogues of pyrimidines, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; means, depressing the function of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; compensator folic acid, such as folinovaya acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alphabetin; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; 2-acylhydrazides; procarbazine; polysaccharide complex PSK®(JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trihlortrietilamin; trichothecenes (especially toxin T-2, verrucarin a, roridin a and unguided); urethane; vindesine (ELDISINE®, FIELDSIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); thiotepa; taxoid, such as paclitaxel (TAXOL®(Bristol-Myers Squibb Oncology, Princeton, N.J.), constructed on the basis of the albumin preparation of nanoparticles of paclitaxel, without cremophor, ABRAXANE™ (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TEXTER®(Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®), oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as the identification of the behaviour of two or more of the above, such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTMin combination with 5-FU and leucovorin.

Also in this definition included antihormone funds, which act through regulation, decreasing, blocking or inhibiting effects of hormones that can stimulate the growth of cancer tumors, and they are often used in the form of systemic treatment or treatment of the whole body. They can be the hormones. Examples include antiestrogens and selective modulators of estrogen receptors (SERM), including, for example, tamoxifen (including tamoxifen NOLVADEX®), EVISTA®, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; antiprogesterone; negative regulators of estrogen receptors (ERD); tools that act, slowing or stopping the functioning of the ovaries, for example, agonists of luteinizing hormone-releasing hormone (LHRH), such as LUPRON®and leuprolide acetate, ELIGARD®, goserelin acetate, buserelin acetate and triptorelin; other anti-androgens such as flutamide, nilutamide and bikalutamid; and aromatase inhibitors that inhibit the enzyme ar is matazu, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutetimid, megestrol acetate MEGAS®, exemestane AROMASIN®, formestane, fadrozole, vorozole REVISOR®, letrozole FEMARA®and anastrozole ARIMIDEX®. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate DIDROCAL®, NE-58095, zoledronicaa acid/zoledronate ZOMETA®, alendronate FOSAMAX®, pamidronate AREDIA®tiludronate SKELID®or risedronate ACTONEL®; and troxacitabine (the analogue of the nucleoside cytosine-based 1,3-dioxolane); antisense oligonucleotides, particularly oligonucleotides that inhibit expression of genes in signaling pathways implicated in impaired cell proliferation, such as, for example,PKC-alpha,Raf,H-Rasand the gene of the receptor for epidermal growth factor (EGF-R); vaccines such as vaccines CERATOP®vaccines and for gene therapy, for example a vaccine ALLOVECTIN®vaccine LEUVECTIN®and vaccine WAXED®; topoisomerase inhibitor 1 LURTOTECAN®; ABARELIX®rmRH; lapatinib ditosylate (low molecular weight dual inhibitor tyrosinekinase ErbB-2 and EGFR, also known as GW572016); and formats whitesky acceptable salt, acids or derivatives of any of the foregoing.

"A means of inhibiting the growth" when used in this specification refers to a compound or composition which inhibits growth of the cell (such as cell expressing OX40L), or in vitro, or in vivo. Thus, a means of inhibiting the growth can be a tool that significantly reduces the percentage of cells (such as cell expressing OX40L) in S-phase. Examples of means, inhibiting growth, include tools that block the passage of the cell cycle (at a different stage compared to S-phase), such as tools, which induce a stop in G1 and stop in M-phase. Classic blockers M-phases include the Vinca alkaloids (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. The means that cause a stop in G1, as well as a side result, cause a stop in S-phase, such as alkylating DNA, 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 p. 13. Taxanes (paclitaxel and docetaxel) are about eparecovery drugs which are both derived from Tissa. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European Tissa, is a semisynthetic analogue of paclitaxel (TAXOL®Bristol-Myers Squibb). Paclitaxel and docetaxel stimulate the Assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

"Doxorubicin" is an anthracycline antibiotic. Full chemical name of doxorubicin is (8S-CIS)-10-[(3-amino-2,3,6-trideoxy-α-L-oxohexanoyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetic)-1-methoxy-5,12-naphthacenedione.

The term "polypeptide containing an Fc region" refers to the polypeptide, such as antibodies or immunoadhesin (see definitions below), which contains the Fc-region. C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during purification of the polypeptide or recombinant engineering of nucleic acid that encodes a polypeptide. Thus, a composition comprising a polypeptide having an Fc-region according to this invention may include polypeptides with K447, all of the remote K447, or a mixture of polypeptides with the K447 residue and without it.

The composition of the invention and methods for their preparation

This invention relates to the positions including pharmaceutical compositions, containing antibodies against OX40L; and polynucleotides comprising sequences encoding antibodies against OX40L. As used in the present description, the compositions contain one or more antibodies that bind to OX40L and/or one or more polynucleotide containing sequences encoding one or more antibodies that bind to OX40L. These compositions can optionally contain suitable carriers, such as pharmaceutically acceptable excipients, including buffers, which are well known in this field.

The invention also includes variants of the implementation with the selected antibodies and polynucleotide. The invention also includes embodiments of essentially pure antibodies and polynucleotide.

Antibodies against OX40L according to the invention are preferably monoclonal. Also in the scope of the invention include fragments presented in this description of OX40L antibodies to Fab, Fab', Fab'-SH and F(ab')2. These antibody fragments can be obtained by conventional methods, such as enzymatic cleavage, or they can be obtained by recombinant methods. Such fragments of antibodies may be chimeric or humanitarianism. These fragments are suitable for diagnostic and therapeutic purposes, as described below.

Monoclonal anti the ate is obtained from the population essentially homogeneous antibodies, i.e. the individual antibodies containing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, modifying the term "monoclonal" indicates the character of the antibodies, because they are not a mixture of separate antibodies.

Monoclonal antibodies against OX40L according to the invention can be obtained using the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or they can be obtained by means of recombinant DNA (U.S. patent No. 4816567).

In the hybridoma method, a mouse or other suitable animal host, such as a hamster, subjected to immunization to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. The induction of antibodies against OX40L, as a rule, carried out in animals, multiple subcutaneous (sc) or intraperitoneal (ip) injection of OX40L and adjuvant. OX40L can be obtained using methods well known in the field, some of which are further described here. For example, recombinant production of OX40L described below. In one of the embodiments of animals subjected to immunization derived OX40L, which contains the extracellular domain (ECD) OX40L fused with the Fc part of the heavy chain of immunoglobulin In the preferred embodiment, animals are subjected to immunization fused protein OX40L-IgG1. Animals initially subjected to immunization immunogenic conjugates or derivatives of OX40L on the basis monophosphoryl-lipid A (MPL)/dicenomicon trehalose (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT) and the solution is injected intradermally in many areas. Two weeks later the animals repeated immunization. 7-14 days later the animals are taken blood and serum examined for the title to OX40L. Animals repeated immunization up until the titer stabilizes.

Alternative lymphocytes can immunize in vitro. Then produce a fusion of lymphocytes with myeloma cells using a suitable substance which causes the merge, such as polyethylene glycol, to obtain a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The thus obtained hybridoma cells were seeded in a suitable culture medium and grown in a medium preferably contains one or more substances that inhibit the growth or survival naturgas merger parental myeloma cells. For example, if the parental myeloma cells lacking the enzyme hypoxanthineguanine (HGPRT or HPRT), the culture medium for the hybridomas typically will include gipoksantin, aminopterin and thymidine (Wednesday HAT), substances that prevent the growth of T-cells with a defect HGPRT.

Preferred myeloma cells are cells that are effectively merge, maintain a stable antibody production at a high level electrovanne producing antibodies cells and are sensitive to the environment, such as environment HAT. Among them, preferred myeloma cell lines are murine myeloma lines, such as lines originating from tumors of mice MOPC-21 and MPC-11, available at the Salk Institute Cell Distribution Center, San Diego, California USA, cells and SP-2 or X63-Ag8-653, available at the American Type Culture Collection, Rockville, Maryland USA. Cell line the human myeloma and heteromyinae mouse-man is also described for obtaining human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma is grown cells, analyze on developing monoclonal antibodies directed against OX40L. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or analysis of binding in vitro, such as radioimmune assay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of monoclonal antibodies, for example, can be determined by analysis of Scatchard by Munson et al., Anal. Biochem., 107:220 (1980).

After the identification of those hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity, the clones can be subclinical by means of serial dilutions and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells can be grown in vivo as ascitic tumors in the animal.

Monoclonal antibodies secreted by the subclones, accordingly isolated from the culture medium, ascites fluid, or serum by conventional methods of purification of immunoglobulins, such as, for example, protein A-sepharose, chromatography with hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

Antibodies against OX40L according to the invention can be obtained by using combinatorial libraries to screen for synthetic clones of antibodies with the desired activity or activities. Essentially clones of synthetic antibodies are selected by screening phage libraries containing the phage, which exposed various fragments of variable regions (Fv) antibody, fused with the protein shell of the phage. Such phage library selected by affinity chromatography with respect to the desired antigen. Chloe is s, expressing Fv-fragments, the ability to communicate with the desired antigen, adsorbed on the antigen and thus are separated from the unbound clones in the library. Bound peroxidase clones then elute with antigen, and can further be enriched by additional cycles of adsorption of the antigen/elution with him. Any of the antibodies against OX40L according to the invention can be obtained by designing a suitable screening method using antigen for selection of interest phage clone then get a clone of the full-size antibodies against OX40L using the Fv sequences from the interest of phage clone and sequence a suitable constant region (Fc), described by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

Antigennegative domain antibodies formed by two variable (V) regions of approximately 110 amino acids, in one area of the light (VL) and heavy (VH) chains, which both give three hypervariable loops or region complementarity determining (CDR). Variable domains can functionally be exposed on the phage, or in the form of single-chain Fv fragments (scFv), in which VH and VL are covalently bound through a short, flexible peptide, or as Fab fragments, in which each of them fused with the constant domain, and they usaimage the point ecovalence, as described in Winter et al., Ann. Rev. Immunol, 12:433-455 (1994). As used in the present description, phage clones encoding scFv, phage clones encoding Fab, collectively referred to as "Fv phage-clones" or "Fv-clones".

The gene repertoires of VH and VL can be individually cloned using polymerase chain reaction (PCR) and randomly recombine in phage libraries, which you can then search antigenspecific clones, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immunized sources give high-affinity antibodies to the immunogen without the need to create a hybrid. Alternative naive repertoire can be cloned to create a single source of human antibodies to a wide range of nauto and proteins without any immunization, as described in Griffiths et al., EMBO J 12:725-734 (1993). In conclusion, not subjected to the library can also be obtained synthetically by cloning nepristoinyi segments V-genes from stem cells and using primers for PCR containing a random sequence to encode vysokopribylnyj CDR3 regions and for the implementation of realignment in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

Filamentous phage are used for exhibiting fragments of antibodies by fusion with the minor protein shell pIII. ragment antibodies can be exhibited in the form of single-chain Fv fragments, in which VH and VL domains are linked by a single polypeptide chain flexible polypeptide spacer, for example, as described in Marks et al., J. Mol. Biol., 222:581-597 (1991), or as Fab fragments, in which one chain is subjected to fusion with pIII and other secreted in periplasm bacterial host cell, where the Assembly structure of the Fab-protein shell, which begins to be exhibited on the surface of phage by the replacement of some membrane proteins of wild-type, for example, as described in Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

Typically, nucleic acids encoding fragments of antibody genes, derived from immune cells isolated from human or animal. If you want a library enriched clones against OX40L, the subject is subjected to immunization through OX40L to obtain a humoral immune response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBL) allocate to create the library. In a preferred embodiment, a library of gene fragments of human antibodies enriched clones against human OX40L, get the induction of humoral immune response against human OX40L transgenic mice carrying a functional set of genes of human immunoglobulins (and lacking a functional system the production of endogenous antibodies) such that immunization OX40L result is to produce B-cells of human antibodies against OX40L. Obtaining transgenic mice that produce human antibodies, as described below.

Additional enrichment of populations of activated cells against OX40L can be obtained by using a suitable screening method for the selection of B-cells expressing specific to OX40L membrane-bound antibodies, for example, by separating cells by affinity chromatography with OX40L or adsorption of cells to labeled fluorochroman OX40L with subsequent fluorescently-activated cell sorting (FACS).

Alternative use of spleen cells and/or B cells or other PBL from an unimmunized donor provides a better representation of the possible repertoire of antibodies, and also allows the creation of a library of antibodies using any kind of animal (human or non-human), in which OX40L is not antigenic. For libraries, including the construction of antibody genes in vitro, stem cells isolated from the subject to obtain nucleic acids encoding nepristoinye segments of antibody genes. Interest of the immune cells can be obtained from many species of animals, such as humans, mice, rats, hares, rabbit, dog, cat, swine, bovine, horse and birds, etc.

Nucleic acid encoding a variable gene segments antibodies (including VH - and VL-CE the cops), allot of interest in cells and amplified. In the case of libraries rebuilt VH and VL genes to the desired DNA can be obtained by selection of the genomic DNA or mRNA from lymphocytes with subsequent polymerase chain reaction (PCR) with primers complementary to the 5'- and 3'-ends rebuilt VH and VL genes, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989), thus creating a varied repertoire of V genes for expression. V-genes can amplify on the basis of cDNA and genomic DNA with a reverse primer at the 5'end of the exon encoding the Mature V-domain, and direct primers located within the J-segment, as described in Orlandi et al. (1989) and Ward et al., Nature, 341:544-546 (1989). However, for amplification with cDNA reverse primers can also be placed in the leader exon, as described in Jones et al., Biotechnol., 9:88-89 (1991), and direct the primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarities primers can include degeneracy, as described in Orlandi et al. (1989) or Sastry et al. (1989). Preferably the diversity of the library to maximize the use of primers for PCR, aimed at every family of V-gene in order to amplify all available VH - and VL-configuration present in the sample nucleic acid from immune cells, for example, as described in the method according to Marks e al., J. Mol. Biol., 222:581-597 (1991), or as described in the method according Orum et al., Nucleic Acids Res., 21:4491-4498 (1993). For cloning of amplified DNA in expressing vectors can be entered rare restriction sites in primer for PCR in the form of a label on one end, as described in Orlandi et al. (1989), or more amplification by PCR with labeled primers as described in Clackson et al., Nature, 352:624-628 (1991).

The repertoire of synthetically reconstructed V-genes can be obtained in vitro, based on segments V-genes. Most segments VH-human genes cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3:88-94 (1993)); these cloned segments (including all the major conformation of the H1 - and H2-loop) can be used to obtain different repertoires of VH genes with primers for PCR, coding H3-loops of different sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH-playlists can also be obtained in a variety of sequences that is concentrated in long-H3-loop the same length, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). Segments Vκ and Vλ human cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:1456-1461 (1993)), and can be used to obtain synthetic repertoires light chain. The repertoire of synthetic V-gene-based range VH - and VL-bends, and L3 and H3-lengths to irout antibodies with considerable structural diversity. After amplification of the DNA encoding the V-gene segments V-genes in the germ line can be rebuilt in vitro according to the methods in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

The repertoires of fragments of antibodies can be designed by combining together repertoires of VH and VL genes in several ways. Each repertoire can be obtained in different vectors, and vector recombine in vitro, for example, as described in Hogrefe et al., Gene, 128:119-126 (1993), or in vivo co-infection, for example, by loxP system described in Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993). In the method of recombination in vivo use of the double-stranded nature of Fab fragments to overcome the limitations of the size of the library, which limit the efficiency of transformationE. coli. "Naive" VH - and VL-clone repertoires separately, one in formigny and another in the phage vector. Two libraries are then combined by ragovoy infections contain fahmida bacteria so that each cell contains a different combination, and the library size is limited only by the number of cells present (approximately 1012clones). Both vectors contain the signals of recombination in vivo such that the VH - and VL-genes recombine into a single replicon together and packaged into phage virions. These huge libraries provide large number of different antibodies with good affinity of the (K d-1approximately 10-8M).

Alternative repertoires can be cloned sequentially into the same vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or to collect together using PCR and then cloned, for example, as described in Clackson et al., Nature, 352:624-628 (1991). Assembly using PCR can also be used to join DNA VH and VL DNA encoding a flexible peptide spacer, for the formation of a repertoire of single-chain Fv (scFv). In another method of Assembly using PCR in the cell" is used for combining VH and VL genes in lymphocytes using PCR and then cloned repertoires connected genes, as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

Antibodies formed naive libraries (or natural or synthetic), may have moderate affinity (Kd-1approximately 106up to 107M-1), but affinity maturation can also be mimicked in vitro by constructing and re-selection based on secondary libraries, as described in Winter et al. (1994), above. For example, the mutation may accidentally enter in vitro using inaccurate polymerase (reported in Leung et al., Technique, 1:11-15 (1989)) in the method according to Hawkins et al., J. Mol. Biol., 226:889-896 (1992) or in the method according to Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). In addition, affinity maturation can be performed random mutagenesis of one or bore the channels at CDR, for example, using PCR with primers carrying a random sequence, covering the interest CDR, the selected individual Fv-clones and screened for clones with higher affinity. In WO 9607754 (published on 14 March 1996) described a method for inducing mutagenesis in a complementarity determining region light chain immunoglobulin to obtain a library of light chain genes. Another effective method is a recombination of VH or VL domains selected using phage display, with repertoires of naturally occurring variants of the V-domain, obtained from unimmunized donors, and screening for higher affinity in a few cycles adjustment circuit, as described in Marks et al., Biotechnol., 10:779-783 (1992). This method allows to obtain antibodies and antibody fragments with affiniscape in the range of 10-9M

The sequence of the nucleic acid encoding OX40L, can be constructed using the amino acid sequence of the desired region OX40L, for example, the extracellular domain. Alternative you can use the cDNA sequence (or its fragments). Additional sequence OX40L further described, for example, SwissProt, inventory number P23510. DNA encoding OX40L, can be obtained in a variety of ways known in this field. These methods include in image quality is as non-limiting examples of chemical synthesis using any of the methods described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989), such as methods based trifiro, phosphite, phosphoramidite and H-phosphonate. In one of the embodiments when constructing DNA encoding OX40L, using codons preferred for expression in the cell host. Alternative DNA encoding OX40L, can be isolated from genomic libraries or cDNA libraries.

After constructing a DNA molecule encoding OX40L, the DNA molecule is functionally connected with the control expression sequence in expressing vector, such as plasmid, where the control sequence is recognized by the cell host transformed by the vector. Generally, plasmid vectors contain replicate and control sequences which are derived from species compatible with the host-cell. Vector source carries replicate plot, as well as sequences that encode proteins that are capable of selection on phenotype in transformed cells. Suitable vectors for expression in prokaryotic and eukaryotic cells-hosts known in this area, and some of them are further described here. You can use eukaryotic organisms such as yeast, or cells derived from multicellular organisms such as mammals.

Optionally, DNA encoding a X40L, functionally associated with a secretory leader sequence resulting in secretion of the expression product the host-cell into the culture medium. Examples of secretory leader sequences include stII, akoten, lamB, GD of herpes, lpp, alkaline phosphatase, invertase, and alpha factor. Also for use in the present description fits 36-amino acid leader sequence of the protein A (Abrahmsen et al., EMBO J., 4:3901 (1985)).

Cell owners transferout and preferably transforms the above expressing or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transfection refers to the capture of expressing the vector in the host-cell, while any coding sequences are in fact expressed or or not. The usual specialist known for the numerous methods of transfection, such as precipitation using a CaPO4and electroporation. Successful transfection, as a rule, determine when any sign of the operation of this vector is in the cell host. Methods of transfection are well known in this area, and some of them are further described here.

the Transformation means introducing DNA into an organism so that DNA is replicated or as an extrachromosomal element, or as an insert into the chromosome. Depending on the host cell transformation carried out using conventional methods suitable for such cells. Methods of transformation are well known in this area, and some of them are further described here.

Prokaryotic cells are the masters used to obtain OX40L, you can cultivate, as broadly described in Sambrook et al., above.

The cells of the host mammal, used to obtain OX40L, can be grown in many environments, which are well known in this area, and some of which are described here.

Cell host, which include the description, include cells for in vitro cultivation, as well as cells that are within an animal host.

Cleaning OX40L can be performed using methods known in this field.

Purified OX40L can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, gels hydroxymethotrexate, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like, for use in the separation of clones of phage display affinity chromatography. The protein accession to OX40L matrices is possible to realize ways, described in Methods in Enzymology, vol. 44 (1976). The conventional method of attaching a protein ligands to the polysaccharide matrix, such as agarose, dextran or cellulose, includes the activation of media changelogname and the subsequent binding of primary aliphatic or aromatic amines peptide ligand to the activated matrix.

Alternative OX40L can be used to coat the wells of adsorption tablets, to Express on the cell-hosts attached to the adsorption tablets, or used in cell sorting, or konjugierte with Biotin for capture streptavidin coated beads, or used in any other method known in this field, for panning libraries phage display.

Samples ragovoy library contact with immobilized OX40L under conditions suitable to bind at least a portion of the phage particles with the adsorbent. Usually conditions, including pH, ionic strength, temperature and the like, are chosen to mimic physiological conditions. Phages associated with the solid phase, washed and then elute the acid, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or alkali, for example, as described in Marks et al., J. Mol. Biol., 222:581-597 (1991), or competition with antigen OX40L, for example, in the way similar to the way competition with antigen in Clackson et al., Nature, 352:624-628 (1991). Phages which you can enrich in 20-1000 times in one cycle of selection. Moreover, the enriched phage can be grown in bacterial culture and be subjected to further cycles of selection.

The effectiveness of selection depends on many factors, including the kinetics of dissociation during washing, and on whether the multiple fragments of antibodies on a single phage simultaneously to contact with the antigen. Antibodies rapid kinetics of dissociation and low affiniscape link) you can keep using short cleaning, polyvalent phage display and high-density coating antigen solid phase. High density not only stabilizes the phage through multivalent interactions, but promotes the re-linking dissociative phage. Selection of antibodies with slow dissociation kinetics (and good affiniscape binding) can facilitate the use of long-term cleaning and monovalent phage display as described in Bass et al., Proteins, 8:309-314 (1990) and in WO 92/09690, and low density coating antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

Possible breeding between OX40L fagbemi antibodies of different affinely, even affinely, which slightly differ. However, random mutation breeding antibodies (for example, in the implementation of some methods of affinity maturation as described above), will likely coord what many mutants, more than just recognize the antigen, and the few with a higher affinity. Given the limited number of OX40L few phages with high affinity can be eliminated. To save all mutants with higher affinity phages can be incubated with excess biotinylated OX40L, but with biotinylated OX40L in concentrations lower polyarnosti than the planned value of molar constant affinity for OX40L. Phages with high affinity binding can then capture on streptavidin coated paramagnetic beads. This "equilibrium capture" enables discriminates antibodies according to their affinity of binding with sensitivity, which allows you to isolate mutant clones with up to two times higher affinity of a large excess of phages with lower affinity. Used washing conditions phages associated with the solid phase, can also be manipulated to separate on the basis of the kinetics of dissociation.

Clones of anti-OX40L can be discriminates activity. In one of the embodiments the invention relates to antibodies against OX40L, which block the binding between the receptor OX40 and OX40L, but do not block the binding between the receptor OX40 and the second protein. Fv clones corresponding to such antibodies against OX40L, you can discriminates (1) selection of clones of anti-OX40L from ragovoy libraries is, as described above, and optional amplification of the selected population of phage clones by growing population in a suitable bacterial host; (2) the choice of OX40L and a second protein against which, respectively, require blocking and non-blocking activity; (3) adsorption of phage clones anti-OX40L on immobilized OX40L; (4) using an excess of the second protein elution any undesired clones that recognize determinants of binding OX40L, which overlap with the determinants of binding of the second protein or coincide with them; and (5) elution of the clones which remain adsorbed during stage 4). Optional clones with the desired properties of the blocking/non-blocking can further enrich the repetition here of the procedures for the selection of one or more times.

DNA encoding obtained from a hybrid monoclonal antibodies or Fv clones of phage display technique according to the invention, is easily isolated and is sequenced using conventional methods (e.g., using oligonucleotide primers designed for specific amplification of interest regions encoding the heavy and light chain from hybridoma or ragovoy matrix DNA). After DNA extraction can be embedded in expressing vectors, which are then transferout in cell host, such to the to cells E. colicells , COS monkey cells Chinese hamster ovary (CHO) or myeloma cells that do not produce another protein immunoglobulin, for the synthesis of the required monoclonal antibodies in the recombinant cell host. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256 (1993) and Pluckthun, Immunol. Revs, 130:151 (1992).

DNA encoding the Fv-clones according to the invention can be combined with known DNA sequences encoding the constant region of the heavy chain and/or light chain (e.g., the appropriate DNA sequence may be obtained from Kabat et al., above), to obtain clones encoding the heavy and/or light chain of the full or partial length. Will be taken into account that you can use for this purpose a constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and that such constant region can be obtained from either human or animal. As used in the present description, Fv-clone originating from the variable domain of the DNA of one species of animal (such as man) and then fused with the constant region DNA of another species of animal, with the formation of the sequence(s), its(their) "hybrid"full size heavy chain and/or light chains include in the definition of "chimeric" and "hybrid" antibodies. the preferred embodiment Fv-clone, originating from variable human DNA, is subjected to the merger with the constant region of human DNA with the formation of the coding(s) sequence(s) for all heavy and/or light chains of a person of full or partial length.

DNA encoding antibodies against OX40L, deriving from hybridoma according to the invention can also be modified, for example, by replacing the sequence encoding the constant domains of the heavy and light chain of a human, instead of the homologous murine sequences derived from clone hybridoma (for example, as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). DNA encoding the antibody or fragment originating from hybridoma or Fv-clone, can be further modified by covalent joining sequence that encodes an immunoglobulin, all or part of a sequence that encodes a polypeptide that is not an immunoglobulin. Thus, you receive a "chimeric" or "hybrid" antibodies that have the binding specificity of the antibodies according to the invention, originating from Fv-clone or clone hybridoma.

Antibody fragments

The present invention relates to fragments of antibodies. In certain circumstances there are advantages to using fragments of antibodies instead of whole antibodies. The smaller size of the fragments provides quick clearance and may lead to improved the Yu access to solid tumors.

To obtain fragments of antibodies have been developed in different ways. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al.Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be obtained directly by recombinant host cells. All Fab, Fv and ScFv fragments of antibodies can be Express inE. coliand they can secretariats fromE. colithus easily obtain large amounts of these fragments. Antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternative fragments, Fab'-SH, you can select directly from theE. coliand chemically combine with the formation of fragments F(ab')2(Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach, the fragments F(ab')2you can select directly from recombinant cell culture-owners. Fragments Fab and F(ab')2with increased half-life ofin vivocontaining residues of the epitope that binds the receptor of salvation described in U.S. patent No. 5869046. Other methods of obtaining fragments of antibodies will be apparent to the skilled technician. In other embodiments, the preferred implementation for selecting the antibody is a single-chain Fv fragment (scF). See WO 93/16185, U.S. patent No. 5571894 and U.S. patent No. 5587458. Fv and sFv are the only types with the intact active centers, which are devoid of constant regions; thus, they are suitable to reduce nonspecific binding when usingin vivo. Merged sFv proteins can be constructed with getting effector protein, fused or N-end or C-end of the sFv. See Antibody Engineering, ed. Borrebaeck, above. Also the fragment of the antibody may be a "linear antibody", e.g., as described in U.S. patent No. 5641870. These linear fragments of antibodies can be monospecificity or especificada.

Humanized antibodies

The present invention relates to humanized antibodies. Various methods of humanizing antibodies, non-human, known in this area. For example, humanitariannet the antibody may possess one or more amino acid residues built into it from a source that is not human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can mainly be carried out in accordance with the method of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 29:1534-1536 (1988)), by replacing the corresponding sequences of human antibodies with sequences of the hypervariable regions. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), where essentially less intact variable domain of the human substituted by the corresponding sequence type, non-human. In practice, humanized antibodies, as a rule, are human antibodies in which some hypervariable residues region and possibly some FR residues substituted by residues from analogous sites of antibodies rodents.

The choice of the variable domains of a man, as light and heavy chains, for use when obtaining humanized antibodies is very important to reduce antigenicity. In accordance with the so-called method of "best match" sequence of the variable domain of the antibody rodent analyze relative to a library of known sequences of the variable domains of a human. Then the sequence of the human being, which is the most similar to the sequence of the rodent, is considered acceptable as the frame area for gumanitarnogo antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). In another method used is isout specific frame region, derived from the 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)).

Moreover, it is important that antibodies were humanitarianism with retention of high affinity towards the antigen and other positive biological properties. To achieve this objective, according to one method, humanized antibodies produced using the process of analysis of the parental sequences and various alleged humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are widely available and well-known experts in this field. Available are computer programs which illustrate and display probable three-dimensional conformational structures of selected prospective sequences of immunoglobulins. The study of these displayed data allows an analysis of the possible role of the residues in the functioning of the anticipated sequences of immunoglobulins, i.e., the analysis of residues that influence the ability of the proposed immunoglobulin has been the substance of its antigen. This way, FR residues can be selected and combined from the recipient and import sequences so that to achieve the desired properties of the antibodies, such as increased affinity for the antigen(s)target(s). Typically, the immediate and most significant effect on the binding of antigen have remnants of the hypervariable region.

Human antibodies

Human antibodies against OX40L according to the invention can be obtained by combining sequence(s) of the variable domain of an Fv clone selected from libraries of phage display derived from the person with the known(s) sequence(s) of the constant domain of a human, as described above. Alternative human monoclonal antibodies against OX40L according to the invention can be obtained hybridoma method. Cell line the human myeloma and heteromyinae mouse-human to obtain human monoclonal antibodies are described, e.g., Kozbor. J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).

Currently you can obtain transgenic animals (e.g. mice)that when immunization capable of producing a full repertoire of human antibodies in the absence of endogenous production of immunoglobulins. For example, it has been described that the homozygous delete the gene region of heavy chain antibodies (J H) chimeric and bearing mutations in the germ line of mice results in complete inhibition of endogenous production of antibodies. The transfer of immunoglobulin genes germline of the person so carrying mutations in the germline of mice with antigenic stimulation will lead to the production of antibodies person. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).

For obtaining human antibodies from non-human antibodies, such as antibodies rodents, where human antibodies have similar appendectomy and specificnosti in relation to the original non-human antibody, it is also possible to use a permutation of genes. According to this method, also referred to as "epitope imprinting", the variable region or a heavy or light chain not owned by the person of a fragment of the antibody, which is produced by means of phage display as described above, replace the repertoire of genes V-domain of a person, receiving a population of chimeric molecules scFv or Fab, consisting of not belonging to/owned by the man chains. Selection against the antigen leads to the secretion of chimeric molecules scFv or Fab, consisting of not belonging to/owned by the man chains, where the chains of man is restored antigennegative ucast is, broken deleting the matching does not belong to the human chain in the primary clone phage display, i.e. the epitope determines (detects) the choice of the partner chain man. Human antibodies were obtained when repeating the process of replacing the remaining not owned by the person chain (see PCT WO 93/06213, published April 1, 1993). Unlike traditional humanizing non-human antibodies by CDR grafting, this method leads to the production of fully human antibodies, which do not contain residues of FR or CDR is not associated with human origin.

Bespecifically antibodies

Bespecifically antibodies are monoclonal, preferably human or humanized antibodies that have specificnosti binding in respect of at least two different antigens. In this case, one of specificdate binding refers to OX40L, and the other refers to any other antigen. Typical bespecifically antibodies can bind to two different epitopes of the protein OX40L. Bespecifically antibodies can also be used for targeted delivery of cytotoxic agents to cells which Express OX40L. These antibodies possess a part to bind to OX40L and part of which is associated with a cytotoxic agent (e.g., caporino, anti-integration is the Feron α, the Vinca alkaloid A-chain of ricin, methotrexate or hapten with a radioactive isotope). Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically antibody F(ab')2).

Methods of obtaining bespecifically antibodies known in the field. Traditionally, the recombinant getting bespecifically antibodies based on simultaneous expression of two pairs of heavy chain-light chain immunoglobulins, where the two heavy chains have different specificnosti (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random Assembly of the heavy and light chains of immunoglobulins such hybridoma (quadroma) produce a mixture of potentially 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which is usually carried out by stages affinity chromatography, is quite difficult, and the product yield is low. Similar methods are described in WO 93/08829, published 13 may 1993, and in Traunecker et al., EMBO J., 10:3655 (1991).

In accordance with another and preferred approach conduct merge variable domains of antibodies with the desired specificnosti binding (the binding sites of the antibody-antigen) with the sequences of the constant domains of immunoglobulins. Merge predpochtitel is on hold with the constant domain of the heavy chain of the immunoglobulin, containing at least part of the hinge regions CH2 and CH3. Preferably, the first constant region of the heavy chain (CH1)containing the site necessary for binding to the light chain was present in at least one of the components to merge. DNA molecules encoding designed to merge the components of the heavy chains of immunoglobulins and, if required, the light chain immunoglobulin is inserted into the individual expressing vectors and implement joint transfection of them fit of the host body. This integration provides significant flexibility in the regulation of the mutual ratios of the three polypeptide fragments in the variants of implementation, where unequal ratios of the three polypeptide chains used in the construction provide the optimum yield. However, you can embed 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 product yield or if the ratios are of no particular value.

In the preferred embodiment, this approach bespecifically antibodies comprise the heavy chain hybrid immunoglobulin single binding specificity in one part and a pair of tie bars is barking chain-light chain hybrid immunoglobulin (having a different binding specificity) in the other part. Found that this asymmetric structure facilitates the separation of the desired especifismo compounds and undesirable combinations of chains of immunoglobulins, as the presence of light chain immunoglobulin in only one half of bespecifically molecules provides an easy way of separation. This method is described in WO 94/04690. For a more detailed description of obtaining bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another method, you can construct a contact area between a pair of antibody molecules to maximize the percentage of heterodimers, which is recovered from recombinant cell culture. The preferred contact area contains at least part of the CH3-domain constant domain of antibodies. In this way one or more small amino acid side chains from the contact region of the first antibody molecules replace the larger side chains (e.g. tyrosine or tryptophan). In the contact region of the second molecule antibodies create compensatory "cavities" of identical or similar with the major(s) side(s) chain(s) size by replacing large amino acid side chains are chains of smaller sizes (e.g., alanine or threonine). This replacement provides a mechanism to enhance the yield of heterodimer what about the comparison with other unwanted end-products, such as homodimer.

Bespecifically antibodies include cross stitched antibodies or heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate you can connect with Avidya, and the other with Biotin. Such antibodies, for example, proposed to target immune system cells against unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be obtained using any suitable methods cross-linkage. Suitable substances for cross-linkage are well known in this field and are described in U.S. patent No. 4676980 together with several ways of cross-linkage.

Methods of obtaining bespecifically antibodies, fragments of antibodies are also described in the literature. For example, bespecifically antibodies can be obtained by using the formation of chemical bonds. In Brennan et al., Science, 229:81 (1985) described the way in which the intact antibody proteoliticeski split to obtain F(ab')2-fragments. These fragments regenerate in the presence of arsenite sodium, a substance that forms complexes with developed, in order to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide. Then, the resulting Fab'-fragments are converted into derivatives of dinitrobenzoate (TNB). Then one of the derivatives of Fab'-TNB postordertraversal in the Fab'-thiol by restoring mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB obtaining especifismo antibodies. Received bespecifically antibodies can be used as a means for the selective immobilization of enzymes.

Recent advances have simplified the direct selection ofE. colifragments, Fab'-SH, which can chemically combine with the formation of bespecifically antibodies. In Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe how to get a fully humanized molecule F(ab')2especifismo antibodies. Carried secretion fromE. colieach Fab'fragment individually and they were subjected to direct chemical bindingin vitrowith the formation of especifismo antibodies. Bespecifically antibody thus obtained, possessed the ability to bind to cells, sverkhekspressiya the HER2 receptor, and normal T-cells, and to run the lytic activity of cytotoxic lymphocytes against targets in breast cancer person.

Also describes the various methods of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, the received bespecifically antibodies using "latinovich lightning", Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The peptides "latinboy lightning" from proteins Fos and Jun were connected with the Fab'portions of two different antibodies by gene fusion. Homodimeric antibodies were restored in the hinge on the region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used to obtain homodimeric antibodies. The way dimeric antibodies described in Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), provides an alternative mechanism for obtaining bespecifically fragments of antibodies. The fragments contain the variable domain of the heavy chain (VH)connected to the variable domain of the light chain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the domains VH and VL of one fragment are forced to pair with complementary domains VL and VH of another fragment, thereby forming two antigenspecific plot. Also described another method of obtaining fragments bespecifically antibodies using dimers of single-chain Fv (sFv). See Gruber et al., J. Immunol., 152:5368 (1994).

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

Multivalent antibodies

Multivalent antibody can be faster than bivalent antibody, internalizacao (and/or metabolizirovannom) a cell expressing the antigen, which bind antibodies. Antibodies of the present invention may be a polyvalent antibodies (which are different from IgM) with t EMA antihistamine sites or more (for example, tetravalent antibodies), which can easily be obtained by recombinant expression of a nucleic acid that encodes a polypeptide chain of an antibody. Polyvalent antibodies may contain dimerization domain, and three or more antigenspecific plot. Preferred dimerization domain contains (or consists of) Fc-region or a hinge region. In this case, the antibody contains a Fc region, and three or more antigenspecific plot from N-Terminus to the Fc-region. Preferred in the present description polyvalent antibody contains (or consists of) three to about eight, but preferably four, antigenspecific plot. Polyvalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide(s) chain(s) contains(at) two or more variable domains. For example, the polypeptide(s) chain(s) may contain VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n represents 0 or 1. For example, the polypeptide(s) chain(s) may contain: chain VH-CH1-flexible linker-VH-CH1-Fc region; or chain VH-CH1-VH-CH1-Fc region. In the present the present description polyvalent antibody preferably further comprises, at least two (and preferably four) of the polypeptide of variable domains of the light chain. In the present description polyvalent antibody, for example, may contain from about two to about eight polypeptides variable domain of the light chain. Polypeptides variable domain of the light chain provided in the present description, contain variable domain light chain and optionally additionally contain the domain CL.

Variants of antibodies

In some embodiments, the implementation provided by(s) modification(s) the amino acid sequences described in this paper antibodies. For example, it may be desirable to improve the affinity of binding and/or other biological properties of the antibodies. Variants of the amino acid sequences of the antibodies get by introducing appropriate nucleotide substitutions in the nucleic acid antibodies, or by peptide synthesis. Such modifications include, for example, deletions of residues, and/or insertion of residues, and/or substitutions of residues of the amino acid sequences of the antibodies. To obtain the final design perform any combination of deletion, insertion and replacement, provided that the final construct possesses the desired properties. Amino acid modifications can be made in this amino acid follow etelnost antibodies during retrieval sequence.

Suitable method of identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning by mutagenesis"as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this paper reveal a residue or group of residues of target proteins (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine)to affect the interaction of amino acids with antigen. Those provisions of amino acids, which are the functional sensitivity to the substitutions then adjust through the introduction of additional or other options in the areas of replacement or instead of them. Thus, despite the fact that the plot for a change in the amino acid sequence is predetermined, there is no need in the immediate certainty of the nature of the mutation. For example, to analyze actions mutations in this site in codon target or area target spend alanine scanning or random mutagenesis and expressed immunoglobulins conduct screening for the presence of desired activity.

Insert amino acid sequences include intended for fusion to the N - and/or C-end of the sequence, oblad the matter of length, in the range from one residue to polypeptides containing a hundred or more residues, as well as to insert into the sequence of one or more amino acid residues. Examples of terminal insertions include an antibody to N-terminal residue of methionine or antibodies, merged with cytotoxic polypeptide. Other options insertions in the antibody molecule include the fusion to the N - or C-Terminus of an antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the half-life of antibodies in the serum.

Another type of amino acid variant antibodies alter the original nature of the glycosylation of antibodies. This change involves deleting one or more carbohydrate groups on the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.

Glycosylation of polypeptides typically represents or N-glycosylation or O-glycosylation. N-glycosylation refers to the attachment of a carbohydrate group to the side chain of an asparagine residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are sequences that are recognized for enzymatic joining of carbohydrate groups to the side chain of asparagine. That is they way the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-glycosylation refers to the attachment of one of the sugars, which represents the N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, as a rule, to serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.

The glycosylation sites you can add to the antibody by modifying the amino acid sequence such that it contains one or more of the above Tripeptide sequences (in the case of sites of N-glycosylation). Change can be accomplished by adding one or more residues of serine or threonine in the sequence of the original antibody or replacing one or more residues of serine or threonine in the sequence of the original antibody (in the case of sites of O-glycosylation).

In that case, if antibodies contain the Fc-region, you can change attached to it is a carbohydrate. For example, antibodies with a Mature carbohydrate structure that lacks fucose attached to the Fc region of the antibodies described in the patent application U.S. No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies containing separating N-acetylglucosamine (GlcNAc) in the carbohydrate, connec nom to the Fc region of antibodies, discussed in WO 2003/011878, Jean-Mairet et al., and U.S. patent No. 6602684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region of the antibodies discussed in WO 1997/30087, Patel et al. See also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) relative to antibodies with altered carbohydrate attached to an Fc region of antibodies. See also US 2005/0123546 (Umana et al.) relatively antigenspecific molecules with modified glycosylation.

In this work, the preferred variant glycosylation contains a Fc region, where the carbohydrate structure attached to the Fc region lacks fucose. Such variants have enhanced function in relation to the ADCC. Optional Fc-region further comprises one or more amino acid substitutions, which further improves ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (residue numbering Eu). Examples of publications related to "deforsirovannym" or "devoid of fucose" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines producing deoksigenirovanii antibodies include cells Lec13 CHO, have violated fokusirovanie protein (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (196); the patent application U.S. No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., in particular, in example 11), and knockout cell lines, such as knockout CHO cells by gene alpha-1,6-fucosyltransferase,FUT8(Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)).

Another type of variant is the variant with the substitution of amino acids. Such variants have at least one amino acid residue in the antibody molecule, which is replaced by another residue. The sites of greatest interest for carrying out mutagenesis with replacement, include the hypervariable sites, but also is expected to change FR. Conservative substitutions are shown in table 1 under the heading of "preferred substitutions". If the result of such substitutions is the change in biological activity, then you can make more substantial changes, indicated in table 1 as "illustrative replacement", or as described below with respect to classes of amino acids, and conduct screening products.

Significant modification of the biological properties of the antibodies is done by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of replacement, such as conformation in the form of a layer or a spiral, (b) the charge or hydrophobicity of the molecule at the site of the target, or (c) the size of the side chain. Meet the I in nature remains in groups based on the similarity of the typical properties of their side chains:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr, asn, gln;

(3) acidic: asp, glu;

(4) basic: his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Nonconservative substitutions lead to the replacement of a member of one of these classes to a member from another class.

One of the variant with the substitution includes replacement of one or more residues of the hypervariable region of the parent antibody (e.g., gumanitarnogo antibodies or human antibodies). Generally, the obtained(s) option(s)selected(s) for further development, has(have) improved biological properties relative to the parent antibody from which it(they) is obtained(s). A convenient way of obtaining such variants with substitutions includes affinity maturation using phage display. In summary, in several sections of hypervariable region (for example, in 6-7 sites) make mutations to obtain all possible amino acid substitutions at each site. The antibodies thus obtained produce the particles of filamentous phages as antibodies, merged with the product of the gene III of M13 packaged within each particle. Then conduct the screening produced in phage variants on their biological activity (e.g. binding affinity of), as the description is about in the present description. In order to identify areas hypervariable region that are candidates for modification, can be performed alanine scanning mutagenesis to identify residues of the hypervariable region, largely involved in the binding to the antigen. Alternative or in addition it may be useful to analyze the crystal structure of the complex antigen-antibody to identify potential areas of contact between the antibody and the antigen. Such forming the contact residues and neighboring residues are candidates for replacement in accordance with the present description means. Upon receipt of such options panel of variants is subjected to screening as described in the present description, and for the further development it is possible to select antibodies with improved properties through one or more suitable methods of analysis.

The nucleic acid molecules encoding amino acid sequence variants of antibodies, produced by many methods known in this field. These methods include as non-limiting examples of selection from a natural source (in the case of naturally occurring amino acid sequence) or the receipt by oligonucleotide-mediated (or site-specific) mutagenesis, mutagenesis using PCR and CAS is to maintain mutagenesis previously obtained which variant or non-variant antibodies.

It may be desirable to make one or more modifications of amino acids in the Fc region of immunoglobulin polypeptides according to the invention, thereby obtaining a variant Fc region. The variant Fc region may contain a sequence of Fc-region of a person (e.g., Fc region of IgG1, IgG2, IgG3, or IgG4 human), containing the amino acid modification (e.g. a substitution) at one or more positions of the amino acids, including the provisions of the hinge cysteine. In accordance with this description and ideas in this area suggest that in some embodiments, the implementation of the antibodies used in the methods according to the invention can contain one or more modifications compared to the same antibody wild-type, for example, in the Fc-region. These antibodies can nevertheless save essentially the same characteristics required for therapeutic use compared with their analogous to wild-type. For example, assume that in the Fc-region, you can make certain modifications that can lead to change (either improvement or deterioration) binding to C1q and/or complementability cytotoxicity (CDC), for example as described in WO 99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. patent No. 5648260; U.S. patent No. 5624821 and WO 94/29351 relative to other examples of variants of the Fc region. In WO 00/42072 (Presta) and WO 2004/05312 (Lowman) described variants of the antibodies with improved or worsened by binding to FcR. The contents of these patent publications, in particular, shown here as a reference. See also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half-life and enhanced binding to the neonatal Fc 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)), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain the Fc region with one or more substitutions that improve the binding of the Fc region with FcRn. Variants of polypeptides with altered amino acid sequences of the Fc-region and increased or reduced ability to bind to C1q described in U.S. patent No. 6194551 B1, WO 99/51642. The contents of these patent publications, in particular, shown here as a reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Derived antibodies

Antibodies of the present invention can be further modified to contain additional non-protein groups, which are known in this area and easily accessible. Preferably groups which are suitable for obtaining derivatives of antibodies, are water-soluble polymers. Non-limiting examples of water-soluble polymers include without limitation polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl with the IRT, pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, a copolymer of ethylene/maleic anhydride, polyaminoamide (or homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, copolymers of polypropyleneoxide/ethylene oxide, polyoxyethylene polyols (e.g. glycerol), polyvinyl alcohol and mixtures thereof. Polyethylenepolypropylene may have advantages in production due to its stability in water. The polymer may have any molecular weight, and may be branched or unbranched. The number attached to the antibodies of the polymers can vary, and if you attach more than one polymer, they may represent the same or different molecules. Typically, the number and/or type of polymers used to obtain the derivatives can be determined on the basis of factors that include as non-limiting examples of specific properties or functions of an antibody that need improvement, the fact will be whether to use a derived antibodies in the treatment of certain conditions, etc.

Screening for antibodies with the desired properties

Antibodies of the present invention can be characterized for their physical/chemical properties and biological f is nccem using different methods of analysis, known in this field. In some embodiments, the implementation of the antibodies are characterized for any one or more of the properties: binding to OX40L, reducing or blocking the activation OX40L, reduction or blocking of molecular signal transmission in the direction from OX40L, reducing or blocking the activation of OX40 receptor, reduction or blocking of molecular signal transmission in the direction from the OX40 receptor, reducing or blocking the binding of OX40 with OX40L and/or multimerization OX40L and/or treatment and/or prevention of immune disorders (such as asthma, allergic rhinitis, atopic dermatitis, multiple sclerosis, GVHF, or systemic lupus erythematosus), and/or treatment and/or prevention of disorders associated with expression and/or activity of OX40L (such as increased expression and/or activity of OX40L).

Purified antibodies can be further characterized in a number of analyses, including as non-limiting examples of N-terminal sequencing, amino acids analysis, exclusion liquid chromatography high pressure (HPLC) in adenocarinoma conditions, mass spectrometry, ion exchange chromatography and papain cleavage.

In specific embodiments the invention, the antibodies obtained in this work, analyze their biological activity. In some embodiments, the OS is enforced antibodies of the present invention are tested for their antigennegative activity. Methods of analysis of binding antigen, which is known in this field and which can be used in this paper include as non-limiting examples, any direct or competitive analysis of binding using methods such as Western blotting, radioimmunoassay methods of analysis, ELISA (enzyme linked immunosorbent assay), ways of "bivalent" immunoassays, methods of analysis, thus, the ways fluorescent immunoassay and immunoassay methods using protein A. Visual analysis of binding of antigen presented below in the "Examples"section.

In some embodiments implementing the invention relates to monoclonal antibodies against OX40L, which compete with antibodies 8E12 and/or antibody 13G5 for binding to OX40L. Such competitive antibodies include antibodies that recognize the epitope OX40L, which coincides with the epitope OX40L, recognizable by antibodies 8E12 and/or 13G5, or overlaps with him. Such competitive antibodies can be obtained by screening hybridoma of supernatants with antibodies against OX40L binding to the immobilized OX40L in competition with labeled antibodies 8E12 and/or 13G5. Hybridoma supernatant containing competitive antibody reduces the number of bound labeled antibodies, detected in this mixture, analyzed on to Kurennoy binding, compared to the number of bound labeled antibodies, detectable in the control mixture, analyzed binding containing extraneous antibodies (or not containing). Any of the methods described here competitive analysis link suitable for use in the above procedure.

Antibodies against OX40L according to the invention, having the properties described here, can be obtained by screening hybridoma clones secreting antibodies against OX40L, which has the required properties in any convenient way. For example, if you want monoclonal antibodies against OX40L, which block or not block binding of the receptor OX40 with OX40L antibodies, which candidates can be tested in the competitive analysis of binding, such as binding in the competitive ELISA, where the wells are covered with OX40L, and the solution of antibodies in excess in relation to the OX40 receptor layer covered with OX40L tablets, and the bound antibodies detected by enzyme reactions, for example, by contacting the linked antibodies with antibodies anti-Ig conjugated with HRP, or biotinylating antibodies anti-Ig, and analysis of color reaction due to HRP, for example, the analysis of tablets by using streptavidin-HRP and/or hydrogen peroxide, and the detection of the color reaction due to HRP, spectrophotometry at 490 nm using spectropho the meter to read the tablets with ELISA.

If you require antibodies against OX40L, which inhibit the activation of OX40L antibodies, which candidates can be tested in the analysis, which detects the signal transmission path associated with OX40L, or signal transmission path associated with OX40, such as the analysis signal NFκB. Such methods of analysis are known in this field.

Other useful methods of analysis to determine the inhibitory ability of OX40L antibodies to known in this field, some of which are shown here as examples.

In some embodiments implementing the present invention relates to altered antibodies, which have some but not all effector functions, which make them required candidates for many applications in which it is important the half-life of antibodies in vivo, however, certain effector functions (such as the binding of complement and ADCC) are unnecessary or harmful. In specific embodiments, the implementation of the measure of activity for Fc produced immunoglobulin to make sure that these are only the required properties. Analysis of cytotoxicity in vitro and/or in vivo can be performed to confirm the reduction/attenuation of activities in relation to CDC and/or ADCC. For example, it is possible to conduct analyses of the binding of Fc-receptor (FcR) to make sure that the antibodies OTS is tstuat binding to FcγR (thus, the probable lack of activity against ADCC), but retained the ability to bind to FcRn. The basic cell for the implementation of the ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Example of analysis ofin vitroto evaluate the activity of interest molecules against ADCC described in U.S. patent No. 5500362 or 5821337. Suitable for such analyses effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer (NK) cells. Alternative or additional activity of interest molecules against ADCC can be evaluatedin vivofor example, in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998). You can also carry out analyses of the binding of C1q to confirm that the antibody is not able to communicate with C1q and, thus, lost its activity against CDC. To assess activation of complement can analyze the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996). The FcRn binding and defining clearance/time-life in vivo can also be carried out using methods known in this field.

In some embodiments implementing the invention relates to altered antibodies is m, which have enhanced effector function and/or increased half-life.

The vectors, cells of the host and recombinant methods

For recombinant generate antibodies according to the invention encoding their nucleic acid is isolated and inserted into can replicate the vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibodies, easy to produce and is sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors. The choice of vector depends in part on the used host cell. Generally, the preferred cell owners have or prokaryotic, or eukaryotic (usually from mammalian) origin. Take into account that for this purpose you can use the constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and that such constant region can be obtained from either human or animal.

a. Obtaining antibodies using prokaryotic host cells:

i. Design vector

Polynucleotide sequences encoding polypeptide components of the antibodies according to the invention, it is possible to get the BL is using conventional recombinant methods. The desired polynucleotide sequence can be extracted and sequenced from producing antibodies cells, such as cell hybrid. Alternative polynucleotide can be synthesized using nucleotide synthesizer or methods based on PCR. After receiving the sequence encoding the polypeptides are inserted into the recombinant vector capable of replication and expression of heterologous polynucleotides in prokaryotic hosts. Many vectors are available and known in this field can be used for the purpose of the present invention. The selection of the appropriate vector will depend largely on the size of nucleic acids which are inserted into the vector and the particular host cell which is transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with a particular cell of the host in which it is located. Vector components generally include, as non-limiting examples: origin of replication, a marker gene for selection, the promoter, the binding site with the ribosome (RBS), signal sequence, the insertion of a heterologous nucleic acid and the sequence termination of transcription.

Generally, plasmid vectors, with the holding of replicon and control sequences, which are derived from species compatible with the host-cell, used in relation to these hosts. Vector, as a rule, is the site of replication and a marker sequence that are capable of breeding on the phenotype of transformed cells. For example,E. coliusually transformed using pBR322, a plasmid derived fromE. coli. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet) and, thus, giving an easy way of identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or they can be modified to contain, promoters which can be used in the microbial organism for expression of endogenous proteins. Examples of derivatives of pBR322, used for the expression of specific antibodies, are described in detail in Carter et al., U.S. patent No. 5648237.

In addition, phage vectors containing replicon and control sequences that are compatible with the microorganism host, can be used as transformation vectors in relation to these hosts. For example, bacteriophage such as λGEM.TM.-11, can be used to obtain a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392.

Expressing the vector according to the invention may contain two or more pairs of the promoter-cistron, each of which encodes the polypeptide components. The promoter is a noncoding regulatory sequence above (5') cistron, which modulates its expression. Prokaryotic promoters, as a rule, belong to two classes, inducible and constitutive. The inducible promoter is a promoter, which stimulates increased levels of transcription of cistron under its control, in response to changes in culture conditions, such as presence or absence of nutrients, or temperature changes.

It is well known a large number of promoters that are recognized by a variety of potential host cells. The selected promoter can functionally associate with castronno DNA encoding the light or heavy chain, highlighting the promoter from the source DNA by cleavage with restriction enzymes and inserting the selected promoter sequence in the vector according to the invention. As a natural promoter sequence and many heterologous promoters may be used to control the amplification and/or expression of target genes. In some embodiments, the implementation of the use of heterologous promoters, that is how they generally provide a higher level of transcription and higher output expressed by the target gene in comparison with the natural promoter of the target polypeptide.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the promoter system β-galactosi and lactose promoter system tryptophan (trp) and hybrid promoters such as the tac promoter or the trc. However, also other suitable promoters that are functional in bacteria (such as other known bacterial or phage promoters). Their nucleotide sequences are published, thus allowing qualified functionally ligitamate them with cisternae coding target light and heavy chains (Siebenlist et al. (1980) Cell 20:269) using linkers or adapters to provide any required restriction sites.

In one aspect of the invention, each cistron in the recombinant vector contains a component of the secretory signal sequence, which directs the movement of expressed polypeptides across the membrane. Typically, the signal sequence may be a component of the vector, or it may be part of the DNA that encodes a target polypeptide, which has been inserted into the vector. The signal sequence selected, for the anointing oil of the present invention, must be recognized by the host-cell and processionals (i.e. to split the signal peptidases) in it. For prokaryotic host cells that do not recognize and do not ProcessInput signal sequence characteristic of heterologous polypeptides, the signal sequence is substituted prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penitsillinazy, Ipp or thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In one of the embodiments of the invention the signal sequence used for both Castronovo expression systems represent the STII signal sequences or their variants.

In another aspect of the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell and, thus, does not require a secretory signal sequences in each cistron. In this case, the light and heavy chains of immunoglobulins are expressed, are minimized and going with the formation of a functional immunoglobulin in the cytoplasm. Some strains hosts (for example, strains ofE. colitrxB) provide conditions in the cytoplasm, which is favorable for the formation of a disulfide bond, thus ensuring the correct folding and the Bork expressed protein subunits, Proba and Pluckthun, Gene, 159:203 (1995).

Prokaryotic cells are the owners that are suitable for expression of the antibodies of the invention include archaebacteria and eubacteria, such as gram-negative or gram-positive organisms. Examples of suitable bacteria includeEscherichia(for example,E. coli),Bacilli(for example,B. subtilis), enterobacteria, the speciesPseudomonas(for example,P. aeruginosa),Salmonellatyphimurium,Serratiamarcescans,Klebsiella,Proteus,Shigella,Rhizobia,VitreoscillaorParacoccus. In one of the embodiments use the gram-negative cells. In one of the embodiments using cellsE. colias hosts for constructions according to the invention. Examples of strains ofE. coliinclude strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; depositing in ATCC No. 27325) and its derivatives, including strain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. patent No. 5639635). Also suitable for other strains and derivatives thereof, such asE. coli294 (ATCC 31446),E. coliB,E. coliλ 1776 (ATCC 31537), andE. coliRV308 (ATCC 31608). These examples are illustrative and not restrictive. The methods of constructing derivatives of any of the above bacteria, possessing certain genotypes, known in this field and are described, for example, Bass et al., Proteins, 8:309-314 (1990). Usually you need is playing the appropriate bacteria, taking into account replenishement replicon cells bacteria. For example, the speciesE. coli,SerratiaorSalmonellacan appropriately be used as a master, if for replication uses well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Typically, a host cell must secrete minimal amounts of proteolytic enzymes, and optionally, you can make additional protease inhibitors in cell culture.

ii. The production of antibodies

Cell owners transform the above expressing vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transformation means introducing DNA into a prokaryotic host such a way that DNA is replicated or as an extrachromosomal element, or as a chromosomal integrant. Depending on the host cell transformation carried out using conventional methods suitable for such cells. The processing of calcium using calcium chloride as a rule, used for bacterial cells, which have a significant barrier in the form of the cell wall. In another way transformationalist the glycol/DMSO. Another method is electroporation.

Prokaryotic cells used for the production of polypeptides according to the invention are grown in environments known in the field and suitable for culturing a selected host cells. Examples of suitable media include environment Luria (LB) plus the necessary nutrients. In some embodiments, the implementation of the environment also contain a substance for making selection based constructs expressing vector to provide selective growth of prokaryotic cells containing expressing vector. For example, ampicillin is added to the media for the growth of cells expressing the gene of resistance to ampicillin.

Any necessary additives, in addition to sources of carbon, nitrogen and inorganic phosphate, can also be included in relevant concentrations, applied separately or in a mixture with other additives or environment, such as a complex nitrogen source. Optional culture medium may contain one or more reducing means selected from the group consisting of glutathione, cysteine, applied, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic cells are the owners of cultivated under suitable temperatures. For the cultivation ofE. colifor example, the preferred is a temperature value varies approximately from 20°C to about 39°C, more preferably from about 25°C to about 37°C, more preferably approximately 30°C. the pH of the medium may be any pH ranging from about 5 to about 9, generally depending on the host body. ForE. colithe pH is preferably from about 6.8 to about 7.4 and more preferably approximately 7,0.

If expressing the vector according to the invention using inducible promoter, expression of the protein induce under conditions suitable for activation of the promoter. In one aspect of the invention using the PhoA promoter for regulating transcription of the polypeptide. Thus, the transformed cell hosts are cultivated in the medium for induction, containing a limited amount of phosphate. Preferably a medium containing a limited amount of phosphate is C.R.A.P (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). You can use many other inductors in the design vector, as is well known in this field.

In one embodiment, the implementation of the expressed polypeptides of the present invention are secreted into periplasm host cells, and distinguish them from it. The selection of protein typically involves the destruction of the microorganism, as a rule, takimotokan, as osmotic shock, sonication or lysis. After the destruction of the cells remains of cells or whole cells can be removed by centrifugation or filtration. Proteins can be further clear, for example, by affinity chromatography on a resin. Alternative proteins can be transferred into culture medium and to select from them. Cells can be removed from the culture, and the culture supernatant filtered and concentrated for further purification of proteins produced. Expressed polypeptides can then be isolated and identified using conventional methods such as polyacrylamide gel electrophoresis (PAGE) and analyzed using Western blotting.

In one aspect of the invention the production of antibodies is carried out in large quantities by fermentation process. Various large-scale fermentation procedure under cultivation with water available for the production of recombinant proteins. Large-scale methods of fermentation include at least a capacity of 1000 liters, preferably capacity of about 1,000 to 100,000 liters. In these fermenters use paddle stirrers for the distribution of oxygen and nutrients, especially glucose (the preferred source of carbon/energy). Small-scale fermentation means, as a rule, EN zymes is the situation in the fermenter, the volumetric capacity of which does not exceed approximately 100 liters and can vary from about 1 liter to about 100 liters.

In the fermentation process the induction of expression of the protein, typically starting after the cells grow under suitable conditions to the desired density, for example OD550approximately 180-220, at this stage the cells are in early stationary phase. You can use a variety of inductors in the design vector, as is well known in this field and described above. Cells can be grown for more than short periods before induction. Cells typically induce approximately 12-50 hours, although you can use a longer or shorter induction time.

To improve the yield and quality of the polypeptides according to the invention can be modified in different fermentation conditions. For example, to improve the proper Assembly and folding of secreted polypeptide antibodies can be used for more vectors, sverkhekspressiya protein chaperones, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptideprophet-CIS,TRANS-isomerase with chaperone activity) co-transformation of prokaryotic host cells. It is shown that protein chaperones promote the proper folding and solubility hetaeras the logical proteins, produced by bacterial cells masters, Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. patent No. 6083715; Georgiou et al., U.S. patent No. 6027888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Environ. 39:199-210.

For minimizing proteolysis of expressed heterologous proteins (in particular proteins, which are sensitive to proteolysis) can be used for the present invention, certain strains hosts with impaired proteolytic enzymes. For example, strains of host cells can be modified to obtain genetic(a) mutation(s) in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and combinations thereof. Some strains ofE. colidisturbed proteases are available and are described, for example, in Joly et al. (1998), above; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:pp.63-72 (1996).

In one embodiment, the implementation of the strains ofE. coliwith impaired proteolytic enzymes and transformed plasmids, sverkhekspressiya one or more proteins, chaperones, are used as host cells in the expression system according to the invention.

iii. Purification of antibodies

You can use the conventional methods of protein purification known in this field. Following the routes are given as examples of suitable methods for cleaning up: fractionation on immunoaffinity or ion-exchange columns, precipitation with ethanol, HPLC with reversed phase chromatography on silica or cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, precipitation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.

In one aspect, the protein A immobilized on a solid phase, for use immunoaffinity purification of full-sized products of the antibodies according to the invention. Protein a is a protein of the cell wall mass of 41 kDa fromStaphylococcusaureasthat binds with high affinity to Fc region of antibodies, Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. Solid phase on which the immobilized protein a, is preferably a column containing glass or silica surface, more preferably a glass column with a controlled pore size or column based on silicic acid. In some embodiments, the implementation of the column cover with a reagent, such as glycerol, to prevent nonspecific adsorption of impurities.

As the first stage of purification of the product obtained from the cell culture, as described above, is placed on a solid phase with immobilized protein A to ensure specific binding interest antibodies with protein A. the Solid phase is then washed to remove impurities, non-contacting solid f the th. In conclusion, interest antibodies are removed from the solid phase by elution.

b. Obtaining antibodies using eukaryotic host cells:

Components of vectors typically include as non-limiting examples of one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a sequence of termination of transcription.

(i) Component signal sequence

Vector for use in eukaryotic cells-the owner may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-end interest of the Mature protein or polypeptide. Heterologous signal sequence is chosen preferably so that it is recognized by the host-cell and processionals (i.e. useplease signal peptidases) in it. For expression in mammalian cell available signal sequence mammals, as well as viral secretory leader sequence, such as gD-signal herpes simplex virus.

DNA encoding such a leader region, are ligated in reading frame to DNA that encodes the antibody.

(ii) the origin of replication

As a rule, a component of originalaktie is not necessary for expressing vectors mammals. For example, the SV40 origin may typically be used only because it contains the early promoter.

(iii) a Component of a gene, providing a selection

Expressing and cloning vectors may contain a gene that provides the selection, also known breeding marker. Typical genes selection, encode proteins that (a) give resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complementary auxotrophic failure in appropriate circumstances, or (c) provide vital nutrients that are not available in complex environments.

In one example of the selection process used medication to stop the growth of the host cell. Those cells that are successfully transformed with a heterologous gene produce a protein which imparts resistance to the drug, and thus survive in the conditions of selection. Examples of such dominant selection includes drugs neomycin, mycophenolate acid and hygromycin.

Another example of suitable breeding markers for mammalian cells are a marker such as DHFR, thymidine kinase, metallothionein-I and-II, preferably genes metallothionein primates, adenoidectomies, unitingcare Selasa etc., which make it possible to identify cells that are competent in respect of the absorption of the nucleic acid that encodes the antibody.

For example, cells transformed by providing a selection of DHFR gene, first identified in the cultivation of all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. When using DHFR wild type corresponding to the host-cell is a cell line of Chinese hamster ovary (CHO) with impaired DHFR activity (e.g., ATCC CRL-9096).

Alternative cell owners (particularly wild-type hosts that contain endogenous DHFR)transformed or co-transformed with DNA sequences encoding the antibodies, protein DHFR wild-type and the other of breeding marker such as aminoglycoside-3'-phosphotransferase (APH)can be selected by cell growth in medium containing ensuring the selection of material for breeding marker such as aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. See U.S. patent No. 4965199.

(iv) promoter Component

Expressing and cloning vectors usually contain a promoter that is recognized by the body is the master and which is functionally linked to a nucleic acid that encodes a polypeptide antibodies. Promoter sequences known the s for eukaryotes. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases above the site of transcription initiation. Another sequence found 70 to 80 bases above from the start of transcription of many genes is an area CNCAAT, where N can be any nucleotide. At the 3'end of most eukaryotic genes is the sequence AATAAA, which may be a signal to add a poly-A tail at the 3'end of the coding sequence. All these sequences are properly inserted into eukaryotic expressing vectors.

Transcription of the polypeptide antibodies on the vectors in the cells of the host mammal control, for example, promoters derived from the genomes of viruses such as virus polyoma, rinderpest virus of birds, adenovirus (such as adenovirus 2), the virus bovine papilloma virus sarcoma birds, cytomegalovirus, a retrovirus, hepatitis B virus and the monkey virus 40 (SV40), from heterologous mammalian promoters, e.g. the actin promoter or promoter of the immunoglobulin from the promoters of heat shock, provided such promoters are compatible with the systems of the host cell.

Early and late promoters of SV40 virus are conveniently obtained as SV40 restriction fragment which also contains the viral origin of replicas and SV40. Pretani the promoter of the human cytomegalovirus is conveniently obtained as a HindIII restriction fragment E. the System for the expression of DNA in mammalian hosts using virus bovine papilloma as a vector described in U.S. patent No. 4419446. Modification of this system is described in U.S. patent No. 4601978. Alternative long terminal repeat of rous sarcoma virus can be used as a promoter.

(v) Component enhancer

Transcription of DNA encoding the polypeptide antibodies according to this invention, in higher eukaryotes is often increased by inserting enhancer sequence into the vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are known at the present time. As a rule, however, use the enhancer of virus eukaryotic cells. Examples include the SV40 enhancer on the late side from the origin of replication (100-270 KBP), enhancer of early cytomegalovirus promoter, enhancer of virus polyoma on the late side from the origin of replication and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) relative to the enhancer elements for activation of eukaryotic promoters. The enhancer can be embedded into a vector in the 5'- or 3'-position with respect to a sequence that encodes a polypeptide antibodies, but preference is sustained fashion to place on the 5'-site from the promoter.

(vi) The termination of transcription

Expressing the vectors used in eukaryotic cells-the owners are usually also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are usually located in the 5'- and sometimes in the 3'untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA that encodes the antibody. One of the suitable components termination of transcription is an area polyadenylation growth hormone bull. See WO94/11026 and described in this work expressing vector.

(vii) Selection and transformation of host cells

Appropriate cell hosts for cloning or expression of the DNA in the vectors in this work include cells of higher eukaryotes, as described in the present work, including cells of vertebrate hosts. Propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of suitable cell lines of mammalian hosts represent a line of monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL 1651); a line of embryonic human kidney (293 or 293 cells, subcloned to grow in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977); the cells of the kidneys of the newborn hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); the Sertoli cells of mice (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); kidney cells monkeys (CV1 ATCC CCL 70); kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); carcinoma cells human cervical (HELA, ATCC CCL 2); cells, dog kidney (MDCK, ATCC CCL 34); liver cells of rats Buffalo (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumor 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 human hepatoma (Hep G2).

Cell owners transform the above expressing or cloning vectors for the production of antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing host cells

Cell owners used to generate antibodies according to this invention, can be grown in many environments. Commercially available medium such as ham's F10 (Sigma), minimal medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Wednesday Needle in the modification of Dulbecco ((DMEM), Sigma) are suitable for culturing the host cells. In addition, as the e culture media for the host cells can be used in any environment, described in 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 90/03430, WO 87/00195 or U.S. patent number Re. 30985. In any of these environments can be added if necessary with hormones and/or other growth factors (such as insulin, transferrin, or a growth factor of the epidermis), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as a drug gentamicin™), trace elements (defined as inorganic compounds usually present at final concentrations of the order of the micromol), and glucose or an equivalent energy source. Any other necessary supplements may also contribute in appropriate concentrations, which are known to experts in this field. Culturing conditions, such as temperature, pH, etc. represent the conditions used for the host cell selected for expression, and visible to an ordinary specialist.

(ix) Purification of antibodies

Using recombinant methods, the antibody can be produced intracellularly or directly to secrete into the environment. If the antibody is produced intracellularly, as a first stage debris in the form of particles or cells of the owners, or lysed fragments, is removed, for example, centrifuge the management or ultrafiltration. If antibodies are secreted into the environment, supernatants from such expression systems are generally first concentrated using a commercially available filter for concentrating the protein, for example set for ultrafiltration Amicon or Millipore Pellicon. You can make a protease inhibitor such as PMSF, on any of the above stages for the inhibition of proteolysis and you can make antibiotics to prevent the growth of accidental contaminants.

The composition of the antibodies obtained from cells can be purified using, for example, chromatography with hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography, where the affinity chromatography is the preferred method of cleaning. The suitability of protein A as an affinity ligand depends on the species and isotype of any Fc-domain of an immunoglobulin, which is the antibody. Protein A can be used for purification of antibodies, which are based on the heavy chain γ1, γ2, or γ4 human (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for ø3 person (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which is attached an affine ligand, most often represents the agarose, but there are other matrices. Mechanically stable matrices such as glass with controlled pore size or poly(Stradivari)benzene, ensuring the Ute higher flow rates and shorter processing time, than those that can be achieved with agarose. When the antibody contains a CH3 domain, suitable for cleaning resin Bakerbond ABX™ (J. T. Baker, Phillipsburg, NJ). Also, depending on the antibody to be the selection that is available other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, HPLC with reversed phase chromatography on silica, chromatography on sepharose™ heparin chromatography on an anion or cation exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After any(s) advance(s) phase(s) of purification of a mixture containing an antibody and impurities, can be subjected to a hydrophobic interaction chromatography with a low pH using a buffer for elution with a pH value of approximately between 2.5 to 4.5, which is preferably performed at low salt concentrations (e.g., approximately 0-0,25 M salt).

Immunoconjugate

The invention also relates to immunoconjugates (interchangeably referred to as "the conjugates of the antibody-drug" or "ADC")that contains any described in this paper antibodies against OX40L, conjugated with a cytotoxic agent such as a chemotherapeutic agent, a drug, a remedy is ingibirovaniya growth toxin (for example, enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments), or a radioactive isotope (i.e radioconjugates).

The use of conjugates of the antibody-drug for local delivery of cytotoxic or cytostatic funds, i.e. drugs, destruction or inhibition of tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. patent No. 4975278) provides targeted delivery of the component drugs to tumors, and intracellular accumulation in them, if systemic administration of these unconjugated drug can lead to unacceptable levels of toxicity to normal cells, as well as when you wish to eliminate tumor cells (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Thus, strive to achieve maximum efficiency and minimal toxicity. It was reported that both polyclonal antibodies and monoclonal antibodies suitable for these methods (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and the wind is in (Rowland et al., (1986), above). Toxins used in the conjugates of the antibody-toxin include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins, such as geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoid (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Toxins can cause cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding or inhibition of topoisomerase. Some cytotoxic drugs have a tendency to inaktivirovanie or become less active during conjugation with major antibodies or protein ligands of receptors.

Of ZEVALIN®(ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioisotope consisting of murine monoclonal antibodies IgG1 kappa directed against the CD20 antigen presented on the surface of normal and malignant B-lymphocytes, and radioisotope111In or90Y associated timesaving chelating linker (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69). Although the ZEVALIN has the activity is against B-cell non-Hodgkin lymphoma (NHL), its introduction causes severe and prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), conjugate antibodies with drug consisting of antibodies to human CD33-related calicheamicin, received approval in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future(2000) 25(7):686; U.S. patents№№ 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen, Inc.), conjugate antibodies with drug consisting of huC242 antibody coupled via a disulfide linker SPP to the component of the medicinal product on the basis of maytansinoid, DM1, is undergoing phase II studies for the treatment of cancers in which to Express CanAg, such as colon cancer, pancreas, stomach, etc. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen, Inc.), conjugate antibodies with drug consisting of monoclonal antibodies against specific for pancreatic membrane antigen (PSMA), is associated with a component of a medicinal product on the basis of maytansinoid, DM1, is under development for the potential treatment of tumors of the pancreas. Auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), synthetic analogs of dolastatin, conjugatively with chimeric monoclonal antibodies cBR96 (specific to Lewis Y on kartz the prefectures) and cAC10 (specific to CD30 on hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21(7):778-784), and they are in therapeutic development.

Chemotherapeutic agents suitable for receiving immunoconjugates described in this paper (e.g., above). Enzymatically active toxins and fragments thereof that can be used include the A-chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin A-chain, exotoxin a (fromPseudomonasaeruginosa), A-chain of ricin A-chain abrina, A-chain medecine, alpha sarcin, proteinsAleuritesfordiiproteins of diantin, proteinsPhytolacaamericana(PAPI, PAPII, and PAP-S), inhibitor ofMomordicacharantiaCurtin, krotin, the inhibitor ofSapaonariaofficinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232, published October 28, 1993. Many radionuclides is available to receive radioconjugates antibodies. Examples include212Bi131I131In90Y and186Re. Conjugates of the antibody and cytotoxic receive funds using a variety of bifunctional substances, consolidating protein, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bi is-(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., Science, 238:1098 (1987). Labelled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is a typical chelating agent for conjugation of radionucleotide with the antibody. See WO94/11026.

Also provided in this paper, the conjugates of the antibody and one or more low molecular weight toxins such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecin and CC1065, and the derivatives of these toxins that have activity of the toxin.

i. Mitanin and maytansinoid

In some embodiments, the implementation immunoconjugate contains antibodies (full-length or fragments) of the invention conjugated to one or more molecules of maytansinoids.

Maytansinoid are inhibitors of mitosis, which act by inhibiting tubulin polymerization. First mitanin was isolated from the East African shrubMaytenus serrata(U.S. patent No. 3896111). Then it was discovered that certain microbes also produce maytansinoid, such as maytansine and the complex is e esters maytansine C-3 (U.S. patent No. 4151042). Synthetic maytansines and its derivatives and analogs are described, for example, in U.S. patents№№ 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533.

Components of the medicinal product on the basis of maytansinoids are attractive components of medicines in the conjugates of antibodies with drug due to the fact that they: (i) on available for fermentation or chemical modification, obtaining derived fermentation products, (ii) suitable for obtaining derivatives with functional groups suitable for conjugation through ridiculing of linkers with antibodies, (iii) stable in plasma, and (iv) effective against many lines of tumor cells.

Immunoconjugate containing maytansinoid, methods for their preparation and their therapeutic use are described, for example, in U.S. patent No. 5208020, 5416064 and in the European patent EP 0425235 B1, described here explicitly as a reference. In Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugate containing maytansinoid, referred to as DM1 associated with the monoclonal antibody C242 directed against colorectal cancer. Found that the conjugate is vysokorychlostnym against cultured cancer cells Tolstoy symptoms such the ICA, and he showed antitumor activity in the analysis of tumor growthin vivo. In Chari et al. Cancer Research 52:127-131 (1992) described immunoconjugate in which maytansinoid conjugatively via a disulfide linker to antibody mouse A7 binding to an antigen on the cell lines of the cancer of the large intestine of a human or other monoclonal antibody mouse TA.1 that are associated with the oncogene HER-2/neu. The cytotoxicity of the conjugate TA.1-maytansinoid tested in vitro on cell lines breast cancer human SK-BR-3, which expresses 3×105surface antigens HER-2 on the cell. Conjugate with the medicinal product has reached the degree of cytotoxicity similar to the free drug on the basis of maytansinoids, which can be increased by increasing the number of molecules maytansinoid molecule antibodies. For conjugate A7-maytansinoid on mice shows low systemic cytotoxicity.

Conjugates of the antibody-maytansinoid get chemically linking an antibody to a molecule maytansinoid without a significant reduction in biological activity or antibodies or molecules maytansinoid. See, for example, U.S. patent No. 5208020 (described here explicitly as a reference). 3-4 molecules maytansinoid conjugate molecule antibodies are effective in strengthening qi is otoxicity against target cells without negative impacts on the feature or the solubility of the antibody, although even one molecule of the toxin to the antibody, as you might expect, enhances cytotoxicity compared using unconjugated antibodies. Maytansinoid well known in this field, and they can be synthesized by known methods or to isolate from natural sources. Suitable maytansinoid described, for example, in U.S. patent No. 5208020 and in other patents and non-patent publications referenced above in this description. Preferred maytansinoid are maytansines and analogues maytansine modified aromatic ring or other provisions of molecules maytansine, such as various esters maytansine.

In this area there are many linking groups for preparation of conjugates of the antibody-maytansinoid, including, for example, the groups described in U.S. patent No. 5208020 or in the European patent 0425235 B1, Chari et al. Cancer Research 52:127-131 (1992), and patent application U.S. No. 10/960602 registered 8 October 2004, the descriptions of which are given here explicitly as a reference. Conjugates of the antibody-maytansinoid containing linker component SMCC, can be obtained as described in patent application U.S. No. 10/960602 registered 8 October 2004. The connecting group include a disulfide group, thioester group unstable to acids group, photolabile GRU is dust, resistant to peptidases group or unstable to the action of esterases group, as described in the above patents, where disulfide and thioester groups are preferred. Additional linking groups are described and shown as examples in the present description.

Conjugates of the antibody and maytansinoid can be obtained using a variety of bifunctional substances to attach proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis-(p-azidobenzoyl)hexanediamine)derived bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). Particularly preferred substances for accession include N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to ensure the formation of disulfide bonds.

The linker can be attached to the molecule maytansinoid in different positions depending on the type of link. For example, the ester bond can be formed by reaction with a hydroxyl group using conventional methods of joining. The reaction may proceed in position C-3 hydroxyl group in position C-14, modified with hydroxymethyl, C-15, modified with hydroxyl group in position C-20 hydroxyl group. In a preferred embodiment, the bond is formed at the position C-3 maytansine or similar maytansine.

ii. Auristatin and dolastatin

In some embodiments, the implementation immunoconjugate contains antibodies of the invention conjugated with dolastatin or peptide analogues and derivatives of dolastatin, auristatin (U.S. patent No. 5635483; 5780588). It is shown that dolastatin and auristatin violate the dynamics of microtubules, the hydrolysis of GTP and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anti-cancer (US 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). A component of a medicinal product on the basis of dolastatin or auristatin can be attached to the antibody via N-Terminus (amino) or C-Terminus (carboxyl) component peptide drugs (WO 02/088172).

Illustrative embodiments of using auristatin include components of the drug with the estva based on the associated N-end of monomethylaniline DE and DF, described in "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Ser. No. 10/983340 registered 5 November 2004, the disclosure of which is explicitly given as a reference in full.

Typically, the components of the medicinal product on the basis of peptides can be obtained by formation of a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, according to the method of liquid-phase synthesis (see E. Schröder and K. Lübke, "The Peptides", volume 1, pp. 76-136, 1965, Academic Press)that is well known in the field of peptide chemistry. Components of the medicinal product on the basis of auristatin/dolastatin can be obtained according to methods: US 5635483; US 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat. Biotechnol. 21(7):778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Ser. No. 10/983340 registered 5 November 2004, are hereby incorporated by reference in full (description, for example, linkers and methods of obtaining monomethylamine compounds, such as MMAE and MMAF conjugated to linkers).

iii. Calicheamicin

In other embodiments, implementation immunoconjugate contains antibodies of the invention conjugated to one or more molecules calicheamicin. Antibiotics family calicheamicin way the s to create double-stranded DNA breaks in subpicomolar concentrations. To obtain conjugates family calicheamicin see U.S. patent 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (all issued by American Cyanamid Company). Structural analogues calicheamicin that can be used include as non-limiting examples γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θI1(Hinman et al. Cancer Research 53:3336-3342 (1993), Lode et al. Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents issued to American Cyanamid). Another anticancer drug, which can be konjugierte antibody is QFA, which is antifolate. As calicheamicin and QFA have intracellular action, and they are not easily pass through the plasma membrane. Thus, the cell capture these substances is mediated by antibody internalization significantly enhances their cytotoxic effects.

iv. Other cytotoxic tools

Other antitumor agents that can be konjugierte with antibodies according to the invention include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of funds, known under the collective name of complex LL-E33288 described in U.S. patent No. 5053394, 5770710 and espiramicina (U.S. patent No. 5877296).

Enzymatically active toxins and fragments thereof that can be used include A-chain d is farinosa toxin, nesviazana active fragments of diphtheria toxin A-chain, exotoxin a (fromPseudomonasaeruginosa), A-chain of ricin A-chain abrina, A-chain medecine, alpha sarcin, proteinsAleuritesfordiiproteins of diantin, proteinsPhytolacaamericana(PAPI, PAPII, and PAP-S), inhibitor ofMomordicacharantiaCurtin, krotin, the inhibitor ofSapaonariaofficinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232, published October 28, 1993.

The present invention also relates to immunoconjugate formed by the antibody and the connection with nucleotidase activity (e.g., a ribonuclease or a DNA-endonuclease, such as desoksiribonukleaza; Ncasa).

For the selective destruction of the tumor antibodies can contain highly radioactive atom. To obtain radioactive conjugated antibodies are available various radioactive isotopes. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu. In the case of the use of the conjugate for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99mor I123or a spin label for image acquisition of nuclear magnetic resonance (NMR) (also known as magnetic resonance imaging, MRI), such ka is iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels can be embedded in the conjugate by known methods. For example, the peptide can biologically synthesize or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors containing, for example, fluorine-19 instead of hydrogen. Labels such as tc99mor I123That Re186That Re188and In111you can join through the cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The way IODOPHENOL (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used for injection of iodine-123. In "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,CRC Press 1989) described other ways.

Conjugates of the antibody and cytotoxic tools can be accessed using a variety of bifunctional substances to attach a protein, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 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-dia is nebenzahl)Ethylenediamine), the diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, the immunotoxin ricin can be obtained as described in Vitetta et al. Science 238:1098 (1987). Labelled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is an illustrative chelating agent for conjugation of the radionuclide to the antibody. See WO94/11026. The linker can be a "biodegradable linker"facilitating release of the cytotoxic drug in the cell. For example, you can use unstable to acids linker sensitive peptidase linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52:127-131 (1992); U.S. patent No. 5208020).

Compounds according to the invention explicitly include as non-limiting examples of the ADC obtained using cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate)which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog.

v. Obtaining conjugates of antibodies with drug

In the conjugates of antibodies with drugs (ADC) from which retenu antibody (Ab) conjugated with one or more components of a drug (D), for example, from about 1 to about 20 components of the drug to the antibody through a linker (L). The ADC of formula I can be obtained in several ways, including reactions of organic chemistry, conditions, and reagents known to specialists in this area, including: (1) the interaction of a nucleophilic group of an antibody with a bivalent linker reagent with the formation of Ab-L, via a covalent bond with the subsequent interaction with a component of a medicinal product D; and (2) the interaction between the nucleophilic group of a component of a medicinal product with a bivalent linker reagent with the formation of D-L, via a covalent bond with the subsequent interaction with the nucleophilic group of an antibody. Additional methods of obtaining the ADC described in this work.

Ab-(L-D)pI

The linker may consist of one or more components of the linker. Typical components of linkers include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe), p-aminobenzeneboronic ("PAB"), N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC") and N-Succinimidyl-(4-iodates)aminobenzoate ("fairs are forthcoming-Siab"). More the positive components of linkers known in this area and some are described in this paper. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Ser. No. 10/983340 registered 5 November 2004, the contents of which are hereby incorporated by reference in full.

In some embodiments, the implementation of the linker may contain amino acid residues. Typical amino acid components of the linker include dipeptide, Tripeptide, tetrapeptide or Pentapeptide. Typical dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Typical tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues that are part of the amino acid linker component, include the remains that exist in nature, as well as minor amino acids not found in nature similar amino acids such as citrulline. The amino acid components of the linker can be designed and optimized in their selectivity with respect to enzymatic cleavage by specific enzymes, for example associated with tumors with protease, cathepsin B, C and D or the protease plasmin.

Nucleophilic groups on the antibodies include as non-limiting examples: (i) N-terminal amino group, (ii) amino side chain, such as lysine, (iii) thiol group on the side chain, for example cysteine, and (iv) a hydroxyl group or amino sugars, if antibodies glycosyl owani. Amino, thiol and hydroxyl groups are nucleophiles and can interact with the formation of covalent bonds with electrophilic groups on the components of the linker, where the linker reagents include: (i) active esters such as NHS esters, esters, HOBt, trechaleidae derivatives and acid halides; (ii) alkyl and benzylchloride, such as haloacetic; (iii) aldehyde, ketone, carboxyl and maleimide group. Some antibodies have recovered megamachine disulphide, i.e. cysteine bridges. Antibodies can be obtained in active form for conjugation with linker reagents processing regenerating reagent such as DTT (dithiothreitol). Each cysteine bridge thus formed, theoretically, of the two active thiol of nucleophiles. Additional nucleophilic groups can be introduced in antibodies with the cooperation of lysine with a 2-aminothiophenol (reagent trout), which leads to the transformation of the amine in the thiol. Active thiol group can be introduced in the antibodies (or fragments thereof) by introducing one, two, three, four or more cysteine residues (e.g., receiving mutant antibodies containing one or more non-natural amino acid cysteine residues).

Conjugates of antibodies with drugs on izobreteny which can also be obtained by modification of antibodies by introducing an electrophilic group, which can react with nucleophilic substituents on the linker reagent or drug. Sugar glycosylated antibodies can oxidize, for example, oxidizing reagents based periodate with the formation of aldehyde or ketone groups that can react with the amino group of the linker reagents or components of the drug. The resulting groups iminovogo Schiff bases can form a stable bond, or they can be recovered, for example, using borohydride of reagents to form stable amine linkages. In one embodiment, the implementation of the interaction of the carbohydrate portion of a glycosylated antibody with either galactosialidosis or metaperiodate sodium can lead to the formation of carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal residues of serine or threonine can interact with metaperiodate sodium, leading to the formation of aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; US 5362852). This aldehyde can react with a component of a medicinal product or a nucleophile linker.

Similarly, the nucleophilic groups on the component l of the drug funds include as non-limiting examples include an amino group, thiol, hydroxyl, hydrazide, Aksinya, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines group capable of reacting with the formation of covalent bonds with electrophilic groups on the components of the linker, where the linker reagents include: (i) active esters such as NHS esters, esters, HOBt, trechaleidae derivatives and acid halides; (ii) alkyl and benzylchloride, such as haloacetic; (iii) aldehyde, ketone, carboxyl and maleinimide group.

Alternative protein containing the antibody and cytotoxic agent, can be obtained, for example, by recombinant methods or peptide synthesis. DNA may contain the corresponding region encoding the two parts of the conjugate, or adjacent to each other, or separated by a region that encodes a linker peptide which does not violate the required properties of the conjugate.

In another embodiment, antibodies can be konjugierte with the "receptor" (such as streptavidin) for utilization in the pre - "the transformation of the tumor in the target, where the conjugate of the antibody-receptor is administered to the patient, followed by removal of unbound conjugate from the blood flow with the use of tools that allow for clearance, and then the introduction of a "ligand" (e.g., avidin), which anywhereman with citato the classical means (for example, radioactively labeled nucleotide).

The pharmaceutical composition

Therapeutic compositions containing the antibodies according to the invention, receive for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000)), in the form of aqueous solutions, lyophilized or dehydrated compositions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers such as phosphate, citrate, his-tag and on the basis of other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chloride hexadecane; benzalkonium chloride, chloride benzathine; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (approximately less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; moosacher the water, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating 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 TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In this paper, the composition may also contain more than one active connection, if required by the specific indication for treatment, preferably an active connection to additional activities that do not have negative effects on each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

The active ingredients can also be incorporated into the microcapsules obtained, for example, ways koatservatsii or by interfacial polymerization, for example, microcapsules based hydroxymethylcellulose or gelatin and microcapsules on the basis of polymethylmethacrylate, respectively, in colloidal systems for delivery of a drug (for example, liposomes, albumen the microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in Remington: The Science and Practice of Pharmacy 20th edition (2000).

Compositions intended is built for use with the introduction of in vivomust be sterile. It is easy to reach by filtration through sterile filtration membranes.

You can get drugs with a slow release. Examples of suitable drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the immunoglobulin according to the invention, where the matrices are in the form with a certain form of particles, e.g. films, or microcapsules. Examples of matrices for sustained release include polyesters, hydrogels (for example, poly-2-hydroxyethylmethacrylate or polyvinyl alcohol), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem the ethylene vinyl acetate, degradiruete copolymers of lactic acid and glycolic acid, such as the LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid and glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxybutiric acid. While polymers such as based on vinyl acetate and lactic acid-glycolic acid capable of releasing molecules within 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they mod the ut denaturing or aggregate under the action of humidity at 37°C, which leads to the loss of biological activity and possible changes in immunogenicity. It is possible to develop rational strategies for stabilization depending on the mechanism involved. For example, if you discovered that the mechanism of aggregation is the formation of intermolecular S-S linkages through the conversion of thiol to disulfide, stabilization can be achieved by modifying sulfhydryl residues, lyophilisation from acidic solutions, controlling the water content, using appropriate additives, and developing specific composition based on a polymer matrix.

Application

Antibodies of the present invention can be applied, for example, in therapeutic methods in vitro, ex vivo and in vivo.

In one aspect the invention relates to methods for treating or preventing an immune disorder, where the methods include the introduction of an effective amount of OX40L antibodies to a subject in need of such treatment. In some embodiments, the implementation of the disorder is an immune disorder. In some embodiments, the implementation of the disorder is an autoimmune disorder. In some embodiments, the implementation of the disorder is a asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, multiple sclerosis, GHD and/or systemic lupus erythematosus. In some embodiments, the implementation of the disorder is a disease caused by a virus, bacteria or other infectious agent. See US 2005/0069548 A1.

Moreover, at least some of the antibodies according to the invention can be contacted with the antigen from other species. Thus, antibodies of the invention can be applied to bind with specific antigenic activity, for example, in the culture of cells containing the antigen in the body or in the body of other mammals, containing the antigen with which the antibody of the invention cross-reacts (e.g. chimpanzee, baboon, igranka,Cynomolgusand rhesus, pig or mouse). In one of the embodiments of the antibodies according to the invention can be used for inhibiting the activity of antigen by contacting the antibody with the antigen thus, what happens in the inhibition of the activity of the antigen. Preferably the antigen is a protein molecule man.

In one of the embodiments of the antibodies according to the invention can be used in the method of binding an antigen in a subject suffering from a disorder associated with increased expression and/or activity of antigen, where the method comprises the administration to a subject the antibody according to the invention in such a way that is binding to the antigen in the subject. Preferably the Academy of Sciences of the Egan is a protein molecule man and the subject is human. Alternative a subject can be a mammal expressing an antigen bound to antibodies according to the invention. In addition, the subject may be a mammal, which introduced the antigen (for example, the introduction of the antigen or by transgenic expression of the antigen). Antibodies according to the invention it is possible to enter a person for therapeutic purposes. Moreover, the antibodies according to the invention it is possible to enter non-human mammal expressing an antigen to which cross-reacts with immunoglobulin (e.g., a Primate, pig or mouse) for veterinary purposes or using it as an animal model for human disease. Regarding the above, these model animals can be useful to assess therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and duration of the course introduction).

Antibodies according to the invention can be used for the treatment, inhibition, pause the progression of, prevent/stop relapse, facilitate or prevent diseases, disorders or conditions associated with expression and/or activity of one or more molecules of the antigen.

In specific embodiments, the implementation immunoconjugate containing antibodies, konyagi is consistent with one or more cytotoxic(their) agent(s), enter the patient. In some embodiments, the implementation immunoconjugate and/or antigen to which it binds, internalizes(are) cell, resulting in increased therapeutic efficacy immunoconjugate in the destruction of target cells with which it is associated. In one embodiment, the implementation of the cytotoxic agent targeted to a nucleic acid target cells or prevents it from functioning. In one embodiment, the implementation of the cytotoxic agent focused on the polymerization of microtubules or hinder. Examples of such cytotoxic agents include any chemotherapeutic agents mentioned in this description (such as maytansinoid, auristatin, dolastatin or calicheamicin), a radioactive isotope, or a ribonuclease or a DNA endonuclease.

Antibodies according to the invention can be used for treatment or as monotherapy or in combination with other compositions. For example, antibodies according to the invention it is possible to enter with other antibodies, steroids (such as steroids for inhalation, systemic or cutaneous injection), chemotherapeutic(s) agent(s) (including cocktails of chemotherapeutic agents), other(mi) cytotoxic(s) agent(s), antiangiogenic(s) agent(s), cytokines, and/or agent(s) of growth inhibition. Such by mentioning is made higher than the combined regimens include the introduction of joint (where two or more funds are part of the same or different compositions) and a separate introduction, when the antibodies according to the invention can be implemented before additional therapy or more types of therapy and/or after it. Effective amounts of therapeutic agents, administered in combination with antibodies anti-EphB4 are at the discretion of the physician or veterinarian. The dose and the adjustment is performed with the aim of achieving the best tactics in the treated States. In addition, the dosage depends on factors such as the type of therapeutic agent and the specific treatment to be patient. In specific embodiments, the implementation of a combination of inhibitors potentiates the efficiency of a single inhibitor. The term "potentiation" refers to improving the effectiveness of a therapeutic agent in its generally accepted or approved dosage.

The antibody according to the invention (and auxiliary therapeutic agent) is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intra-lungs and intranasal, and, if required for local treatment, intranidus introduction. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, antibodies appropriately injected pulse infusion, particularly with declining doses of the antibody. Introduction d the C can be accomplished in any suitable way, for example injections, such as intravenous or subcutaneous injection, partly depending on whether the introduction of short-term or chronic.

The composition of the antibodies according to the invention are dosed and administered in accordance with good medical practice. Factors for consideration in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery agent, the method of introduction, the scheme of administration and other factors known to practitioners. Antibodies do not necessarily vary in composition with one or more tools, currently used for the prevention or treatment considered disorders. The effective amount of such other funds depends on the number of antibodies according to the invention is present in the composition, the type of disorder or treatment, and other factors discussed above. They usually use in the same doses and routes of administration, which is used earlier in this description, or from about 1 to 99% from the previously used dosages.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used in monotherapy or in combination with other agents) head of the sieves of the type subject to treatment, the type of antibody, the severity and course of the disease, from being injected whether antibodies for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. Antibodies are suitably administered to the patient at one time or during the course of treatment. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of antibody is an initial possible dosage for administration to the patient or, for example, in the form of one or more separate injections or a continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. With repeated introductions for several days or more, depending on the condition, treatment continues until, until the desired attenuation of the symptoms of the disease. One exemplary dosage of the antibody is in the range from about 0.05 mg/kg to about 10 mg/kg Thus, the patient can enter a single or multiple doses of approximately 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof). Such doses can be entered periodically, for example every week or every three weeks (e.g. such that the patient receives approximately otdyh to about twenty, for example, about six doses of the antibody). You can enter a higher initial loading dose, followed by one or more lower doses. An exemplary dosing regimen comprises the introduction of an initial loading dose of approximately 4 mg/kg followed by a supporting dose for weeks at a level of approximately 2 mg/kg of antibody. However, you can use other dosing regimens. The progress of this treatment is easy to monitor by conventional methods and analysis.

Antibodies against OX40L according to the invention is suitable for analyses that detects the expression of OX40L (such as diagnostic or prognostic assays) in specific cells or tissues, where antibodies are tagging, as described below, and/or immobilized on an insoluble matrix.

In another aspect the invention relates to methods of detection, OX40L, where the methods include the detection of complex OX40L-OX40L antibody to in the sample. The term "detection"as used in this description, includes qualitative and/or quantitative detection (measurement levels) compared with the control or without it.

In another aspect the invention relates to methods of diagnosis of a disorder associated with expression and/or activity of OX40L, where the methods include the detection of complex OX40L-OX40L antibody to" in a biological sample from the human patient, having a disorder or suspected of possession. In some embodiments, the implementation of the expression of OX40L is an increased expression or abnormal (junk) expression.

In another aspect the invention relates to any antibodies against OX40L, described in the present work, where antibodies against OX40L contain detektiruya tag.

In another aspect the invention relates to a complex of any of the described in this paper antibodies to OX40L and OX40L. In some embodiments, the implementation of complex exists in vivo or in vitro. In some embodiments, the implementation of the complex contains a tumor cell. In some embodiments, the implementation of antibodies against OX40L been labelled with a detectable label.

Antibodies against OX40L can be used for the detection of OX40L in any of a number of well known ways of detecting analysis. For example, the biological sample can be analyzed in respect of OX40L by obtaining the sample from a desired source, mixing the sample with the antibodies against OX40L, to allow the antibody to form a complex of the antibody/OX40L with any OX40L present in the mixture, and detecting any complex antibody/OX40L, is present in the mixture. The biological sample can be obtained for analysis known in this field in ways that are fit for a particular sample. The methods of mixing the sample with antibodies and with the person detection antibody complex/OX40L choose according to the type of analysis. Such analyses include immunohistochemistry, competitive and dyadic analyses and analyses of steric inhibition.

All analytical methods for OX40L use one or more of the following reagents: labeled analogue OX40L, immobilized similar OX40L, labeled antibodies against OX40L, immobilized antibodies against OX40L and steric conjugates. Labeled reagents also known as "indicators".

Used label is any detektiruya activity which does not interfere with the binding of OX40L antibodies anti-OX40L. Numerous labels are known for use in immunoassay, where examples include components that can be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as components, such as enzymes, that must react, or you must obtain their derivatives for detection. Examples of such labels include the radioisotopes32P,14C,125I3H and131I, fluorophore, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as Firefly luciferase and bacterial luciferase (U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saridakis is, for example, glucose oxidase, galactosidase, and glucose-6-phosphatedehydrogenase, heterocyclic oxidases such as uricase and the xanthine oxidase-related enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microbiocides, Biotin/avidin, spin labels, the labels on the basis of bacteriophages, stable free radicals, etc.

For covalent binding of these labels to proteins or polypeptides of the available conventional methods. For example, for labeling antibodies of the above-described fluorescent, chemiluminescent, and enzyme labels, you can use the consolidating substances such as dialdehyde, carbodiimide, timelinelite, bis-imidate, bis-diazotized benzidine, and the like, See, for example, U.S. patent No. 3940475 (fluorometry) and 3645090 (enzymes); Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Methods, 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982). In the present work, the preferred labels include enzymes, such as horseradish peroxidase and alkaline phosphatase. The conjugation of such marks, including enzymes, antibodies is a common method of handling for an ordinary specialist in the immunoassay methods. See, for example, O'sullivan et al., "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. JJ. Langone and H. Van Vunais, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166.

For certain methods of analysis necessary immobilization of the reagents. Immobilization leads to the separation of antibodies anti-OX40L from any OX40L, which remains free in solution. Immobilization conventional method is carried out or transfer to an insoluble form antibodies against OX40L or similar OX40L before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. 3720760), covalent binding (e.g., using cross-linking with glutaraldehyde) or transfer into an insoluble form antibodies against OX40L or similar OX40L subsequently, for example, immunoprecipitate.

The expression of proteins in the sample can be investigated using immunohistochemistry and staining. It is shown that immunohistochemical staining of tissue sections is a reliable way to measure or detect the presence of proteins in the sample. In the methods of immunohistochemistry ("IHC") uses antibodies to bind antigens in cells and their visualization in situ, usually by using chromogenic or fluorescent methods. For a sample you can use a sample of tissue or cells from a mammal (typically, the individual who is the man). The sample can be obtained in a variety of ways known in this area, including as the e non-limiting examples of surgical resection, aspiration or biopsy. The tissue may be fresh or frozen. In one of the embodiments the sample is fixed and pour in Paraty or other tissue Sample may be fixed (i.e. save) the conventional ways. An ordinary person skilled in the art will take into account that the choice of a fixative is determined proceeding from the purpose for which conduct histological staining or other analysis of the sample. An ordinary person skilled in the art will also take into account that the duration of fixation depends on the size of the tissue sample and used the lock.

IHC can be performed in combination with additional means, such as morphological staining and/or fluorescent in situ hybridization. There are two main ways IHC: direct and indirect analyses. According to the first type of analysis of antibody binding to a target antigen (for example, OX40L) is determined directly. This direct analysis using labeled reagent, such as a fluorescent label or enzyme labeled primary antibodies, which can be visualized without the use of additional interaction of antibodies. In a typical indirect analysis of unconjugated primary antibodies bind to the antigen, and then labeled secondary antibody binds to primary antibody. If the secondary antibody is conjugated to a farm is nteu label, the chromogenic or fluorogenic substrate added to provide visualization of the antigen. The signal amplification is due to the fact that more secondary antibodies can interact with different epitopes on the primary antibody.

Primary and/or secondary antibodies used for immunohistochemistry, as a rule, mark detectable by the group. Available numerous labels, which can generally be grouped into the following categories:

Regardless of the methods of obtaining the sample, discussed above, may require additional processing of the tissue section prior to IHC, during or after it. For example, you can implement the methods of translation of the epitope to the desired state, such as heating the tissue sample in citrate buffer (see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996)).

After an optional stage driving slice of tissue is exposed to primary antibody for a sufficient period of time and under suitable conditions so that the primary antibody binds to a protein target antigen in a tissue sample. The appropriate conditions to achieve this, you can determine when conducting conventional experiments. The degree of binding of the antibody with the sample determined using any of the detectable labels discussed above. Preferably the label, not only is em an enzyme label (e.g., HRPO), which catalyzes a chemical alteration of the chromogenic substrate, such as the Chromogen 3,3'-diaminobenzidine. Preferably the enzyme label kongugiruut with antibodies that specifically bind with the primary antibodies (for example, primary antibodies are rabbit polyclonal antibodies and secondary antibodies represent a goat anti-rabbit antibody).

The thus obtained samples can be prepared for microscopic examination and cover with cover glass. Assessment of microscopic preparation is then carried out, for example, using a microscope, and you can use the criteria in the intensity of staining, which is used in common practice.

Other methods of analysis, known as competitive or bivalent types of analysis are common, and they are widely used in the commercial production of diagnostic kits.

Types of competitive analysis is based on the ability of a labeled analogue of OX40L to compete with the test OX40L in the sample for a limited amount of antigenspecific sites on the antibodies against OX40L. Antibodies against OX40L, usually translated into an insoluble form before conducting competitive analysis, or after him, and then label and OX40L associated with antibodies against OX40L, is separated from unbound label and OX40L. This Department is done by the keys decanting, where participants linking previously been converted into an insoluble form) or by centrifugation (where the participant binding precipitiously after the competitive reaction). The number of test sample OX40L inversely proportional to the amount of bound label, which is measured by the amount of marker substance. Get curves from dose to known amounts of OX40L and compared with the test results to quantitatively determine the amount of OX40L, present in the tested sample. These methods of analysis are called systems ELISA, if, as detectable markers using enzymes.

In another form of competitive analysis, called "homogeneous" analysis does not require the separation of the phases. It receives the enzyme conjugate with OX40L and used in such a way that, when antibodies against OX40L contact OX40L, antibodies against OX40L modifies the enzyme activity. In this case, OX40L or its immunologically active fragments kongugiruut by a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates selectrow for use with antibodies against OX40L so that antibody binding to OX40L inhibits or stimulates the enzymatic activity of the label. This method, as such, is widely used called EMIT.

Steric conjugates using sposobah on the basis of steric hindrance in a homogeneous assay. These conjugates are synthesized by covalent binding of low-molecular-weight hapten to a small fragment of OX40L so that antibody to hapten is essentially not able to contact the conjugate simultaneously with antibodies against OX40L. In this way analysis OX40L, present in the tested sample, binds to the antibodies against OX40L, thus allowing the antibody to the hapten contact conjugate, which leads to changes in the characteristics of conjugated hapten, such as a change in fluorescence when the hapten is a fluorophore.

Types of divalent analysis, in particular suitable for determining OX40L or antibodies to OX40L. In the sequential views of divalent analysis of immobilized antibodies against OX40L use to bind to OX40L in the test sample, the test sample is removed by washing, the bound OX40L use to associate with the second labeled antibodies against OX40L, and bound peroxidase molecules are then separated from unbound label. The amount of bound label is directly proportional to the number of OX40L in the tested sample. In "simultaneous" kinds of divalent analysis of the test sample is not separated before adding the labeled antibody to OX40L. Sequential dyadic analysis using monoclonal antibodies against OX40L as some antibodies and polyclonal antibodies against OX40L in cachestorage suitable for testing samples against OX40L.

The above is provided only as examples of assays for the detection of OX40L. Other developed now or in the future, the ways in which use antibodies against OX40L to determine OX40L, are included in the scope of the present invention, including the described species of bioanalysis.

Product

In another embodiment, the invention relates to a product containing substances suitable for the treatment, prevention and/or diagnosis of the above disorders. The product includes a container and a label or an insert in the packaging, combined with the container. Suitable containers include, for example, bottles, vials, syringes, etc. of the Container can be formed from various materials, such as glass or plastic. The container contains a composition which by itself or when combined with other(them) composition(s) is effective for the treatment, prevention and/or diagnosis of the condition and may have a hole for sterile access (for example, the container may be a package for intravenous solution or vial having a stopper, permeable to hypodermic needles). At least one active substance in the composition is an antibody according to the invention. On the label or an insert in the packaging indicates that the composition is used to treat specific is Oceania, such as asthma. Moreover, the product may contain (a) a first container containing a composition, where the composition comprises the antibody according to the invention; and (b) a second container containing a composition. The product in this embodiment of the invention may further comprise an insert in the packaging, which indicated that the first and second compositions with antibodies can be used to treat a specific condition, such as asthma. Alternative or additionally, the product may contain a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-saline buffer, ringer's solution and dextrose. In addition, it may contain other necessary commercial and consumer point of view the materials, including other buffers, diluents, filters, needles and syringes.

Examples of methods and compositions according to the invention. It should be understood that it is possible to apply various other ways of implementation based on the above General description.

EXAMPLES

Example 1: Receiving murine monoclonal antibodies 8E12 and 13G5 against OX40L

Antibodies to the extracellular sequence of human OX40L was obtained as follows. Five Balb/c mice (Charles River Laboratories, Wilmington, DE) was hyperimmunoglobulin recombi is based by OX40L polypeptide-flag man (the extracellular domain of OX40 ligand person, fused with flag peptide and expressed by CHO cells, Genentech, Inc., South San Francisco, CA) in Freund Ribi (Ribi Immunochem Research, Inc., Hamilton, MO). Carried out the merger of B-cells from mice with high titers of antibodies against OX40L, with myeloma cells of mice (pu1.Anan.22.4, Genentech, Inc., South San Francisco, CA). After a few days supernatant collected and subjected to screening for the production of antibodies by direct enzyme-linked immunosorbent assay (ELISA). IgG from media to grow positive clones were purified by affinity chromatography with protein A (fast protein liquid chromatography, Pharmacia (Pharmacia, Uppsala, Sweden)). Purified antibody preparations were sterilized by filtration (pore size 0.2 μm; Nalgene, Rochester NY) and stored at 4°C in phosphate buffer saline (PBS).

Analyses based on flow cytofluorimetry carried out in media for the cultivation of positive ELISA of clones for screening for monoclonal antibodies that compete with recombinant OX40R-Fc (receptor OX40 person, fused with Fc fragment of IgG1expressed by CHO cells, Genentech, Inc.) for binding to human OX40L expressed on CHO cells. Cells CHO-OX40L pre-incubated with OX40R-Fc followed by the addition of media to grow. After incubation, the cells were washed in phosphate-buffered saline and then stained artemisinin IgG, conjugated with FIC what erythrina, for detection of the number corresponding mouse monoclonal antibodies to human OX40L. Defined secondary index fluorescence (MFI). Monoclonal antibodies (Mab), designated as 8E12 and 13G5, had a good performance in these tests.

Total RNA was extracted from cells hybridomas producing antibodies 8E12 and 13G5, using conventional methods. Variable domain light chain (VL) variable domain and the heavy chain (VH) amplified using RT-PCR with degenerate primers for the heavy and light chains, as indicated below. Direct primers had a specificity to N-terminal amino acid sequence of the VL region and a VH. The reverse primers for LC and HC designed so that they were annealed in order to a region in the constant domain of the light chain (CL) and constant domain 1 heavy chain (CH1), respectively, which are highly conserved in different species. Amplificatoare region VL and VH was cloned in expressing vectors mammals and polynucleotide sequence of the inserts was determined using conventional sequencing methods. Amino acid sequences of VL and VH 8E12 shown in Fig. 1. Amino acid sequences of VL and VH 13G5 shown in Fig. 3.

Example 2: ELISA Analysis of binding of Mab 8E12 and 13G5 with OX40L protein

Competitive ELISA was performed, as is shown next, to study the binding of Mab 8E12 and 13G5 to OX40L with OX40L in solution and to study the binding expressed on the cell surface OX40L. In some experiments, 96-well microwell tablets maxiSorp (Nunc, Roskilde, Denmark) were coated overnight at 4°C with antibodies anti-FlagM2 at the rate of 2 µg/ml (Sigma) in 50 mm carbonate buffer, pH 9,6. The tablets were washed in PBS containing 0.05% Polysorbate-20, and scored with 0.5% bovine serum albumin, 10 hours/million Proclin-300 (Supelco, Bellefonte, PA) in PBS at room temperature for 1 hour. The tablets were washed and added to 0.5 μg/ml of OX40L polypeptide ECD-Flag person. After incubation for one hour tablets washed. Was added a mixture of OX40-IgG-bio of a person and serial dilutions of antibodies in PBS containing 0.5% bovine serum albumin, 0.05% Polysorbate-20, 10 hours/Proclin 300 million. The plates were incubated for 2 h at room temperature and washed. Associated OX40-IgG-bio of a person were detected by adding streptavidin-HRP (Amdex, Copenhagen, Denmark) followed by the addition of 3,3',5,5'-tetramethylbenzidine (Kirkegaard &Perry Laboratories, Gaithersburg, MD) as substrate. Tablets left for the development of staining and the reaction was stopped by adding 1 M phosphoric acid. The absorption was measured at 450 nm on a plate spectrophotometer Titertek (ICN, Costa Mesa, CA). Titration curves for built using a curve approximation four the parametric regression (KaleidaGraph, software Synergy, Reading, PA). Mab 8E12 and 13G5 to OX40L linked human OX40L approximately EC50 of 1.4 nm and 1.1 nm, respectively. In some experiments, the analysis was performed in the same way as above, except that the tablets for ELISA were covered with 0.5 μg/ml OX40-IgG man, a mixture of OX40-Flag man and serial dilutions of antibody were incubated on tablets and associated OX40-Flag person were detected using antibodies anti-Flag-bio with the subsequent addition of streptavidin-HRP. Mab 8E12 and 13G5 to OX40L linked human OX40L approximately EC50 of 1.9 nm and 0.46 nm, respectively.

In other experiments, CHO cells expressing full-OX40L man, raised in an environment of 50:50 F12/DMEM with addition of 2 mm L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (medium with additives) and 5% FBS (Gibco BRL Life Technologies, Gaithersburg, MD) (culture medium) in a humid incubator with 5% CO2at 37°C. the Cells were separated from the culture of tablets using Accutase (ICN) and added to 96-well microwell tablets based 300,000 cells/well. A mixture of OX40-IgG person and serial dilutions of the antibodies in the culture medium was added to the tablets. After incubation for one hour on ice, the cells were washed by centrifugation tablets and removal of supernatant. Associated OX40-human IgG were detected using an anti-Fc-HRP. Certain Also 20 and 130 nm for 8E12 and 13G5, respectively. In some experiments OX40-IgG-bio of a man used instead of OX40-IgG person. Associated OX40-IgG-bio of a person were detected using streptavidin-HRP. The development of the color reaction in the tablets, and the measurement was carried out as described above. Mab 8E12 and 13G5 been associated with human OX40L approximately EC50 of about 11 nm and 60 nm, respectively.

These analyses showed that Mab 8E12 and 13G5 contact with human OX40L in solution and with human OX40L expressed on CHO cells.

To determine contact whether Mab 8E12 and 13G5 with OX40L mouse, tablets for ELISA covered OX40L mouse. Added serial dilution 8E12 or 13G5 (0,004-4 µg/ml). For detection used goat antimachine Fc-HRP. Observed no binding. In another embodiment, the tablets for ELISA covered 8E12 or 13G5. Added serial dilution OX40L-Flag mouse. For detection used biotinylated anti-Flag followed by the addition of streptavidin-HRP. Observed no binding.

Example 3: Analysis of Mab 8E12 and 13G5 using surface plasmon resonance

The kinetics of binding of mouse antibodies to OX40L with human OX40L was measured using surface plasmon resonance using a Pharmacia BIAcore®3000 (BIAcore AB, Uppsala, Sweden) at room temperature (Karlsson et al., 1994; Morton & Myszka, 1998). Antibodies anti-Flag (anti-FlagM2, Sigma, St. Louis, MO) was immobilized on the sensor chip (CM) via primary amine groups. The high flow rate used to minimize the effects of mass transfer. Carboxymethylamino surface matrix of sensor chip was activated by injection of 20 μl of a mixture of 0.025 M N-hydroxysuccinimide and 0.1 M N-ethyl-N'-(dimethylaminopropyl)carbodiimide at 5 ál/min 7 µl of 50 µg/ml of anti-Flag in 10 mm sodium acetate, pH 4.5, was injected with 5 μl/min two times. After joining the unoccupied sites on the chip were blocked by injection of 20 μl of 1 M ethanolamine, pH 8.5. Work buffer consisted of PBS containing 0.05% Polysorbate-20. 5 μl of 4 μg/ml OX40L-Flag were injected with 30 μl/min for binding OX40-Flag with anti-Flag on the chip. For kinetics measurements twofold serial dilutions (6,2-50 nm) mouse antibodies to human OX40 8E12 (PUR 9333) and 13G5 (PUR 9306) in working buffer were injected with in the flow cell for 2 minutes at a flow rate of 30 µl/min and gave the opportunity related antibodies against OX40L to dissociate for 20 minutes. Surface binding was regenerated by injection of 30 μl of 10 mm glycine-HCl (pH of 1.5). Flow cell, which contains immobilized antibodies, anti-Flag, but it did not add OX40L-Flag was used as a control cuvette. Data were analyzed using model binding 1:1 using a global approximation. The rate constants of Association and dissociation were approximatively simultaneously (software is BIAevaluation).

The apparent rate constants of Association was 2.6×106and 4.8×105(1/Ms) for IgG antibodies 8E12 and 13G5, respectively. Due to the very slow kinetics of the rate of dissociation rate constants of dissociation was impossible to determine (<5×106/s). The calculated apparent values of Kdwas <0,019 nm and <0,010 nm for IgG antibodies 8E12 and 13G5, respectively.

Example 4: Antibodies against OX40L 8E12 and 13G5 recognize different determinants of binding OX40L

Experiments on the binding of Scatchard was carried out to determine, arbitrate whether mAb to OX40L 8E12 and 13G5 with each other for binding with similar or overlapping epitopes on human OX40L expressed on CHO cells, and to determine the affinity of binding of antibodies. 50 μg of mouse monoclonal antibodies against OX40L 8E12 and 13G5 were subjected to jodirovannuju using the conventional way with lactoperoxidase. Antibodies 8E12 and 13G5 serially diluted 1:4 in buffer media (F12:DMEM, Dulbecco, 1% bovine serum albumin, 0.05% of sodium azide and 25 mm Hepes buffer, pH 7,2), starting from an initial concentration of 200 nm. Carried out two sets of serial dilutions (50 μl) in two Parallels in 96-microwell tablet. From each set of dilutions to the wells contributed 50 ál of iodized or 8E12, or 13G5 (final concentration of 0.25 nm). The contents of the tablet was stirred and then the total smesiteli in 96-well plate for tissue culture, containing 30000 OX40L-expressing CHO cells per well, which were sown on the tablet in the previous day. The tablets were covered and incubated overnight at 4°C. the next day the liquid from the wells (containing cells) was aspirated and the wells washed twice with buffer for environments. 200 μl of 1N NaOH was added to each well for lizirovania cells were shaken for 10 minutes and then transferred to 96-hole tripod for microtubes. To each tube was counted number of pulses in scintillation gamma counter Wallac. The number of pulses corresponding concentrations of antibodies were made by the analysis program of Scatchard New Ligand for application to graph number of related antibodies relative ratio related/free and to determine the affinity of binding and concentration of the receptor. For competition experiments, the data were collected and put on a graph using Kaleidagraph. Curves were drawn using 4-parametric approximation.

Mab 8E12 been associated with human OX40L with Kdto 0.92 nm. Mab 13G5 been associated with human OX40L with Kd1,09 nm. 8E12 and 13G5 not competed with each other for binding to OX40L expressed on CHO cells, indicating that monoclonal antibodies against OX40L 8E12 and 13G5 recognize different determinants of binding OX40L on human rights.

Example 5: OX40L Antibodies to inhibit the interaction the interaction human OX40L and OX40 person

Experiments on competitive binding was carried out to determine, arbitrate whether mAb to OX40L 8E12 and 13G5 with iodinated OX40-Fc (extracellular domain of OX40 receptor, fused with the Fc domain) for binding to OX40L expressed on CHO cells. 50 μg of ligand OX40L-Fc was subjected to jodirovannuju using the conventional way with lactoperoxidase. Antibodies 8E12 and 13G5 serially diluted 1:4 in buffer media (F12:DMEM, Dulbecco, 1% bovine serum albumin, 0.05% of sodium azide and 25 mm Hepes buffer, pH 7,2), starting from an initial concentration of 200 nm. Dilutions (50 μl) was carried out in two Parallels in 96-microwell tablet. To each well was brought to 50 μl of iodized OX40L-Fc (final concentration of 0.25 nm)were mixed and then the total mixture was transferred into a 96-well plate for tissue culture containing 30000 OX40L-expressing CHO cells per well, which were sown on the tablet in the previous day. The tablets were covered and incubated overnight at 4°C. the next day the liquid from the wells (containing cells) was aspirated and the wells washed twice with buffer for environments. 200 μl of 1N NaOH was added to each well for lizirovania cells were shaken for 10 minutes and then transferred to 96-hole tripod for microtubes. To each tube was counted number of pulses in scintillation gamma counter Wallac. Data were collected and put on gra the IR using Kaleidagraph. Curves were drawn using 4-parametric approximation.

The results of this experiment are presented in Fig. 3. Mab 8E12 (light triangles) and 13G5 (dark squares) competed with the OX40 receptor for binding to OX40L expressed on CHO cells, indicating that the antibodies inhibit the binding of OX40 receptor human with human OX40L.

Example 6: Treatment with antibodies against OX40L inhibits the proliferation of T-cells in cell analysis

Analyses of the reaction of mixed lymphocytes (MLR) was performed to determine inhibits the proliferation of T-cells by blocking the interaction of OX40L-OX40 by mab to OX40L. In this cell the experiment analyze the ability of OX40L antibodies to inhibit OX40L mediated signal transmission. In summary, plasmacytoid dendritic cells (pDC) were isolated from human blood using beads BDCA-4 MACS according to the manufacturer's instructions (Miltenyi Biotec). The cells were placed at a rate of 1×106cells/ml in medium containing RPMI/10% fetal bovine serum/2 mm L-glutamine, and stimulated for 3 days by 2.5 µg/ml of soluble protein CD40L person (R & D Systems) and 10 ng/ml protein IL-3 (R & D Systems) to activate pDC. Activated pDC then washed three times with phosphate-buffered saline/3% fetal bovine serum, and then together Kul who was rivervale with "naive" T cells CD45RA+, which were isolated from human blood using MACS beads according to the manufacturer's instructions (Miltenyi Biotec). Joint culture were sown at the rate of pDC:T-cells 1:200, at a rate of 1×105T-cells per well, in 96-well plate with a U-shaped bottom for 1 week in the presence of the indicated reagents. Was carried out by pulse labeling cells by 1 µci methyltriclosan thymidine per well for the last 6 hours of cultivation, collected on the filter plates and counted the number of pulses.

The results of the two productions of experiments, designated as "dnrl and dnr2"shown in Fig. 4. Processing each of the mab to OX40L 8E12 (light squares) and mab to OX40L 13G5 (light circles) significantly inhibited the proliferation of T-cells, indicating that both antibodies inhibit OX40L mediated signal transmission. At the same time, the control mouse IgG1 (crosses) are not inhibited the proliferation of T-cells. Control levels of proliferation of T-cells showing bright diamonds, and control levels of proliferation of T-cells, mixed with activated cells, shown as dark squares. The value of IC50 for inhibition of proliferation of T-cells by mab to OX40L varied from donor to donor in the range from 0.5 to 10 nm.

Example 7: Treatment with antibodies against OX40L inhibits the generation of IL-2 T-cell memory

Expressed otcu IL-2 T-cell memory was analyzed to determine inhibits whether blocking the interaction of OX40L-OX40 by mab to OX40L the production of IL-2. In this experiment tested the ability of OX40L antibodies to inhibit OX40L mediated signal transmission. In brief, T cells memory CD45RO+ were isolated from human blood using MACS beads according to the manufacturer's instructions (Miltenyi Biotec). The cells were placed at a rate of 1×106cells/ml in medium containing RPMI/10% fetal bovine serum/2 mm L-glutamine, and stimulated for 16 hours by 1 μg/ml of antibodies anti-CD3 (BD Pharmingen) and 10 nm soluble protein OX40L person (R & D Systems). Antibodies against OX40L or control mIgG1 antibody was added in the indicated concentrations. The levels of IL-2 in supernatant was measured using ELISA kit according to manufacturer's instructions (R & D Systems).

The results of the two productions of experiments, designated as "dnr5" and "dnr6"shown in Fig. 5. Processing each of the mab 8E12 (light squares) and mab 13G5 (light circles) significantly inhibited the production of IL-2 T-cell memory. At the same time adding a control mouse IgG1 (crosses) are not inhibited the production of IL-2. The production levels of IL-2 by T-cells of memory when processing only antibodies anti-CD3 shown by dashed squares in the processing of antibodies anti-CD3 and human OX40L shown as dark squares, or T-cell memory without processing p is cauldrons bright diamonds.

Example 8: Treatment with antibodies against OX40L inhibits the survival of activated T-cells

Survival of activated T-cells were analyzed to determine inhibits whether blocking the interaction of OX40L-OX40 by mab to OX40L survival. In this cell the experiment analyze the ability of OX40L antibodies to inhibit OX40L mediated signal transmission. In summary, plasmacytoid dendritic cells (pDC) were isolated from human blood using beads BDCA-4 MACS according to the manufacturer's instructions (Miltenyi Biotec). The cells were placed at a rate of 1×106cells/ml in medium containing RPMI/10% fetal bovine serum/2 mm L-glutamine, and stimulated for 3 days by 2.5 µg/ml of soluble protein CD40L person (R & D Systems) and 10 ng/ml protein IL-3 (R & D Systems). Activated pDC CD123+BDCA2 is then selected using FACS and were co-cultured with activated T-cells. T cells were isolated by negative selection from human blood using MACS beads according to the manufacturer's instructions (Miltenyi Biotec) and activated within 3 days by 0.1 µg/ml anti-CD3/1 μg/ml anti-CD28 (BD Pharmingen). Joint culture were sown in the ratio pDC:T-cells 1:50 on the basis of 1×106T-cells per ml in 6-well plate in the presence of the indicated reagents (concentrations of 1 nm or 10 nm). Kolichestvennye analyzed over time with the exception Trypanosoma blue.

The results of the experiments shown in Fig. 6. Processing by each mab to OX40L 8E12 (light squares) and mab to OX40L 13G5 (light circles) significantly inhibited the survival of activated T-cells. At the same time adding a control mouse IgG1 ("mIgG1"; crosses) are not inhibited survival of activated T-cells. Control untreated T cells ("restimulative") shows bright diamonds, and control T cells with activated pDC ("activated pDC") is shown as dark squares.

Although the above invention is described in detail by bringing the illustrations and examples for purposes of clear understanding, the description and examples should not be interpreted as limiting the scope of invention.

1. The selected antibody to OX40L, containing:
(a) a light chain containing (i) HVR-L1 containing the sequence RSSQSIVHGNGNTYLE (SEQ ID NO:1) or RSSQSPVHSNGNTYLH (SEQ ID NO:2); (ii) HVR-L2, containing the sequence RVSNRFS (SEQ ID NO:3) or KVSNRFS (SEQ ID NO:4); and (iii) HVR-L3, containing the sequence FQGSHVPYT (SEQ ID NO:5) or SQSTHIPWT (SEQ ID NO:6); and
(b) a heavy chain containing (i) HVR-H1 containing the sequence SYWLN (SEQ ID NO:7) or SYWMH (SEQ ID NO:8); (ii) HVR-H2 containing the sequence MIDPSDSETHYNQVFKD (SEQ ID NO:9) or EIDPSNGRTNYNEKFKS (SEQ ID NO:10); and (iii) HVR-H3 containing the sequence GRGNFYGGSHAMEY (SEQ ID NO:11) or ERSPRYFDV (SEQ ID NO:12),
where OX40L antibody to bind to OX40L people the century.

2. Antibody to OX40L according to claim 1, where the antibody contains:
(a) a light chain containing (i) HVR-L1 containing the sequence RSSQSIVHGNGNTYLE (SEQ ID NO:1); (ii) HVR-L2, containing the sequence RVSNRFS (SEQ ID NO:3); and (iii) HVR-L3, containing the sequence FQGSHVPYT (SEQ ID NO:5); and
(b) a heavy chain containing (i) HVR-H1 containing the sequence SYWLN (SEQ ID NO:7); (ii) HVR-H2 containing the sequence MIDPSDSETHYNQVFKD (SEQ ID NO:9); and (iii) HVR-H3 containing the sequence GRGNFYGGSHAMEY (SEQ ID NO:11).

3. Antibody to OX40L according to claim 1, where the antibody contains:
(a) a light chain containing (i) HVR-L1 containing the sequence RSSQSPVHSNGNTYLH (SEQ ID NO:2); (ii) HVR-L2, which contains a sequence of KVSNRFS (SEQ ID NO:4); and (iii) HVR-L3, containing the sequence SQSTHIPWT (SEQ ID NO:6); and
(b) a heavy chain containing (i) HVR-H1 containing the sequence SYWMH (SEQ ID NO:8); (ii) HVR-H2 containing the sequence EIDPSNGRTNYNEKFKS (SEQ ID NO:10); and (iii) HVR-H3 containing the sequence ERSPRYFDV (SEQ ID NO:12).

4. Antibody to OX40L according to claim 1, where the antibody contains a variable domain light chain having the sequence:

5. Antibody to OX40L according to claim 1, where the antibody contains a variable domain of a heavy chain having the sequence:

6. Antibody to OX40L according to claim 1, where the antibody contains:
(a) a variable domain of light chain having the sequence:
br/> and
(b) a variable domain of a heavy chain having the sequence:

7. Antibody to OX40L according to claim 1, where the antibody contains a variable domain light chain having the sequence:

8. Antibody to OX40L according to claim 1, where the antibody contains a variable domain of a heavy chain having the sequence:

9. Antibody to OX40L according to claim 1, where the antibody contains:
(a) a variable domain of light chain having the sequence:

and
(b) a variable domain of a heavy chain having the sequence:

10. The antibody according to any one of claims 1 to 3, 6 and 9, where the antibody is a monoclonal antibody.

11. The antibody according to any one of claims 1 to 3, 6 and 9, where the antibody is selected from the group consisting of chimeric antibodies, gumanitarnogo antibodies, affinity-Mature antibodies, human antibodies and especifismo antibodies.

12. The antibody according to any one of claims 1 to 3, 6 and 9, where the antibody is an antibody fragment.

13. Polynucleotide encoding the antibody according to any one of claims 1 to 3, 6 and 9.

14. The expression vector containing polynucleotide on item 13.

15. A host cell that can Express the exogenous nucleic acid that contains the vector by 14.

16. A host cell according to item 15, where CL is TKA-host is prokaryotic.

17. A host cell according to item 15, where a host cell is eukaryotic.

18. A host cell according to item 15, where a host cell is a cell of a mammal.

19. A method of producing antibodies against OX40L, and the method includes culturing the host cell containing nucleic acid encoding the antibody according to any one of claims 1 to 3, 6 and 9, so that the nucleic acid is expressed.

20. The method according to claim 19, where a host cell is prokaryotic.

21. The method according to claim 19, where a host cell is eukaryotic.

22. Method for the treatment or prevention of an immune disorder, the method includes introducing an effective amount of antibodies against OX40L according to any one of claims 1 to 3, 6 and 9 to a subject in need of such treatment.

23. The method according to item 22, where the immune disorder is an autoimmune disorder.

24. The method according to item 22, where the immune disorder is an asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, the disease is graft versus host disease, multiple sclerosis or systemic lupus erythematosus.

25. The method according to paragraph 24, where the immune disorder is an asthma, atopic dermatitis or allergic rhinitis.

26. The method according to claim 19, further comprising the selection of antibodies from the culture of the host cell.

27. The method according to p, where the antibody is recovered from the Reda cultivation of the host cell.

28. The method according to p, additionally comprising combining the selected antibody with a pharmaceutically acceptable carrier or excipients for preparation of pharmaceutical composition containing the antibody.



 

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39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.

EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.

29 cl, 15 dwg, 6 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: binding molecule represents a CD45RO and CD45RB chimeric antibody. The molecule contains two domains with consistent hypervariable sites CDR1, CDR2 and CDR3, and CDR1', CDR2' and CDR3', CDR1 has amino acid sequence NYIIH, CDR2 has amino acid sequence YFNPYNHGTKYNEKFKG, and CDR3 has amino acid sequence SGPYAWFDT. CDR1' has amino acid sequence RASQNIGTSIQ, CDR2' has amino acid sequence SSSESIS, and CDR3' has amino acid sequence QQSNTWPFT. Related coding polynucleotide is described.

EFFECT: use of the invention allows to induce immunosuppression, to inhibit T-cell response and primary lymphocyte reaction in the mixed culture, to prolong survival time in mice with severe combined immunodeficiency SCID.

6 cl, 5 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of an angiopoietin-2 (Ang-2) specific antibody and a pharmaceutical antibody composition for treatment of various diseases associated with angiopoietin-2 overexpression. Also there are described methods of inhibition, modulation and treatment of various diseases mediated by angiopoietin-2 activity. There are offered: coding nucleic acid, an expression vector and a vector-transformed cell, as well as a method for producing antibodies.

EFFECT: use of the invention ensures new high-cytotoxicity antibodies (according to ELISA analysis IC50=0,35 nM) comparable with a common antibody Ab536 that further can find application in medicine.

22 cl, 2 dwg, 11 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is claimed isolated human antibody or its fragment, which binds to human EGFR. Antibody contains corresponding CDR areas of light and heavy chain. Its conjugate with anti-neoplastic means or marker is described. Also described are: coding nucleic acid, expression vector, recombinant cell-host for obtaining antibodies and method of inhibiting growth of tumor, expressing EGFR on the basis of antibody.

EFFECT: application of invention provides antibodies with affinity comparable or higher, than in IMC-C225, which neutralises EGFR activation, what can be applied in medicine for treatment of tumours.

36 cl, 14 dwg, 6 tbl, 13 ex

FIELD: chemistry.

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

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

131 cl, 44 dwg, 12 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: what is described is new NRP1 antibodies including a variable region of a light chain containing CDRL1 which includes amino acid sequence RASQSISSYLA (SEQ ID NO:123), CDRL2 which includes amino acid sequence GASSRAS (SEQ ID NO:124) and CDRL3 which includes QQYMSVPIT (SEQ ID N0:125), and a variable region of a heavy chain containing CDRH1 which includes amino acid sequence GFSFSSEPIS (SEQ ID NO:126), CDRH2 which includes amino acid sequence SSITGKNGYTYYADSVKG (SEQ ID NO:127); and CDRH3 which includes amino acid sequence WGKKVYGMDV (SEQ ID NO:128). Also, application of the antibodies in research, diagnostic and therapeutic purposes is described.

EFFECT: invention allows extending the range of product for controlling the disorders associated with pathological angiogenesis.

35 cl, 11 dwg, 5 tbl, 3 ex

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