ES2348087T3 - USE OF ANTI-CD40 ANTI-BODIES FOR THE TREATMENT OF INFLAMMATORY AND AUTOIMMUNE DISEASES AND ORGAN TRANSPLANT REJECTION. - Google Patents

Abstract

An anti-CD40 human monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a human cell expressing CD40, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, for use in a method for treating a human subject of an inflammatory disease or an autoimmune disease, the method comprising administering to said subject an effective amount of the human anti-CD40 monoclonal antibody, said human anti-CD40 monoclonal antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the dep hybridoma cell line ossified at the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

Description

FIELD OF THE INVENTION

The invention relates to methods for treating autoimmune and inflammatory diseases using anti-CD40 monoclonal antibodies.

BACKGROUND OF THE INVENTION

CD40 is a 55 kDa cell surface antigen present on the surface of normal B cells and human neoplastic B cells, dendritic cells, other antigen presenting cells (APC), endothelial cells, monolithic cells, CD8 + T cells and epithelial cells . The CD40 antigen is also expressed in activated T cells, activated platelets, inflamed vascular smooth muscle cells, eosinophils, synovial membranes in rheumatoid arthritis, dermal fibroblasts and other types of non-lymphoid cells. Depending on the type of cell that expresses CD40, the ligation can induce intercellular adhesion, differentiation, activation and proliferation. For example, the binding of CD40 to its cognate ligand, CD40L (also referred to as CD154), stimulates B cell proliferation and differentiation in plasma cells, antibody production, isotype change and memory generation in B cells. During cell differentiation B, CD40 is expressed in pre-B cells but is lost in differentiation in plasma cells.

The CD40 ligand has been identified on the surface of activated B cells (Fenslow et al. (1992) J. Immunol. 149: 655; Lane et al. (1992) Eur. J. Immunol. 22: 2573; Noelle et al. . (1992) Proc. Natl. Acad. Sci. USA 89: 6550), but is not generally expressed in resting human T cells. DC40L is a type I transmembrane glycoprotein with homology to TNF-α (Armitage et al. (1992) Nature 357: 80 and Spriggs et al. (1992) J. Exp. Med. 176: 1543). The extracellular domain of CD40L contains two arginine residues proximal to the transmembrane region, providing a potential proteolytic cleavage site that causes a soluble form of the ligand (sCD40L). Overexpression of CD40L causes autoimmune diseases similar to systemic lupus erythematosus in rodent models (Higuchi et al. (2002) J. Immunol. 168: 9-12). In contrast, the absence of functional CD40L in activated T cells causes X-linked hyper-IgM syndrome (Allen et al. (1993) Science 259: 990; and Korthauer et al. (1993) Nature 361: 539). Additionally, blocking the CD40 / CD40L interaction can prevent transplant rejection in nonhuman primate models.

See, for example, Wee et al. (1992) Transplantation 53: 501-7.

The expression of CD40 in APC plays an important costimulatory role in the activation of these cells. For example, agonist anti-CD40 monoclonal antibodies (mAbs) have been shown to mimic the effects of helper T cells in activating B cells. When present in adherent cells expressing FcγRII, these antibodies induce B cell proliferation (Banchereau et al. (1989) Science 251: 70). In addition, agonist anti-CD40 monoclonal antibodies can replace the T-signal helper for the secretion of IgM, IgG and IgE in the presence of IL-4 (Gascan et al. (1991) J. Immunol. 147: 8). In addition, agonist anti-CD40 mAbs can prevent programmed cell death (apoptosis) in B cells isolated from lymph nodes.

These and other observations support the current theory that the interaction of CD40 and CD40L plays a fundamental role in regulating both humoral immune responses and cell-mediated immune responses. More recent studies have revealed a much broader role of CD40 / CD40L interaction in various physiological and pathological procedures.

The signal transduction path of CD40 depends on the coordinated regulation of many intracellular factors. Like other membranes in the TNF receptor family, CD40 reacts with TRAF proteins (proteins associated with TNF receptor factors) such as TRAF2 and TRAF3, which mediate an intracellular signal after binding of CD40 with CD40L (either solid-phase CD40L or well soluble CD40L). TRAFs translude a signal into the nucleus by means of map kinases such as NIK (NK-κB inducing kinase) and Ikappa B kinases (IKK α / β), definitely activating the transcription factor NK-κB (Young et al. ( 1998) Immunol. Today 19: 502-06). Signaling via Ras and the MEK / ERK route has been demonstrated in a subset of B cells. Additional routes involved in CDF40 cell signaling include PI3k / Akt pathway or P30 MAPK pathway (Craxton et al. (1998) J. Immunol .5: 439-447). Signaling via CD40 has been shown to prevent cell death from apoptosis (Makus et al. (2002) J. Immunol. 14: 973-982). Apoptotic signals are necessary to induce programmed cell death in a coordinated manner. Cell death signals may include intrinsic stimuli from inside the cell such as endoplasmic reticulum stress or external stimuli such as FasL receptor binding or TNFα. The signaling pathway is complex, involving activation of caspases such as caspases 3 and 9 and poly (ADP-ribose) polymerase (PARP). During the cascade, anti-apoptotic signaling proteins, such as Mcl-1 and BCLx, and members of the IAP family prtoteins, such as X-linked Inhibitor of Apoptosis (XIAP), are regulated downwards (Budihardjo and col. (1999) Annu. Rev. Cell Dev. Biol. 15: 269-90). For example, in dendritic cells, CD40 cell signaling can block apoptosis signals transduced by FasL (Bjorck et al. (1997) Int’l Immunol. 9: 365-372).

Accordingly, binding of CD40 by CD40L and subsequent activation of CD40 signaling are necessary steps for normal immune responses; however, deregulation of CD40 signaling can lead to disease. The CD40 signaling pathway has been showing to be involved in autoimmune disease (Ichikawa et al. (2002) J. Immunol. 169: 2781-7 and Moore et al. (2002) J. Autoimmun. 19: 139-45). Additionally, the CD40 / CD40L interaction plays an important role in inflammatory procedures. For example, both CD40 and CD40 ligand are overexpressed in human atherosclerosis and experimental atherosclerosis lesions. CD40 stimulation induces expression of matrix degrading enzymes and induces tissue factor expression in cell types associated with atheroma, such as endothelial cells, smooth muscle cells and macrophages. Additionally, CD40 stimulation induces production of proinflammatory cytokines such as IL-1, IL-6 and IL-8, and adhesion molecules such as ICAM-1, Eselectin and VCAM. The inhibition of the interaction of CD40 / CD40L prevents atherosclerosis in animal models. In transplant models, blocking the CD40 / CD40L interaction prevents inflammation. It has been shown that the CD40 / CD40L binding acts synergistically with the Alzheimer's beta-amyloid peptide to promote microglial activation, thus leading to neurotoxicity.

In patients with rheumatoid arthritis (RA), CD40 expression is increased in articular chondrocytes, therefore, CD40 signaling probably contributes to the production of harmful cytokines and matrix metalloproteinases. See, Gotoh et al. (2004) J. Rheumatol. 31: 1506-12. Additionally, this has shown that the amplification of the synovial inflammatory response takes place by activation of mitogen-activated protein kinases (MAP) and the kappaB nucleus factor (NFκB) by means of ligation of CD40 into CD14 + synovial cells of RA patients. (Harigai et al. (2004) Arthritis. Rheum., 50: 2167-77). In an experimental model of RA, anti-CD40L antibody treatment prevented disease induction, inflammation of the joints, and production of anti-collagen antibodies (Durie et al. (1993) Science 261: 1328). Finally, in clinical trials, it has been shown that depleting CD20 + B positive cells from RA patients by administering RITUXAN® (usually indicated by B cell lymphoma) improves symptoms (Shaw et al. (2003) Ann. Deum. Dis. 62: ii55-ii59).

It has also been shown that blocking CD40 / CD40L interactions during antigen presentation to T cells induces T cell tolerance. Therefore, blocking CD40 / CD40L interaction prevents initial T cell activation as well as induces long term tolerance for re-exposure. to the antigen.

WO 01/83755, WO 02/28480 and WO 02/28904 describe antibodies capable of binding to CD40, methods for producing these antibodies and methods for their use.

Ellmark et al. (Immunology 2002, 106, 456-463) give a report on the modulation of the CD40-CD40 ligand interaction using human single chain anti-CD40 antibody fragments.

BRIEF SUMMARY OF THE INVENTION

The invention provides a human monoclonal antibody that is capable of specifically binding to the human CD40 antigen expressed on the surface of cells expressing human CD40, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell. , for use in a method for treating a human subject of an inflammatory disease or an autoimmune disease, the method comprising administering to said subject an effective amount of the human anti-CD40 monoclonal antibody, said human anti-CD40 monoclonal antibody being selected from the group Constituted by:

a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

The invention also provides the use of an effective amount of a human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40 in the preparation of a medicament for treating a human subject for an inflammatory disease or autoimmune disease, said monoclonal antibody being free of significant antagonistic activity when bound to the CD40 antigen expressed on the surface of said cell, said human anti-CD40 monoclonal antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

The invention provides a human monoclonal antibody that is capable of specifically binding to the human CD40 antigen expressed on the surface of cells expressing human CD40, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell. , for use in a method for treating a human transplant rejection subject, the method comprising administering to said subject an effective amount of the human anti-CD40 monoclonal antibody, said human anti-CD40 monoclonal antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

The invention also provides the use of an effective amount of a human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40 in the preparation of a medicament for treating a human transplant rejection subject, said monoclonal antibody being free of significant antagonistic activity when bound to the CD40 antigen expressed on the surface of said cell, said human antiCD40 monoclonal antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can be obtained from the deposited hybridoma cell line in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

The invention also provides a human monoclonal antibody that is capable of specifically binding to the human CD40 antigen expressed on the surface of cells expressing human CD40, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, for use in a method for treating a human subject of rheumatoid arthritis, the method comprising administering to said subject an effective amount of the human anti-CD40 monoclonal antibody, said human anti-CD40 monoclonal antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

The invention also provides the use of an effective amount of a human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40 in the preparation of a medicament for treating a human subject of rheumatoid arthritis, said monoclonal antibody being free of significant antagonistic activity when bound to the CD40 antigen expressed on the surface of said cell, said human anti-CD40 monoclonal antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

Other aspects of the invention are presented in the appended claims.

BRIEF SUMMARY OF THE REVELATION

Methods for treating a human being with an autoimmune disease and / or an inflammatory disease are disclosed, the subject comprising administering an anti-CD40 antibody or an antigen-binding fragment thereof that is free of significant agonist activity where it binds. to a CD40 antigen in a human cell that expresses CD40. Methods for inhibiting the response of cells expressing CD40 antigen are also disclosed. Anti-CD40 antagonist antibodies suitable for use in the present invention have a strong affinity for CD40. These monoclonal antibodies and antigen binding fragments thereof are capable of specific binding to human CD40 antigen expressed on the surface of a human cell. They are free of significant agonist activity but exhibit antagonistic activity when they bind to the CD40 antigen in human cells. In one embodiment, the anti-CD40 antibody or fragment thereof exhibits antagonistic activity when they bind to the CD40 antigen in antigen presenting cells such as B cells.

Antagonist antibodies are especially useful in preventing, improving or treating diseases that comprise an autoimmune and / or inflammatory component. These diseases include but are not limited to autoimmune diseases such as systemic lupus erythematosus (SLE), discoid lupus, lupus nephritis, sarcoidosis, inflammatory arthritis, including, but not limited to, juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome. , ankylosing spondylitis and gouty arthritis, rejection of an organ or tissue transplant, hyperacute, acute or chronic rejection and / or graft versus host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory disease of the intestine, Crohn's disease, celiac disease (gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Graves disease, hasimotoid thyroiditis, complex disease immune, chronic fatigue syndrome and immune dysfunction (CFS DI), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris, interstitial pulmonary fibrosis, sarcoidosis, diabetes mellitus Type I and Type II, delayed type hypersensitivity of type 1, 2, 3 and 4, allergy or allergic disorders , unwanted / unwanted immune responses to therapeutic proteins, asthma, Churg-Strauss syndrome (allergic granulomatosis), atopic dermatitis, allergic and irritating contact dermatitis, urticaria, IgE-mediated allergy, atherosclerosis, vaculitis, idiomatic inflammatory myopathies, hemolytic disease , Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the amino acid sequences for the light and heavy chains of the CHIR-12.12 mAb. The leading regions (residues 1-20 of SEQ ID NO: 2), variable (residues 21-132 of SEQ ID NO: 2), and constant (residues 133-239 of SEQ ID NO: 2) of the light chain are shown in Figure 1A. The leading regions (residues 1-19 of SEQ ID NO: 4), variable (residues 20-139 of SEQ ID NO: 4), and constant (residues 140-469 of SEQ ID NO: 4) of the heavy chain are shown in Figure 1B. The alternative constant region for the CHIR-12.12 mAb heavy chain shown in Figure 1B reflects a substitution of an alanine residue for a serine residue at position 153 of SEQ ID NO: 4. The complete sequence for this heavy chain variant of the CHIR-12.12 mAb is presented in SEQ ID NO: 5. Figure 2 shows the coding sequence for the light chain (Figure 2A; SEQ ID NO: 1) and the heavy chain (Figure 2B; SEQ ID NO: 3) for the CHIR-12.12 mAb. Figure 3 shows the amino acid sequences for the light and heavy chains of the CHIR-5.9 mAb. The leading regions (residues 1-20 of SEQ ID NO: 6), variable (residues 21-132 of SEQ ID NO: 6), and constant (residues 133-239 of SEQ ID NO: 6) of the light chain are shown in Figure 3A. The leading regions (residues 1-19 of SEQ ID NO: 7), variable (residues 20-144 of SEQ ID NO: 7), and constant (residues 145-474 of SEQ ID NO: 7) of the heavy chain are shown in Figure 3B. The alternative constant region for the CHIR-5.9 mAb heavy chain shown in Figure 3B reflects a substitution of an alanine residue with a serine residue at position 158 of SEQ ID NO: 7. The complete sequence for this heavy chain variant of mAB CHIR-5.9 is presented in SEQ ID NO: 8. Figure 4 shows the coding sequence (Figure 4A; SEQ ID NO: 9) for the short isoform of human CD40 (amino acid sequence shown in Figure 4B; SEC ID No.: 10), and the coding sequence (Figure 4C; SEQ ID NO: 11) for the long isoform of human CD40 (amino acid sequence shown in Figure 4D). Figure 5 shows the melting temperature of CHIR-12.12 in formulations of different pH measured by differential scanning calorimetry (DSC).

DETAILED DESCRIPTION

The present disclosure relates to methods for treating autoimmune and anti-inflammatory diseases, using antibodies that have a strong affinity for the CD40 cell surface antigen. These anti-CD40 antibodies and antibody binding fragments thereof are free of significant agonist activity when they bind to CD40 in cells expressing CD40.

"Antibodies" and "immunoglobulins" (Igs) are glycoproteins that have the same structural characteristics. While antibodies exhibit binding specificity for an antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter type are produced, for example, at low levels by the lymphatic system and at levels increased by myelomas.

The term "antibody" is used in the broadest sense and encompasses fully structured antibodies, antibody fragments that can bind to the antigen (eg, Fab ', F' (ab) 2, Fv, single chain antibodies, divalent fragments or diabodies), and recombinant peptides comprising the above.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for the possible mutations that occur in nature. which may be present in smaller amounts.

"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two light chains

(L) identical and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each light and heavy chain also has regularly spaced intra-chain disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the variable domains of the light and heavy chains.

The term "variable" refers to the fact that certain parts of the variable domains differ widely in sequence between the antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not distributed evenly across the variable domains of the antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both light chain and heavy chain domains. The most highly conserved parts of the variable domains are called frame regions (FR). The variable domains of the native light and heavy chains each comprise four FR regions, which largely adopt a β-sheet configuration, connected by three CDRs, which form loops that connect the structure of the β-sheet, and in some cases are part Of the same. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antibody antigen binding site (see Kabat et al. (1991) NIH Publ. Nº: 91-3242, Vol. I, pages 647-669).

Constant domains do not directly participate in the binding of an antibody to an antigen, but exhibit various effector functions, such as FC receptor binding (FcR), antibody involvement in antibody-dependent cellular toxicity, opsonization, initiation of complement-dependent cytotoxicity and mast cell degranulation.

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues of a "complementarity determining region" or "CDR" (ie residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th ed. , Public Health Service, National Institute of Health, Bethesda, MD) and / or waste from a “hypervariable loop” (ie, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3 ) in the variable domain of the light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Clothia and Lesk (1987) J. Mol. Biol 196: 901-917) The residues of the "frame" or "FR" are the residues of the variable domain other than the residues of the hypervariable region.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antibody binding region or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ’, F (ab’) 2 and Fv fragments; divalent fragments; linear antibodies (Zapata et al. (1995) Protein Eng. 8 (10): 10571062); single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of the antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects their ability to crystallize easily. Pepsin treatment gives an F (ab ’) 2 fragment that has two combination sites with the antigen and is still capable of forming a cross-link with the antigen.

"Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. In a two-chain Fv species, this region is constituted by a dimer of a heavy chain and a light chain variable domain in close association, not covalent. In a single chain Fv species, a heavy chain and a light chain variable domain can be covalently linked by a flexible peptide linker such that light and heavy chains can be associated in a "dimeric" structure analogous to that of a Fv species of two chains. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, the six CDRs confer the antigen binding specificity for the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind to the antigen, although with a lower affinity than the complete binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab ’fragments by the addition of a few residues at the carboxy terminus of the CH1 domain of the heavy chain that includes one or more cysteines from the hinge region of the antibody. Fab’-SH is the denomination in this document for Fab ’in which the cysteine residue (s) of the constant domains has a free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab ’fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of the antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly different 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 assigned to different classes. There are five main classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The constant domains of the heavy chains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The three-dimensional structures and configurations of the subunits of the different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 and IgG3 isotypes mediate antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

The word "marker" when used herein refers to a detectable compound or composition that conjugates directly or indirectly to antibodies to generate a "labeled" antibody. The label can be detectable by itself (for example, radioisotope markers or fluorescent markers) or, in the case of an enzymatic label, it can catalyze the chemical alteration of a compound or substrate composition that is detectable.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or totally blocks, inhibits or neutralizes a biological activity of a native target disclosed herein or its transcription or translation.

"Vehicles" as used herein includes pharmaceutically acceptable carriers, excipients or stabilizers, which are non-toxic to the cell or mammal that is exposed thereto in the doses and concentrations used. Frequently the physiologically acceptable vehicle is an aqueous solution of buffered pH. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, succinate and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (with less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN, polyethylene glycol (PEG) and Pluronics. Administration "in combination with" other or other therapeutic agents includes simultaneous administration (at the same time) and consecutive administration in any order.

A "host cell," as used herein, refers to a microorganism.

or to a cultured eukaryotic cell or cell line as a unicellular entity that can be used, or has been used, as a receptor for a recombinant vector or other transfer polynucleotides, and includes the progeny of the original cell that has been transfected. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in complement of genomic or total DNA to the original, by natural, accidental or deliberate mutations.

"Human effector cells" are leukocytes that express one or more FcR and that perform effector functions. Preferably, the cells express at least FcγRIII and perform the effector function of antigen-dependent cell-mediated cytotoxicity (ADCC). Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural cytolytic cells (NK), monocytes, macrophages, eosinophils and neutrophils, with PBMC and NK cells being preferred. Antibodies that have ADCC activity are typically of the IgG1 or IgG3 isotype. Note that in addition to isolating IgG1 and IgG3 antibodies, such antibodies that mediate ADCC can be generated by modifying a variable region of an antibody that does not mediate ADCC or a variable region fragment forming a constant region of the IgG1 or IgG3 isotype.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes the receptors of the subclasses FcγRI, FcγRII and FcγRIII, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (an "activator receptor") and FcγRIIB (an "inhibitor receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The FcγRIIA activator receptor contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain. The FcγRIIB inhibitor receptor contains a tyrosine-based immunoreceptor inhibition (ITIM) motif in its cytoplasmic domain (see Daeron (1997) Annu. Rev. Immunol. 15: 203-234). FcRs are compiled in Ravetch and Kinet (1991) Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al. (1994) Immunomethods 4: 25-34; and from Haas et al. (1995) J. Lab. Clin. Med. 126: 330-341. Other FcRs, including those that will be identified in the future, are covered by the term “FcR” in this document. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al. (1976) J. Immunol. 117: 587 and Kim et al. (1994) J. Immunol. 24 : 249 (1994)).

There are several ways to produce human antibodies. For example, secretory cells can be immortalized by infection with the Epstein-Barr virus (EBV). However, EBV infected cells are difficult to clone and usually produce only relatively low immunoglobulin yields (James and Bell (1987) J. Immunol Methods 100: 540). In the future, the immortalization of human B cells could possibly be achieved by introducing a defined combination of transforming genes. Such a possibility is highlighted by a recent demonstration that the expression of the telomerase catalytic subunit together with the large SV40 oncoprotein and an H-ras oncogenic allele resulted in the tumorigenic conversion of normal human fibroblast and epithelial cells ( Hahn et al. (1999) Nature 400: 464-468). It is now possible to produce transgenic animals (eg, mice) that are capable, after immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production (Jakobovits et al. (1993) Nature 362: 255-258; Lonberg and Huszar (1995) Int. Rev. Immunol.

13: 65-93; Fishwild et al. (1996) Nat. Biotechnol. 14: 845-851; Mendez et al. (1997) Nat. Genet.

15: 146-156; Green (1999) J. Immunol. Methods 231: 11-23; Tomizuka et al. (2000) Proc. Natl Acad. Sci. USA 97: 722-727; compiled in Little et al. (2000) Immunol. Today 21: 364-370). For example, it has been described that homozygous deletion of the heavy chain (JH) binding region of the antibody in chimeric mice and germline mutants results in complete inhibition of endogenous antibody production (Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2551-2555). Transfer of the set of human germline immunoglobulin genes in such germline mutant mice results in the production of human antibodies after challenge with antigens (Jakobovits et al. (1993) Nature 362: 255-258). Mendez et al. (1997) (Nature Genetics 15: 146156) have generated a line of transgenic mice that, when challenged with an antigen, generate fully human antibodies of high affinity. This was achieved by the integration in the germline of the megabase heavy chain and human light chain loci in mice with deletion in the endogenous JH segment as described above. These mice (XenoMouse® II technology (Abgenix; Fremont, California)) house 1,020 kb of the human heavy chain locus that contains approximately 66 VH genes, complete DH and JH regions, and three different constant regions, and also houses 800 kb of the Human κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. The antibodies produced in these mice closely resemble those seen in humans in all aspects, including genetic reorganization, structure and repertoire. Human antibodies are preferably expressed on endogenous antibodies by deletion in the endogenous segment that prevents genetic reorganization in the murine locus. Such mice can be immunized with an antigen of particular interest.

Sera from such immunized animals can be examined for antibody reactivity against the initial antigen. Lymphocytes can be isolated from lymph nodes or spleen cells and can also be selected for B cells by selecting CD138 negative and CD19 positive cells. In one aspect, such B-cell cultures (BCC) can be fused to myeloma cells to generate hybridomas as detailed above.

In another aspect, such B-cell cultures may also be selected for reactivity against the initial antigen, preferably. Such selection includes ELISA with the target protein / antigen, a competition assay with known antibodies that bind the antigen of interest, and in vitro binding to CHO cells or other transiently transfected cells expressing the target antigen.

The present disclosure relates to compositions and procedures for treating human subjects who have autoimmune diseases and / or inflammatory diseases. The methods involve treatment with an anti-CD40 antibody described herein, or one of its antigen binding fragments, wherein administration of the antibody or its antigen binding fragment promotes a positive therapeutic response in the patient who is undergo this therapy procedure. The procedures and compositions are especially useful in treating diseases that include, but are not limited to, autoimmune diseases, systemic lupus erythematosus (SLE), lupus discoid, lupus nephritis, sarcoidosis, inflammatory arthritis, including, but not limited to, juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis and gouty arthritis, rejection of an organ or tissue transplant, hyperacute, acute or chronic rejection and / or graft versus host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, celiac disease (gluten sensitivity enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, disease of Graves, hasimotoid thyroiditis, complex disease in mune, chronic fatigue syndrome and immune dysfunction (SFCDI), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris, interstitial pulmonary fibrosis, sarcoidosis, Type I and Type II diabetes mellitus, and the like. Additionally, these anti-CD40 antagonist antibodies and antigen-binding fragments thereof anti-CD40 antagonists are especially useful for diseases associated with inflammation, including but not limited to delayed type hypersensitivity of type 1, 2, 3 and 4, allergy. or allergic disorders, unwanted / unwanted immune responses to therapeutic proteins (see, for example, US Patent Application No. US 2002/0119151 and Koren et al. (2002) Curr. Pharm. Biotechnol. 3: 349- 60), asthma, Churg-Strauss syndrome (allergic granulomatosis), atopic dermatitis, allergic and irritant contact dermatitis, urticaria, IgE-mediated allergy, atherosclerosis, vaculitis, idiomatic inflammatory myopathies, hemolytic disease, Alzheimer's disease, inflammatory demyelinating polyneuropathy Chronic, and the like.

Anti-CD40 antibodies suitable for use in the methods of the invention specifically bind to the human CD40 antigen expressed on the surface of a human cell and are free of significant agonist activity, but exhibit antagonistic activity when they bind to the CD40 antigen in a cell. human expressing CD40. These anti-CD40 antibodies and their antigen binding fragments are referred to herein as "anti-CD40 antagonist antibodies." Such antibodies include, but are not limited to, the fully human CHIR-5.9 and CHIR-12.12 monoclonal antibodies described below and the monoclonal antibodies have the binding characteristics of the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described. then too.

Antibodies that have the binding characteristics of CHIR monoclonal antibodies

5.9 and CHIR-12.12 include antibodies that interfere competitively with binding to CD40 and / or bind to the same epitopes as CHIR-5.9 and CHIR-12.12. An expert could determine whether an antibody interferes competitively with CHIR-5.9 or CHIR-12.12 using conventional procedures.

When these antibodies bind to exposed CD40 on the surface of human cells, such as human B cells, T cells, dendritic cells, endothelial cells, activated platelets, inflamed vascular smooth muscle cells, eosinophils, synovial membranes, dermal fibroblasts and the like , the antibodies are exempt from significant agonist activity; In some embodiments, their binding to exposed CD40 on the surface of human cells results in the inhibition of proliferation and differentiation of these human cells. Accordingly, anti-CD40 antagonist antibodies suitable for use in the methods of the invention include monoclonal antibodies that can exhibit antagonistic activity towards normal and malignant human cells that express the CD40 cell surface antigen.

Anti-CD40 Antagonist Antibodies

The monoclonal antibodies CHIR-5.9 and CHIR-12.12 represent anti-CD40 antagonist antibodies suitable for use in the methods of the present invention. CHIR-5.9 and CHIR-12.12 antibodies are fully human anti-CD40 monoclonal antibodies of the IgG1 isotype produced from hybridoma cell lines 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.D10.D6.12.12 (referred to herein as cell line 12.12). These cell lines were created using splenocytes from immunized xenotypic mice containing the human IgG1 heavy chain locus and the human K chain locus (XenoMouse® technology (Abgenix; Fremont, California)). Spleen cells were fused with mouse SP2 / 0 myeloma cells (Sierra BioSource). The resulting hybridomas were subcloned several times to create stable monoclonal cell lines

5.9 and 12.12. Other antibodies of the invention can be prepared similarly using transgenic mice for human immunoglobulin loci or by other methods known in the art and / or described herein.

The amino acid sequences for the leader, variable and constant regions for the light chain and heavy chain for the CHIR-12.12 mAb are presented in Figures 1A and 1B, respectively. See also SEQ ID NO: 2 (complete sequence for the CHIR-12.12 mAb light chain), SEQ ID NO: 4 (complete sequence for the CHIR-12.12 mAb heavy chain) and SEQ ID NO: 5 (complete sequence for a heavy chain variant for the CHIR-12.12 mAb presented in SEQ ID NO: 4, where the variant comprises a substitution of the alanine residue with serine at the 153 position of SEQ ID NO: 4). Nucleotide sequences encoding the light chain and heavy chain for the CHIR-12.12 mAb are presented in Figures 2A and 2B, respectively. See also SEQ ID NO: 1 (coding sequence for the light chain for the CHIR-12.12 mAb) and SEQ ID NO: 3 (coding sequence for the heavy chain for the CHIR-12.12 mAb). The amino acid sequences for the leader, variable and constant regions for the light chain and heavy chain of the CHIR-5.9 mAb are presented in Figures 3A and 3B, respectively. See also SEQ ID NO: 6 (complete sequence for the light chain of the CHR-5.9 mAb), SEQ ID NO: 7 (complete sequence for the heavy chain of the CHIR-5.9 mAb) and SEQ ID NO: 8 (complete sequence for a variant of the heavy chain of the CHIR-5.9 mAb presented in SEQ ID NO: 7, where the variant comprises a substitution of the alanine residue for serine at the position of 158 of SEQ ID NO: 7). In addition, the hybridomas expressing the CHIR-5.9 and CHIR-12.12 antibodies have been deposited in the ATCC with a patent deposit designation of PTA-5542 and PTA-5543, respectively.

The monoclonal antibodies CHIR-5.9 and CHIR-12.12 bind to soluble CD40 in ELISA type assays, prevent binding of the CD40 ligand to the CD40 of the cell surface, and displace the previously bound CD40 ligand, as determined by assays of flow cytometry. The CHIR-5.9 and CHIR-12.12 antibodies compete with each other for binding to CD40 but not with 15B8, the anti-CD40 monoclonal antibody described in WO 02/28904.

When the effects on B cell proliferation of normal human subjects are tested in vitro, CHIR-5.9 and CHIR-12.12 act as anti-CD40 antagonist antibodies. In addition, CHIR-5.9 and CHIR-12.12 do not induce strong proliferation of human lymphocytes from normal subjects. The binding affinity of CHIR-5.9 for human CD40 is 1.2 x 10-8 M and the binding affinity of CHIR-12.12 is 5 x 10-10 M, as determined by the Biacore ™ assay.

Anti-CD40 antagonist antibodies suitable for use in the methods of the present invention exhibit a strong single site binding affinity for the CD40 cell surface antigen. The monoclonal antibodies of the invention exhibit a dissociation equilibrium constant (KD) for CD40 of at least 10-5 M, at least 3 X 10-5 M, preferably at least 10-6 M to 10-7 M, of more preference at least 10-8 M to about 10-12 M, measured using a conventional assay such as Biacore ™. Biacore analysis is known in the art and details are provided in the "BIAapplications handbook". The procedures described in WO 01/27160 can be used to modulate binding affinity.

By "CD40 antigen", "CD40 cell surface antigen", "CD40 receptor" or "CD40" is a transmembrane glycoprotein belonging to the family of tumor necrosis factor (TNF) receptors (see, for example, Patents No. 5,674,492 and 4,708,871; Stamenkovic et al. (1989) EMBO 8: 1403; Clark (1990) Tissue Antigens 36: 33; Barclay et al. (1997) The Leucocyte Antigen Facts Book (2nd ed. ; Academic Press, San Diego)). Two isoforms of human CD40 have been identified, encoded by transcription variants with alternative splices of this gene. The first isoform (also known as the "long isoform" or "isoform 1") is expressed as a 277 amino acid precursor polypeptide (SEQ ID NO: 12 (first reported in GenBank with accession No. CAA43045, and identified as isoform 1 in GenBank with accession number NP 001241), encoded by SEQ ID NO: 11 (see GenBank accession number X60592 and NM 001250)), which has a signal sequence represented by the first 19 residues. The second isoform (also known as the "short isoform" or "isoform 2") is expressed as a 203 amino acid precursor polypeptide (SEQ ID NO: 10 (GenBank Accession No. NP 690593), encoded by SEQ ID NO: 9 (GenBank Accession No. NM 152854)), which also has a signal sequence represented by the first 19 residues. The precursor polypeptides of these two isoforms of human CD40 share in common their first 165 residues (i.e. residues 1-165 of SEQ ID NO: 10 and SEQ ID NO: 12). The short isoform precursor polypeptide (shown in SEQ ID NO: 10) is encoded by a transcription variant (SEQ ID NO: 9) that lacks an encoding segment, which results in a change of the translation framework; The resulting CD40 isoform contains a shorter and different C-terminal end (residues 166-203 of SEQ ID NO: 10) than is contained in the long CD40 isoform (C-terminal end shown in residues 166-277 of SEQ ID Nº: 12). For the purposes of the present invention, the term "CD40 antigen", "cell surface CD40 antigen", "CD40 receptor" or "CD40" encompasses both the short and long isoforms of CD40. The anti-CD40 antibodies of the present invention bind to a human CD40 epitope that is in the same situation within the short isoform or the long isoform of this cell surface antigen as noted hereinbelow.

The CD40 antigen is exposed on the surface of a variety of cell types, as described elsewhere herein. By "exposed on the surface" and "expressed on the surface" is meant that all or part of the CD40 antigen is exposed to the outside of the cell. The exposed or expressed CD40 antigen may be totally or partially glycosylated.

By "agonist activity" is meant that the substance functions as an agonist. An agonist combines with a receptor in a cell and initiates a reaction or activity that is similar to that initiated by the receptor's natural ligand. A CD40 agonist induces, but is not limited to, any or all of the following responses: cell proliferation and differentiation, ascending regulation of intercellular adhesion by means of molecules such as ICAM-1, E-selectin, VCAM and the like; secretion of proinflammatory cytokines such as IL1, IL-6, IL-8, IL-12, TNF and the like, signal transduction by the CD40 receptor by routes such as TRAF (eg, TRAF2 and / or TRAF3), MAP kinases such as N1K (NF-κB inducing kinase), I-kappa B kinase (IKK α / β), transcription factor NF-κB, Ras and the MEK / ERK route, the PI3K / Akt route, the P38 MAPK route, and the like; transduction of an antiapoptotic signal by molecules such as XIAP, Mcl-1, BCLx, and the like; generation of memory cells B and / or memory cells T; B cell antibody production; B cell isotype change; ascending regulation of cell surface expression of MHC class II and CD80 / 86 and the like. By "antagonist activity" is meant that the substance functions as an antagonist. For example, a CD40 antagonist prevents or reduces the induction of any of the responses induced by the binding of the CD40 receptor to an agonist ligand, in particular CD40L. The antagonist may reduce the induction of one or more of the agonist binding responses by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50 %, 55%, 60%, more preferably 70%, 80%, 85% and most preferably 90%, 95%, 99% or 100%. Methods for measuring binding specificity and antagonist activity of anti-CD40 antibodies and CD40 ligands are known to those skilled in the art and include, but are not limited to, standard competitive binding assays, secretion control assays. of immunoglobulins by B cells, B cell proliferation assays, B-cell proliferation assays analogous to Banchereau, T-cell helper assays for antibody production, costimulation cell proliferation assays and assays for ascending regulation of markers of B cell activation

By "significant" antagonist activity is meant an agonist activity of at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the agonist activity induced by a neutral substance or a negative control as measured in a bioassay such as a B-cell response assay. Preferably, the "significant" agonist activity is an agonist activity that is at least 2 times greater or at least 3 times greater than the agonist activity induced by a neutral substance or a negative control as measured in the assay of a B cell response. Therefore, for example, when the B cell response of interest is the B cell proliferation, the “significant” agonist activity would be the induction of a level of B cell proliferation that is at least 2 times greater or at least 3 times greater than the level of B cell proliferation induced by a neutral substance or a negative control In one embodiment, a non-specific immunoglobulin, for example IgG1, that does not bind to CD40 serves as the negative control. A substance "free of significant agonist activity" will exhibit an agonist activity of no more than about 25% greater than the agonist activity induced by a neutral substance

or a negative control, preferably no more than about 20% greater, 15% greater, 10% greater, 5% greater, 1% greater, 0.5% greater, or even no more than approximately 0.1% greater than the agonist activity induced by a neutral substance or a negative control as measured in an assay of a B-cell response. Anti-CD40 antagonist antibodies useful in the methods of the present invention are free of significant agonist activity as noted above when bind to a CD40 antigen in a human cell. In one embodiment of the invention, the anti-CD40 antagonist antibody is free of significant agonist activity in a B cell response. In another embodiment of the invention, the anti-CD40 antagonist antibody is free of significant agonist activity in assays of more than a B cell response (for example, proliferation and differentiation, or proliferation, differentiation and antibody production).

As used herein "anti-CD40 antibody" encompasses any antibody that specifically recognizes the cell surface CD40 antigen, including polyclonal antibodies, monoclonal antibodies, single chain antibodies, and their fragments such as Fab, F (ab ') 2, Fv, and other fragments that retain the antigen binding function of the original anti-CD40 antibody. Of particular interest to the present invention are anti-CD40 antagonist antibodies that share the binding characteristics of the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described above.

Therefore, in addition to the monoclonal antibodies CHIR-5.9 and CHIR-12.12, other antibodies that would be useful in carrying out the invention described herein include, but are not limited to, the following: (1) monoclonal antibodies produced by the hybridoma cell lines designated 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.D10.D6.12.12 (referred to herein as line cell 12.12), deposited in the ATCC as Patent Deposit No. PTA-5542 and Patent Deposit No. PTA-5543, respectively; (2) a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO: 2, the sequence shown in SEQ ID NO: 4, the sequence shown in SEQ ID NO: 5, both sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and for both sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5; (3) a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO: 6, the sequence shown in SEQ ID NO: 7, the sequence shown in SEQ ID NO: 8, both sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, and for both sequences shown in SEQ ID NO: 6 and SEQ ID NO: 8; (4) a monoclonal antibody having an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence shown in SEQ ID No.: 3, and both nucleotide sequences shown in SEQ ID NO: and SEQ ID NO: 3; (5) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line

5.9 or the hybridoma cell line 12.12; (6) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the amino acid sequence shown in SEQ ID NO: 10

or SEQ ID NO: 12; (7) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; and (8) a monoclonal antibody that is a fragment of the monoclonal antibody CHIR-12.12 or CHIR-5.9 that binds to the antigen

or the previous monoclonal antibodies in the above points (1) - (7), in which the fragment retains the ability to specifically bind to the human CD40 antigen. Those skilled in the art recognize that antagonist anti-CD40 antibodies and fragments of Antigen binding of these antibodies suitable for use in the methods disclosed herein include anti-CD-40 antagonist antibodies and antigen binding fragments thereof that are recombinantly produced using methods well known in the art and described below, and include, for example, monoclonal antibodies CHIR-5.9 and CHIR-12.12 that have been recombinantly produced.

Production of anti-CD40 antibodies

Anti-CD40 antagonist antibodies for use in the present invention can be produced using any of the methods well known to those skilled in the art. In general, a solution containing the CD40 antigen is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits or goats are preferably for the preparation of polyclonal sera due to the volume of serum that can be obtained and the availability of labeled anti-rabbit and anti-goat antibodies.

Polyclonal sera can be prepared in a transgenic animal, preferably a mouse carrying human immunoglobulin loci. In a preferred embodiment, Sf9 cells expressing CD40 are used as the immunogen. Immunization can also be carried out by mixing or emulsifying the solution containing the antigen in saline solution, preferably in an adjuvant such as Freund's complete adjuvant, and injecting intravenously the mixture or emulsion parenterally (usually subcutaneously or intramuscular). Typically a dose of 50-200 µg / injection is sufficient. Immunization is usually stimulated 2-6 weeks later with one or more injections of the protein in saline, preferably using incomplete Freund's adjuvant. Alternatively, antibodies can also be generated by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to immunization in vivo. Polyclonal antisera are obtained by extracting blood from the immunized animal in a glass or plastic container, incubating the blood at 25 ° C for one hour, followed by incubation at 4 ° C for 2-18 hours. The serum is recovered by centrifugation (for example, 1,000 g x for 10 minutes). Approximately 20-50 ml can be obtained by drawing blood from rabbits.

The production of Sf9 cells (Spodoptera frugiperda) is disclosed in US Patent No. 6,004,552. In summary, sequences encoding human CD40 in a baculovirus were combined using transfer vectors. Plasmids were cotransfected with wild-type baculovirus DNA in Sf9 cells. Sf9 cells infected with recombinant baculovirus were identified and purified as clones.

Preferably the antibody is monoclonal in nature. By "monoclonal antibody" is meant an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in smaller amounts. The term is not limited with respect to the species or source of the antibody. The term encompasses complete immunoglobulins, as well as fragments such as Fab, F (ab ') 2, Fv and others that retain the antigen binding function of the antibody. Monoclonal antibodies are very specific, being directed against a single antigenic site, that is, the cell surface CD40 antigen in the present invention. In addition, unlike conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. The "monoclonal" modifier indicates the character of the antibody since it is obtained from a substantially homogeneous population of antibodies, and should not be construed as producing the antibody by any particular procedure. For example, the monoclonal antibodies used according to the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495,

or they can be produced by recombinant DNA methods (see, for example, US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al. (1991) Nature 352: 624-628; Marks et al. (1991) J Mol. Biol. 222: 581-597; and in U.S. Patent No. 5,514,548.

By "epitope" is meant the part of the antigenic molecule for which an antibody is produced and to which the antibody will bind. The epitopes may comprise linear amino acid residues (ie, residues in the epitope are sequentially organized one after the other in a linear fashion), nonlinear residues (referred to herein as "nonlinear epitopes"; these epitopes are not organized consecutively), or amino acid residues both linear and non-linear.

The term "epitope of the CD40 antigen" as used herein refers to a molecule of three-dimensional structure (either linear or conformational) that is capable of immunoreactivity with the anti-CD40 monoclonal antibodies of this invention, excluding itself CD40 antigen. The epitopes of the CD40 antigen may comprise proteins, protein fragments, peptides, carbohydrates, lipids and other molecules, but for the purposes of the present invention are more commonly proteins, short oligopeptides, oligopeptide mimetics (i.e., organic compounds that mimic the antibody binding properties of the CD40 antigen), or combinations thereof. Suitable oligopeptide mimetics are described, among others, in PCT application US 91/04282.

Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256: 495-496, or a modification thereof. Typically, a mouse is immunized with a solution containing the antigen. Immunization can be carried out by mixing

or by emulsifying the solution containing the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally. Any immunization procedure known in the art can be used to obtain monoclonal antibodies of the invention. After immunization of the animal, the spleen is removed (and optionally, several large lymph nodes) and the individual cells are separated. Spleen cells can be examined by applying a suspension of cells to a plate or well coated with the antigen of interest. B cells expressing the membrane-bound immunoglobulin specific for the antigen bind to the plate and are not cleared. The fusion of the resulting B cells, or all cells separated from the spleen, is then induced with myeloma cells to form hybridomas, and cultured in a selective medium. The resulting cells are plated by serial dilution and the production assay of antibodies that specifically bind the antigen of interest (and that do not bind unrelated antigens) is performed. Hybridomas secreting the selected monoclonal antibody (mAb) are then cultured in vitro (for example, in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

When the anti-CD40 antagonist antibodies of the invention must be prepared using recombinant DNA methods, the DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (for example, by the use of oligonucleotide probes that are capable of specifically binding to genes that encode the light and heavy chains of murine antibodies). The hybridoma cells described herein serve as a preferred source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells or non-myeloma cells otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Collection articles on recombinant expression in DNA bacteria encoding the antibody include Skerra et al. (1993) Curr. Opinion in Immunol. 5: 256 and Phickthun (1992) Immunol. Revs 130: 151. As an alternative to the use of hybridomas, antibody can be produced in a cell line such as a CHO cell line, as disclosed in US Pat. Nos. 5,545,403; 5,545,405; and 5,998,144. In summary, the cell line is transfected with vectors capable of expressing a light chain and a heavy chain, respectively. By transfecting the two proteins into separate vectors, chimeric antibodies can be produced. Another advantage is the correct glycosylation of the antibody.

In some embodiments, the anti-CD40 antagonist antibody, for example the CHIR antibody

12.12 or CHIR-5.9, or its antigen-binding fragment is produced in CHO cells using the GS gene expression system (Lonza Biologics, Portsmouth, New Hampshire), which uses glutamine synthetase as a marker. See also US Patent No. 5,122,464; 5,591,639; 5,658,759; 5,770,359; 5,827,739; 5,879,936; 5,891,693; and 5,981,216.

Monoclonal antibodies against CD40 are known in the art. See, for example, sections dedicated to B cell antigens in McMichael, ed. (1987; 1989). Leukocyte Typing III and IV (Oxford University Press, New York); U.S. Patent No. 5,674,492; 5,874,082; 5,677,165; 6,056,959; WO 00/63395; International Publication Documents No. WO 02/28905 and WO 02/28904; Gordon et al. (1988) J. Immunol. 140: 1425; Valle et al. (1989) Eur. J. Immunol. 19: 1463; Clark et al. (1986) PNAS 83: 4494; Paulie et al. (1989) J. Immunol. 142: 590; Gordon et al. (1987) Eur. J. Immunol. 17: 1535; Jabara et al. (1990) J. Exp. Med. 172: 1861; Zhang et al. (1991) J. Immunol. 146: 1836; Gascan et al. (1991) J. Immunol. 147: 8; Banchereau et al. (1991) Clin. Immunol Spectrum 3: 8; and Banchereau et al. (1991) Science 251: 70. Of particular interest to the present invention are the anti-CD40 antagonist antibodies disclosed herein that share the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR

12.12 described above.

Additionally, the term "anti-CD40 antibody" as used herein encompasses anti-CD40 chimeric antibodies; Such chimeric anti-CD40 antibodies for use in the methods of the invention have the binding characteristics of the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described herein. By "chimeric" antibodies are meant antibodies that are most preferably obtained using recombinant deoxyribonucleic acid techniques and which comprise both human components (including immunologically "related" species, for example, chimpanzees) and nonhuman. Rituxan® is an example of a chimeric antibody with a murine variable region and a human constant region. For purposes of the present invention, the constant region of the chimeric antibody is most preferably substantially identical to the constant region of a natural human antibody; The variable region of the most preferred chimeric antibody is obtained from a non-human source and has the desired antigen specificity for the CD40 cell surface antigen. The non-human source can be any vertebrate source that can be used to generate antibodies against a human CD40 cell surface antigen or material comprising a human CD40 cell surface antigen. Such non-human sources include, but are not limited to, rodents (e.g., rabbit, rat, mouse, etc .; see, for example, U.S. Patent No. 4,816,567) and non-human primates (e.g., monkey old world, ape, etc .; see, for example, US Patent Nos. 5,750,105 and 5,756,096). As used herein, the phrase "immunologically active" when used in reference to chimeric anti-CD40 antibodies means a chimeric antibody that binds to human CD40.

Humanized anti-CD40 antibodies represent additional anti-CD40 antibodies suitable for use in the present invention. By "humanized" are meant forms of anti-CD40 antibodies that contain a minimal sequence derived from non-human immunoglobulin sequences. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues of a hypervariable region (also known as complementarity determining region or CDR) of the receptor are replaced by residues of a hypervariable region of a non-human species (antibody donor) such as mouse, rat, rabbit or non-human primate that has the desired specificity, affinity and capacity. The term "complementarity determining region" refers to the amino acid sequences that together define the affinity and binding specificity of the natural Fv region of a native immunoglobulin binding site. See, for example, Chothia et al. (1987) J. Mol. Biol. 196: 901-917; Kabat et al. (1991) U.S. Dept of Health and Human Services, NIH Publication No. 91-3242). The term "constant region" refers to the part of the antibody molecule that confers effector functions. In previous studies aimed at the production of non-immunogenic antibodies for use in therapy of human diseases, mouse constant regions were replaced by human constant regions. The constant regions of the subject's humanized antibodies were derived from human immunoglobulins. However, these humanized antibodies still facilitated an unwanted and potentially dangerous immune response in humans and there was loss of affinity. Humanized anti-CD40 antibodies for use in the methods of the present invention have binding characteristics similar to those exhibited by the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described herein.

Humanization can be essentially performed following the procedure of Winter et al. (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239 : 1534-1536), replacing sequences of a human antibody with the corresponding CDR or CDR sequences of rodents or mutant rodents. See also US Patent No. 5,225,539; 5,585,089; 5,693,761;

5,693,762 and 5,859,205. In some cases, residues within the framework regions of one or more variable regions of the human immunoglobulin are replaced by the corresponding non-human residues (see, for example, US Pat. Nos. 5,585,089; 5,693,761; 5,693. 762; and 6,180,370). In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the antibody performance (for example, to obtain the desired affinity). In general, in humanized antibody it will comprise substantially all of at least one, and typically two, variable domains, in which all

or substantially all hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of a constant region (Fc) of immunoglobulin, typically that of a human immunoglobulin. For more details, see Jones et al. (1986) Nature 331: 522-525; Riechmann et al. (1988) Nature 332: 323-329; and Presta (1992) Curr. Op. Struct. Biol. 2: 593-596. Accordingly, such "humanized" antibodies may include antibodies in which substantially less of an intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are replaced by residues of similar sites in rodent antibodies. See, for example, US Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also US Patent No. 6,180,370, and International Publication WO 01/27160, in which humanized antibodies and techniques for producing humanized antibodies that have better affinity for a predetermined antigen are disclosed.

Also encompassed by the term anti-CD40 antibodies are xenogeneic or modified anti-CD40 antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by endogenously inactivated immunoglobulin (Ig) loci. In such transgenic animals, the endogenous genes competent for the expression of the light and heavy subunits of host immunoglobulins become nonfunctional and are replaced with human immunoglobulin analog loci. These transgenic animals produce human antibodies in the substantial absence of light or heavy subunits of host immunoglobulin. See, for example, U.S. Patent No. 5,877,397 and 5,939,598.

Preferably, fully human antibodies against CD40 are obtained by immunizing transgenic mice. One such mouse is obtained using XenoMouse® technology (Abgenix; Fremont, California), and is disclosed in US Pat. Nos. 6,075,181,

6,091,001 and 6,114,598. To produce the antibodies disclosed herein, transgenic mice were immunized for the human IgG1 heavy chain locus and the human K light chain locus with Sf9 cells expressing human CD40. Mice can also be transgenic for other isotypes. Fully human antibodies useful in the methods of the present invention are characterized by having binding properties similar to those exhibited by the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein.

Fragments of the anti-CD40 antibodies are suitable for use in the methods of the invention as long as they retain the desired affinity of the full length antibody. Accordingly, a fragment of an anti-CD40 antibody will retain the ability to bind to the surface CD40 antigen of B cells. Such fragments are characterized by having properties similar to the corresponding full length antagonistic anti-CD40 antibody, that is, the fragments they will specifically bind to a human CD40 antigen expressed on the surface of a human cell, and are free of significant agonist activity but exhibit antagonistic activity when they bind to a CD40 antigen in a human cell that expresses CD40. These fragments are referred to herein as "antigen binding" fragments.

Suitable fragments of an antibody that bind to the antigen comprise a part of a full-length antibody, usually the antigen-binding region or one of its variable regions. Examples of antibody fragments include, but are not limited to, Fab, F (ab ’) 2 and Fv fragments and single chain antibody molecules. By "Fab" is meant a monovalent fragment of an immunoglobulin that binds to the antigen that is composed of the light chain and part of the heavy chain. F (ab ’) 2 means a bivalent fragment of an immunoglobulin that binds to the antigen that contains both light chains and part of both heavy chains. By "single chain Fv" or "sFv" fragments of the antibody is meant fragments comprising the VH and VL domains of an antibody, in which these domains are present in a single polypeptide chain. See, for example, U.S. Patent Nos. 4,946,778, 5,260,203, 5,455,030 and 5,856,456. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun (1994) in The Pharmacology of Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pages 269

315. Fragments of the anti-CD40 antagonist antibodies that bind to the antigen disclosed herein may also be conjugated to a cytotoxin to produce lysis of the target cancer cells, as described hereinbelow.

Antibodies or antibody fragments can be isolated from phage antibody libraries generated using the techniques described in, for example, McCafferty et al. (1990) Nature

348: 552-554 (1990) and in US Patent No. 5,514,548. Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Later publications describe the production of high affinity human antibodies (of the order of nM) by random chain mixing (Marks et al. (1992) Biol / Technology 10: 779-783), as well as by combinatorial infection and recombination in vivo as a strategy to build very large phage libraries (Waterhouse et al. (1993) Nucleic, Acids Res. 21: 22652266). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al. (1992) Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al. (1985) Science 229: 81). However, these fragments can now be produced directly by means of recombinant host cells. For example, antibody fragments can be isolated from the phage antibody library discussed above. Alternatively, Fab’-SH fragments can be recovered directly from E. coli and chemically coupled to form F (ab ’) 2 fragments (Carter et al. (1992) Bio / Technology 10: 163-167). According to another approach, F (ab ’) 2 fragments can be isolated directly from recombinant host cell culture. Another technique for the production of antibody fragments will be apparent to the person skilled in the art.

Anti-CD40 antagonist antibodies useful in the present invention include the monoclonal antibodies CHIR-5.9 and CHIR 12.12 disclosed herein as well as antibodies that differ from these antibodies but retain CDRs; and antibodies with one or more additions, deletions or substitutions, in which the antagonistic activity is measured by inhibition of proliferation and / or differentiation of B cells. The invention also encompasses deimmunized anti-CD40 antagonist antibodies, which may occur. as described in, for example, International Publication Documents No. WO 98/52976 and WO 0034317. In this manner, the residues within the anti-CD40 antagonist antibodies of the invention are modified to make the antibodies non-immunogenic or less immunogenic for human beings while maintaining their antagonistic activity towards human cells expressing CD40, in which such activity is measured by the tests indicated elsewhere in this document. Also included within the scope of the claims are fusion proteins comprising an antagonistic antiCD40 antibody of the invention, or one of its fragments, whose fusion proteins can be synthesized or expressed from corresponding polynucleotide vectors, as known in the invention. technique. Such fusion proteins are described with reference to antibody conjugation as noted below.

The antibodies of the present invention may have sequence variations produced using the procedures described in, for example, Patent Publication Documents No. EP 0 983 303 Al, WO 00/34317 and WO 98/52976. For example, it has been shown that sequences within the CDR can cause an antibody to bind to MAC Class II and trigger an unwanted helper T cell response. A conservative substitution may allow the antibody to retain binding activity and still lose its ability to trigger an unwanted T-cell response. Any such conservative or non-conservative substitutions may be made using methods recognized in the art, such as those set forth elsewhere herein, and the resulting antibodies will be within the scope of the invention. Variant antibodies can be routinely tested to verify their antagonistic activity, affinity and specificity using the procedures described herein.

Anti-CD40 antagonist antibodies useful in the practice of the invention may have one

or many mechanisms of action. An antibody produced by any of the procedures described above, or by any other procedure not disclosed herein, will be within the scope of this description if it has at least one of the following biological activities in vitro and / or in vivo. : inhibition of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by cells expressing CD40L or soluble CD40 ligand (sCD40L); inhibition of intra apoptotic intracellular "survival" signals in any cell stimulated by solid phase sCD40L or CD40L; and; inhibition of CD40 signal transduction in any cell after binding with solid phase sCD40L or CD40L, deletion, anergy and / or induction of tolerance of target cells carrying CD40 or of cells carrying cognate ligands for CD40 including, but not limited to, T cells and B cells, induction of expansion or activation of CD4 + CD25 + regulatory T cells (see, for example, rejection of donor alloantigen specific tissues by means of CD40-CD40L interference, van Maurik et al. (2002 ) J. Immunol. 169: 5401-5404), cytotoxicity by any mechanism (including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), descending proliferation regulation, and / or apoptosis in target cells), modulation of cytokine secretion and / or expression of molecules on the cell surface in target cells and combinations thereof. Assays for such biological activities can be performed as described in the Examples herein. See also the tests described in Schultze et al. (1998) Proc. Natl Acad Sci. USA 92: 8200-8204; Denton et al. (1998) Pediatr. transplant 2: 6-15; Evans et al. (2000) J. Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13: 12; Kwekkeboom et al. (1993) Immunology

79: 439-444; and U.S. Patent Nos. 5,674,492 and 5,847,082.

A representative assay for detecting anti-CD40 antagonist antibodies specific for the epitopes of the CD40 antigen identified herein is a "competitive binding assay." Competitive binding assays are serological assays in which the unknown is detected and quantified by its ability to inhibit the binding of a known labeled ligand to its specific antibody. This is also known as a competitive inhibition assay. In a representative competitive binding assay, labeled CD40 polypeptide is precipitated by candidate antibodies in a sample, for example, in combination with monoclonal antibodies raised against one or more epitopes of the monoclonal antibodies of the invention. Anti-CD40 antibodies that react specifically with an epitope of interest can be identified by selecting a series of antibodies prepared against a CD40 protein or protein fragment comprising the particular epitope of the CD40 protein of interest. For example, for human CD40, epitopes of interest include epitopes comprising linear or non-linear amino acid residues of the short isoform of human CD40 (see GenBank Accession No. NP_690593) presented in Figure 4B (SEQ ID NO: 10), encoded by the sequence presented in Figure 4A (SEQ ID NO: 9; see also GenBank Accession No. MN_152854), or the long isoform of human CD40 (see GenBank Accession No. CAA43045 and NP_001241) presented in Figure 4D (SEC ID No.: 12), encoded by the sequence presented in Figure 4C (SEQ ID NO: 11; see GenBank Accession No. X60592 and MN_001250). Alternatively, competitive binding assays with suitable anti-CD40 antagonist antibodies identified above could be used to select monoclonal antibodies comparable to the antibodies identified above.

The antibodies used in such immunoassays may be labeled or unlabeled. Unlabeled antibodies can be used in agglutination; labeled antibodies can be used in a wide variety of assays, using a wide variety of markers. The detection of the formation of an antigen-antibody complex between an anti-CD40 antibody and an epitope of interest can be facilitated by attaching a detectable substance to the antibody. Suitable detection media include the use of markers such as radionuclides, enzymes, coenzymes, fluorescent, chemiluminescent, chromogens, substrate enzyme complexes or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; An example of a fluorescent material is luminol; Examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive material include 125I, 131I, 35S or 3H. Such labeled reagents can be used in a variety of known assays, such as radioimmunoassays, enzyme immunoassays, for example, ELISA fluorescent immunoassays and the like. See, for example, US Patents No. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.

Any of the anti-CD40 antagonist antibodies or their antibody fragments described previously may be conjugated before use in the methods of the present invention. Methods for producing conjugated antibodies are known in the art. Accordingly, the anti-CD40 antibody can be labeled using indirect labeling or an indirect labeling approach. Suitable markers include fluorophores, chromophores, radioactive atoms (especially 32P and 125I), electron-dense reagents, enzymes and ligands that have specific binding partners.

Enzymes are normally detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3 ’, 5,5 ′ tetramethylbenzidine (TMB) into a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding to a ligand molecule with high specificity, such as in the case of an antigen and a specific monoclonal antibody for it. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not intended to categorize the various markers into different classes, since the same marker can serve in several different ways. For example, HRP can serve as an enzyme or as an antigen for a mAb. In addition, various markers can be combined for the desired effect. For example, mAbs and avidin also require markers in the practice of this invention: therefore, a mAb could be labeled with biotin, and its presence detected with avidin

125I,

labeled with or with an anti-biotin mAb labeled with HRP. Other permutations and possibilities will be readily apparent to those skilled in the art, and are considered as equivalent within the scope of the present invention.

In addition, an antibody (or its fragment) can be conjugated to a therapeutic moiety such as a therapeutic agent, for example. The rest of the drug can be a protein or a polypeptide that has a desired biological activity. Such proteins may include, for example, a protein such as CTLA4-Ig, an antibody or any other protein, or biological response modifiers such as, for example, lymphokines, tumor necrosis factor, alpha interferon, beta interferon, nerve growth, platelet-derived growth factor, tissue plasminogen activator, BLys (Lymphocyte Stimulator B), interleukin 1 ("IL-1"), interleukin 2 ("IL-2"), interleukin-6 ( "IL-6"), granulocyte and macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("GCSF") or other growth factors.

Techniques for conjugating such therapeutic moieties to antibodies are well known. See, for example, Amon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs and Cancer Therapy", ed. Reisfeld et al. (Alan R. Liss, Inc.), pages 243-256; ed. Hellstrom et al.

(1987), Controlled Drug Delivery, ed. Robinson et al. (2nd ed; Marcel Dekker, Inc.), pages 623653; Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies ’84: Biological and Clinical Applications, ed. Pinchera et al. pages 475506 (Editrice Kurtis, Milan, Italy, 1985); Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pages 303-316; and Thorpe et al. (1982) Immunol. Rev. 62: 119-158.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in US Patent No. 4,676,980. In addition, connectors between the markers and the antibodies of the invention can be used (see US Patent No. 4,831,175). Antibodies or their antigen binding fragments can be labeled directly or indirectly (US Patent No. 5,595,721). The treatment may consist of a combination of treatment with conjugated and unconjugated antibodies administered simultaneously or consecutively (WO 00/52031 and WO 00/52473).

Anti-CD40 antagonist antibody variants

Variants of suitable biologically active antagonist anti-CD40 antibodies can be used in the methods of the present invention. Such variants will retain the desired binding properties of the original anti-CD40 antagonist antibody. Generally, methods for producing antibody variants are available in the art.

For example, amino acid sequence variants of an anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-5.9 or CHIR-12.12 described herein, can be prepared by mutations in the cloned DNA sequence encoding the antibody of interest Procedures for mutagenesis and alterations of nucleotide sequences are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl Acad Sci. USA 82: 488-492; Kunkel et al. (1987) Methods Enzymol. 154: 367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); U.S. Patent No. 4,873,192; and the references cited therein. In the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.) guidance for suitable amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found. Conservative substitutions, such as exchanges of one amino acid with another having similar properties, may be preferred. Examples of conservative substitutions include, but are not limited to, Gly⇔Ala, Val⇔Ile⇔Leu, Asp⇔Glu, Lys⇔Arg, Asn⇔Gln and Phe⇔Trpo⇔Tyr.

In the construction of variants of the polypeptide of interest of the anti-CD40 antagonist antibody, the modifications are made such that the variants continue to have the desired activity, that is, similar binding affinity and are capable of specifically binding to a human CD40 antigen. expressed on the surface of a human cell and are free of significant agonist activity but that exhibit antagonistic activity when bound to a CD40 antigen in a human cell that expresses CD40. Obviously, any mutations made in the DNA encoding the polypeptide variant should not place the sequence outside the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See Patent Application Publication No. 75,444.

In addition, the constant region of an anti-CD40 antagonist antibody can be mutated to alter effector function in a number of ways. For example, see US Patent No. 6,737,056B1 and Patent Application Publication No. 2004 / 0132101A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.

Preferably, variants of a reference antagonist anti-CD40 antibody have amino acid sequences that have at least 70% or 75% sequence identity, preferably at least 80% or 85% sequence identity, more preferably at minus 90%, 91%, 92%, 93%, 94% or 95% sequence identity with the amino acid sequence for the reference antagonist anti-CD40 antibody molecule, for example, the CHIR-5.9 or CHIR monoclonal antibody -12.12 described herein, or with a shorter part of a reference antibody molecule. More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence identity. For the purposes of the present invention, the percent sequence identity is determined using the Smith-Waterman identity search algorithm using a related gap search with open hole penalty parameters of 12 and gap extension penalty of 2 , BLOSUM matrix of 62. The Smith-Waterman identity search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math 2: 482-489. A variant may, for example, differ from the reference antagonist anti-CD40 antibody in as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

With respect to the optimal alignment of two amino acid sequences, the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence. The contiguous segment used for comparison with the reference amino acid sequence will include at least 20 contiguous amino acid residues and may have 30, 40, 50

or more amino acid residues. Corrections can be made for sequence homology associated with gaps or conservative substitutions of residues (see Smith-Waterman's search algorithm).

The exact chemical structure of a polypeptide capable of specifically binding to CD40 and retaining antagonistic activity, particularly when it binds to the CD40 antigen in malignant B cells, depends on a number of factors. Since ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide can be obtained as an acidic or basic salt, or in a neutral form. All preparations that retain their biological activity when placed under suitable environmental conditions are included in the definition of anti-CD40 antagonist antibodies as used herein. In addition, the main amino acid sequence of the polypeptide can be increased by derivatization using sugar moieties (glycosylation) or by other complementary molecules such as lipids, phosphate, acetyl groups and the like. It can also be increased by conjugation with saccharides. Certain aspects of such an increase are carried out through post-translational processing systems of the producer host; Other such modifications may be introduced in vitro. In any case, such modifications are included in the definition of an anti-CD40 antibody used herein as long as the anti-CD40 antibody antagonist properties are not destroyed. It is expected that such modifications can quantitatively or qualitatively affect the activity, either by increasing or decreasing the activity of the polypeptide, in the various assays. In addition, individual amino acid residues in the chain can be modified by oxidation, reduction or other derivatization, and the polypeptide can be cleaved to obtain fragments that retain activity. Such alterations that do not destroy the antagonistic activity do not remove the polypeptide sequence from the definition of anti-CD40 antibodies of interest as used herein.

The technique provides substantial guidance regarding the preparation and use of polypeptide variants. In the preparation of anti-CD40 antibody variants, one skilled in the art can easily determine which modifications to the nucleotide or amino acid sequence of the native protein will result in a variant that is suitable for use as a therapeutically active component of a Pharmaceutical composition used in the procedures disclosed herein.

Therapy methods using the anti-CD40 antagonist antibodies of the invention

The procedures disclosed herein are directed to the use of anti-CD40 antagonist antibodies to treat subjects (ie patients) who have an autoimmune disease and / or an inflammatory disease, or a predisposition to develop an autoimmune disease and / or an inflammatory disease, in which the disease and / or inflammation is mediated by CD40 signaling mediated by CD40 ligand in cells expressing CD40. By the term "cells expressing CD40", the cells expressing the CD40 antigen are understood. Methods for detecting CD40 expression in cells are well known in the art and include, but are not limited to, PCR techniques, immunohistochemistry, flow cytometry, Western blotting, ELISA and the like.

The procedures disclosed herein are especially useful for treating inflammatory diseases and / or autoimmune diseases in which CD40 stimulation mediated by CD40 ligand is involved. The compositions of the invention can be administered prophylactically or therapeutically or in a combination thereof.

Inflammatory diseases are characterized by inflammation and tissue destruction, or by a combination thereof. "Inflammatory disease" includes any process mediated by the inflammatory immune system where the initiating event or the objective of the imune response involves antigen or antigens that are not autoantigens, including, for example, alloantigens, xenoantigens, viral antigens, bacterial antigens, unknown antigens or allergens

Additionally, for purposes of the present invention, the term "inflammatory disease (s)" includes "autoimmune disease (s)". As used herein, the term "autoimmunity" is generally understood to encompass inflammatory immune system mediated processes that involve "autoantigens." In autoimmune diseases, the autoantigen (s) activate host immune responses.

In addition, the present disclosure includes inflammation treatment associated with tissue transplant rejection. "Transplant rejection" or "graft rejection" refers to any immune response made by the host against a graft including but not limited to HLA antigens, blood group antigens and the like.

The invention can also be used to treat graft versus host disease, such as that associated with bone marrow transplantation, for example. In such graft versus host disease, the donor's bone marrow includes lymphocytes and cells that mature in lymphocytes. Therefore, as used herein, "graft versus host disease" or "graft versus host reaction" refers to a T-cell mediated immune response in which donor lymphocytes react with host antigens.

The anti-CD40 antagonist antibodies and the antigen-binding fragments thereof described herein can be used in accordance with the invention to treat autoimmune and / or inflammatory disorders that include, but are not limited to, systemic lupus erythematosus ( SLE), discoid lupus, lupus nephritis, sarcoidosis, inflammatory arthritis, including, but not limited to, juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis and gouty arthritis, rejection of an organ or tissue transplant, hyperacute, acute or chronic rejection and / or graft versus host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, celiac disease (gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, thromboc purpura autoimmune pathogenic, autoimmune thyroiditis, Graves' disease, hasimotoid thyroiditis, complex immune disease, chronic fatigue syndrome and immune dysfunction (SFCDI), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris, pulmonary interstitial fibrosis, sarcoidosis, diabetes Type I and Type II, delayed type hypersensitivity of type 1, 2, 3 and 4, allergy or allergic disorders, unwanted / unwanted immune responses to therapeutic proteins, asthma, Churg-Strauss syndrome (allergic granulomatosis), dermatitis atopic, allergic and irritating contact dermatitis, urticaria, IgE-mediated allergy, atherosclerosis, vaculitis, idiomatic inflammatory myopathies, hemolytic disease, Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, and the like. In some other embodiments, antagonist anti-CD40 antibodies of the invention are useful in treating pulmonary inflammation including but not limited to pulmonary graft rejection, asthma, sarcoidosis, emphysema, cystic fibrosis, idiomatic pulmonary fibrosis, chronic bronchitis, allergic rhinitis and allergic lung diseases such as hypersensitivity pneumonitis, eosinophilic pneumonia, obliterating bronchiolitis due to bone marrow transplantation and / or of the lung, or other causes, graft atherosclerosis / graft flebosclerosis, as well as pulmonary fibrosis resulting from collagen, vascular and autoimmune diseases such as rheumatoid arthritis and lupus erythematosus.

"Treatment" is defined herein as the application or administration of an anti-CD40 antagonist antibody or its antigen binding fragment to a subject, or the application or administration of an anti-CD40 antagonist antibody or its binding fragment to antigen to an isolated tissue or cell line of a subject, where a subject who has an autoimmune disease and / or an inflammatory disease, a symptom of an autoimmune disease and / or an inflammatory disease or a predisposition towards an autoimmune disease and / or a inflammatory disease, where the goal is to cure, heal, soothe, relieve, alter, remedy, improve, make improvements in or affect autoimmune disease and / or inflammatory disease, any symptoms of autoimmune disease and / or inflammatory disease or predisposition to the development of autoimmune disease and / or inflammatory disease. By "treatment" is also understood the application or administration of a pharmaceutical composition comprising an anti-CD40 antagonist antibody or an antigen binding fragment thereof to a subject, or the application or administration of a pharmaceutical composition comprising an anti antibody -CD40 antagonist or an antigen binding fragment thereof to an isolated tissue or a cell line of the subject, where the subject has an autoimmune disease and / or an inflammatory disease, a symptom associated with an autoimmune disease and / or an inflammatory disease , or a predisposition towards an autoimmune disease and / or an inflammatory disease, the objective being to cure, heal, calm, relieve, alter, remedy, improve, make improvements in or affect autoimmune disease and / or inflammatory disease, the symptoms of autoimmune disease and / or inflammatory disease or predisposition towards autoimmune disease and / or disease i inflammatory.

"Anti-inflammatory activity" means a reduction or prevention of inflammation. Therapy with at least one anti-CD40 antibody (or its antigen binding fragment) as defined elsewhere herein causes a physiological response that is beneficial with respect to the treatment of an autoimmune disease and / or an inflammatory disease. , where the disease involves cells that express the CD40 antigen. It is recognized that the procedures described herein may be useful in preventing phenotypic change in cells such as proliferation, activation and the like.

In accordance with the methods disclosed herein, at least one anti-CD40 antagonist antibody (or its antigen binding fragment) as defined elsewhere in this document is used to promote a positive therapeutic response with regarding the treatment or prevention of an autoimmune disease and / or an inflammatory disease. By "positive therapeutic response" with respect to an autoimmune disease and / or an inflammatory disease is meant an improvement in the disease associated with the anti-inflammatory activity of these antibodies or their antigen-binding fragments, and / or an improvement in the associated symptoms. With the disease That is, an antiproliferative effect can be observed, the prevention of additional proliferation of cells expressing CD40, a reduction in the inflammatory response that includes but is not limited to reduced secretion of inflammatory cytokines, adhesion molecules, proteases, immunoglobulins (in cases where the cell carrying CD40 is a B cell), combinations thereof, and the like, increased production of anti-inflammatory proteins, a reduction in the number of self-reactive cells, an increase in immune tolerance, inhibition of self-reactive cell survival, and / or a decrease in one or more symptoms mediated by stimulation of cells expressing CD40. Such positive therapeutic responses are not limited by the route of administration and may comprise administration to the donor, to donor tissue (such as organ perfusion), to the host, any combination thereof and the like.

The clinical response can be evaluated using examination techniques such as magnetic resonance imaging (MRI), x-ray imaging, computerized tomography (CT) scan, flow cytometry or fluorescence-activated cell sorter analysis. (FACS), histology, macroscopic pathology and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography and the like. In addition to these positive therapeutic responses, the subject undergoing therapy with the anti-CD40 antagonist antibody or with its antigen binding fragment may experience the beneficial effect of an improvement in the symptoms associated with the disease.

By "therapeutically effective dose or amount" or "effective amount" is meant an amount of anti-CD40 antagonist antibody or its antibody binding fragment which, when administered, results in a positive therapeutic response with respect to the treatment of a subject with a disease that involves the stimulation of cells expressing CD40. In some embodiments, a therapeutically effective dose of an anti-CD40 antibody or its fragment is in the range from about 0.01 mg / kg to about 40 mg / kg, from about 0.01 mg / kg to about 30 mg / kg , from about 0.1 mg / kg to about 30 mg / kg, from about 1 mg / kg to about 30 mg / kg, from about 3 mg / kg to about 30 mg / kg, from about 3 mg / kg to about 25 mg / kg, from about 3 mg / kg to about 20 mg / kg, from about 5 mg / kg to about 15 mg / kg, or from about 7 mg / kg to about 12 mg / kg. It is recognized that the treatment method may comprise a single administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of the anti-CD40 antagonist antibody or its antigen binding fragment.

Another embodiment of the description is the use of anti-CD40 antagonist antibodies for the diagnostic control of protein levels in the tissue as part of a clinical testing procedure, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; An example of a fluorescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125I, 131I 35S or 3H.

Anti-CD40 antagonist antibodies and their antigen binding fragments can be used in combination with any known therapies for autoimmune diseases.

or inflammatory, including any agent or combination of agents that are known to be useful, or that have been used or are currently being used, for the treatment of autoimmune and inflammatory diseases. Such therapies and therapeutic agents include, but are not limited to, surgery or surgical procedures (for example, splenectomy, hepatectomy, lymphadenectomy, leukophoresis, cell, tissue or organ transplantation, intestinal procedures, organ perfusion and the like); radiation therapy; therapy such as steroid therapy and non-steroidal therapy, hormone therapy, cytokine therapy, therapy with dermatological agents (for example, topical agents used to treat skin conditions such as allergies, contact dermatitis and psoriasis), immunosuppressive therapy and distinct and similar monoclonal antibody anti-inflammatory therapy. Thus, the anti-CD40 antagonist antibodies described herein, or the antigen-binding fragments thereof are administered in combination with at least one other therapy, including but not limited to, surgery, organ perfusion, therapy. radiation, steroid therapy, nonsteroidal therapy, antibiotic therapy, antifungal therapy, hormone therapy, cytokione therapy, therapy with dermatological agents (for example topical agents used to treat skin conditions such as allergies, contact dermatitis and psoriasis ), immunosuppressive therapy, anti-inflammatory therapy of different monoclonal antibodies, combinations thereof and the like. Therefore, where the combination therapies comprise administration of an anti-CD40 antagonist antibody or its antibody binding fragment in combination with administration of another therapeutic agent and with steroids as an example, the methods disclosed herein comprise co-administration, using different formulations or an individual pharmaceutical formulation and consecutive administration in any order.

Where the methods disclosed herein comprise combined therapeutic regimens, these therapies can be given simultaneously, that is, the anti-CD40 antagonist antibody or its antigen binding fragment is administered simultaneously or within the same period of time that the other therapy (that is, the therapies take place simultaneously, but the anti-CD40 antagonist antibody or its antigen binding fragment is not administered precisely at the same time as the other therapy). Alternatively, the anti-CD40 antagonist antibody of the present invention or its antigen binding fragment can also be administered before, or after the other therapy. The sequential administration of the different cancer therapies can be carried out regardless of whether the treated subject responds to the first course of therapy to reduce the possibility of remission or relapse.

In some embodiments of the disclosure, the anti-CD40 antagonist antibodies described herein, or their antigen binding fragments, are administered in combination with immunosuppressive drugs or anti-inflammatory drugs, in which the antibody and the agent (s) ) therapeutic (s) can be administered sequentially, in any order, or simultaneously (that is, concurrently or within the same time frame). Examples of suitable immunosuppressive drugs that can be administered in combination with the antagonist anti-CDE40 antibodies of the invention include, but are not limited to, methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, such as, for example, aerosolized cyclosporine (see , United States Patent Application Publication No. US20020006901), tacrolimus (FK506, ProGraf ™), mycophenolate mofetil and azathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxyspergualine, leflunomon and its analogues of maltyl; and immunosuppressive proteins, including, for example, anti-CTLA4 antibodies and Ig condensations, anti-lymphocyte B-stimulating antibodies (for example LYMPHOSTAT-B ™) and Ig condensations (BLyS-Ig), anti-CD80 antibodies and etanercept (Enbrel®) , as well as anti-T cell antibodies such as anti-CD3 (OKT3), antiCD4 and the like. Examples of suitable anti-inflammatory agents include, but are not limited to, corticosteroids such as, for example, clobetasol, halobetasol, hydrocortisone, triamcyclolone, betamethasone, flucinocol, fluocinonide, prednisone, prednisolone, methylprednisolone, non-steroidal anti-inflammatory drugs (NSAIDs) such as NIDs (NSAIDs) such as NSAIDs, such as steroids for example, sulfasalazine, medications containing mesalamine (known as 5-ASA agents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen, oxaprozine, piroxyaprococcal, pheroxyaproxin, piroxyaproxin tomentine; anti-inflammatory antibodies such as adalidumab (HUMIRA®, a TNF-α antagonist) and infliximab (Remicade®, a TNF-α antagonist), and the like.

Transplant rejection and graft versus host disease can be hyperacute (humoral), acute (mediated by T cells), or chronic (unknown ethylogy), or a combination thereof. Accordingly, anti-CD40 antagonist antibodies of the invention are used in some embodiments to prevent and / or improve rejection and / or symptoms associated with hyperacute, acute, and / or chronic transplant rejection of any tissue, including, but not limited to, liver, kidney, pancreas, islet cells of the pancreas, small intestine, lung, heart, corneas, skin, blood vessels, bone, heterologous or antologous bone marrow and the like. The graft tissues can be obtained from any donor and can be transplanted into any recipient host and thus the transplant procedure may include transplanting animal tissue to humans (e.g., xenografts), transplanting tissue from one human being to another human being. (for example, allografts), and / or transplant tissue from one part of a human being to another (for example, autografts). Treatment with the antibodies of the invention may also reduce transplant sequelae such as fever, anorexia, hemodynamic abnormalities, leukopenia, infiltration of white cells from the transplanted tissue / organ, as well as opportunistic infections.

In some embodiments, the anti-CD40 antagonist antibodies of the invention can be used alone or in combination with immunosuppressive drugs to treat and / or prevent transplant rejection such as hyperacute, acute or chronic transplant rejection and / or graft disease versus host. Accordingly, in some embodiments where the anti-CD40 antagonist antibodies of the invention are used to treat and / or prevent transplant rejection, the antibodies may be used in combination with suitable immunosuppressive drugs, including, but not limited to, methotrexate, cyclophosphamide. , mizoribine, chlorambucil, cyclosporine, such as, for example, aerosolized cyclosporine (see, US Patent Application Publication No. US20020006901), tacrolimus (FK506, ProGraf ™), mycophenolate mofetil and azathioprine (6-mercaptopurine ), sirolimus (rapamycin), deoxiespergualine, leflunomide and their malononitriloamine analogues; and immunosuppressive proteins, including, for example, anti-CTLA4 antibodies and Ig condensations, anti-lymphocyte B-stimulating antibodies (for example LYMPHOSTAT-B ™) and Ig condensations (BLyS-Ig), anti-CD80 antibodies and etanercept (Enbrel®) , as well as T-cell antibodies such as anti-CD3 (OKT3), anti-CD4 and the like.

It is contemplated as such that the compositions and procedures described herein are used in combination with other drugs to further improve symptoms and consequences in transplant recipients, such as those receiving pulmonary grafts, for example. Accordingly, in some embodiments, the anti-CD40 antagonist antibodies of the invention were used to treat transplant rejection (such as, for example, hyperacute, acute and / or chronic rejection or graft versus host disease in lung transplant recipients). alone or in combination with cyclosporine administered parenterally and / or non-parenterally, including, for example, oral cyclosporine, injectable cyclosporine, aerosolized cyclosporine (eg, inhaled) and combinations thereof. In some embodiments where at least one component of the therapy is aerosolized cyclosporine, cyclosporine is administered to the recipient's lung by cyclosporine inhalation in the form of an aerosol spray using, for example, a pressurized delivery device or a nebulizer. Cyclosporine can be administered either in dry powder form or in wet form.

In some other embodiments, the anti-CD40 antagonist antibodies of the invention can be used alone or in combination with immunosuppressive drugs to treat and / or prevent rheumatoid arthritis. Accordingly in some embodiments where the anti-CD40 antagonist antibodies of the invention are used to treat rheumatoid arthritis. Accordingly in some embodiments where the anti-CD40 antagonist antibodies of the invention can be used to treat rheumatoid arthritis, the antibodies can be used in combination with suitable immunosuppressive drugs, including, but not limited to methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine. , tacrolimus (FK506, ProGraf ™), mycophenolate mofetil and azathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxiespergualine, leflunomide and their malononitriloamine analogues; and immunosuppressive proteins, including, for example, anti-CTLA4 antibodies and Ig condensations, anti-lymphocyte B-stimulating antibodies (for example LYMPHOSTAT-B ™) and Ig condensations (BLyS-Ig), anti-CD20 antibodies (for example RITUXAN®) , the fully human HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab (Bexxar®), ibritumomab tituxetan (Zevalin®) antibody; anti-CD80 antibodies, and etanercept (ENBREL®), as well as anti-T cell antibodies such as antiCD3 (OKT3), anti-CD4 and the like. As discussed above, the effectiveness of the treatment was evaluated using any means and includes, but is not limited to, effectiveness as measured by clinical responses defined by the American Collage of Rheumatology criteria, the European League of Rheumatism criteria or any others. criteria See for example, Nelson et al. (1995) Rheum arthritis. 38: 727-35 and van Gestel et al. (1996) Arthritis Rheum. 39: 34-40.

In still other embodiments, the anti-CD40 antagonist antibodies of the invention can be used alone or in combination with immunosuppressive drugs to treat multiple sclerosis, the antibodies can be used in combination with immunosuppressive drugs, including, but not limited to, methotrexate, cyclophosphamide. , mizoribine, chlorambucil, cyclosporine, tacrolimus (FK506, ProGraf ™), mycophenolate mofetil and azathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxiespergualin, leflunomide and their malononitrile analogues; and immunosuppressive proteins, which include, for example, anti-CTLA4 antibodies and Ig condensations, anti-lymphocyte B stimulating antibodies (for example LYMPHOSTATB ™) and Ig condensations (BLyS-Ig), anti-CD20 antibodies (for example RITUXAN®), the fully human HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab (Bexxar®), ibritumomab tituxetan (Zevalin®) antibody; anti-CD80 antibodies, and and etanercept (ENBREL®), as well as anti-T cell antibodies such as anti-CD3 (OKT3), antiCD4 and the like.

Pharmaceutical formulations and modes of administration

The anti-CD40 antagonist antibodies of this invention are administered in a concentration that is therapeutically effective for preventing or treating autoimmune diseases and / or anti-inflammatory diseases. To achieve this goal, antibodies can be formulated using a variety of acceptable excipients known in the art. Typically, antibodies are administered by injection, for example, either intravenously, either intraperitoneally or subcutaneously. The procedures for carrying out this administration are known to those skilled in the art. It is also possible to obtain compositions that can be administered topically or orally, or that may be capable of transmission through mucous membranes.

Intravenous administration is preferably performed by means of infusion over a period of about 1 to about 10 hours, more preferably for about 1 to about 8 hours, more preferably still for about 2 to about 7 hours, even more preferably during about 4 to about 6 hours, depending on the anti-CD40 antibody that is administered. The initial infusion with the pharmaceutical composition can be administered over a period of about 4 to about 6 hours with subsequent infusions administered more quickly. Subsequent infusions can be administered over a period of about 1 to about 6 hours, including, for example, about 1 to about 4 hours, about 1 to about 3 hours, or about 1 to about 2 hours.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion), oral and pulmonary (e.g., inhalation), nasal, transdermal administration (topical), transmucosal and rectal. The solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, non-volatile oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabenos; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride

or dextrose The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparation can be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Anti-CD40 antagonist antibodies are typically provided by conventional techniques in a pharmaceutically acceptable buffer, for example, sterile saline, sterile buffered water, propylene glycol, combinations of the above, etc. Procedures for preparing agents that can be administered parenterally are described in Remington’s Pharmaceutical Sciences (18th ed .; Mack Publishing Company, Eaton, Pennsylvania, 1990). See also, for example, WO 98/56418, which describes pharmaceutical formulations of stabilized antibodies suitable for use in the methods described in the present invention.

The amount of at least one anti-CD40 antagonist antibody or its fragment to be administered will be readily determined by someone of ordinary skill in the art without undue experimentation. Factors that influence the mode of administration and the respective amount of at least one anti-CD40 antagonist antibody (or fragment thereof) include, but are not limited to, the particular disease undergoing therapy, the severity of the disease, the history. of the disease, and the age, height, weight, health and physical condition of the individual undergoing therapy. Similarly, the amount of anti-CD40 antagonist antibody or its fragment to be administered will depend on the mode of administration and whether the subject will be subjected to one or multiple doses of this agent. Generally, as the weight of the subject under treatment increases, a higher dose of the anti-CD40 antibody or its fragment is preferably preferred. The dose of the anti-CD40 antibody or its fragment to be administered is in the range of about 0.003 mg / kg to about 50 mg / kg, preferably in the range of 0.01 mg / kg to about 40 mg / kg. Therefore, for example, the dose may be 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg , 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg or 50 mg / kg.

In another embodiment of the disclosure, the method comprises the administration of multiple doses of anti-CD40 antagonist antibody or its fragment. The method may comprise the administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more therapeutically effective doses of a pharmaceutical composition comprising a anti-CD40 antagonist antibody or its fragment. The frequency and duration of administration of the multiple doses of the pharmaceutical compositions comprising anti-CD40 antibody or its fragment can easily be determined by one skilled in the art without undue experimentation. In addition, treatment of a subject with a therapeutically effective amount of an antibody may include a single treatment or, preferably, may include a series of treatments. In a preferred example, the subject is treated with anti-CD40 antagonist antibody or its antigen binding fragment in the range of about 0.1 to 20 mg / kg body weight, once a week for about 1 to 1 10 weeks, preferably about 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Treatment can be carried out annually to prevent relapse or after indication of relapse. It will also be appreciated that the effective dose of antibody or its antigen binding fragment used for the treatment may increase or decrease in the course of a particular treatment. Dosage changes may be the result and may be apparent from the results of diagnostic tests as described herein. Accordingly, in one embodiment, the dosage regimen includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or its fragment on days 1, 7, 14 and 21 of a treatment period. In another embodiment, the dosage regimen includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or its fragment on days 1, 2, 3, 4, 5, 6 and 7 of a week in a period of treatment. Additional embodiments include a dosage regimen having a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or its fragment on days 1, 3, 5 and 7 of a week in a treatment period; a dosage regimen includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or its fragment on days 1 and 3 of a week in a treatment period; and a preferred dosage regimen that includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or its fragment on day 1 of a week in a treatment period. The treatment period may comprise 1 week, 2 weeks, 3 weeks, one month, 3 months, 6 months or one year. The treatment periods can be consecutive or they can be separated from each other by a day, a week, 2 weeks, a month, 3 months, 6 months or a year.

It ranges from about 0.003 mg / kg to about 50 mg / kg, from about 0.01 mg / kg to about 40 mg / kg, from about 0.01 mg / kg to about 30 mg / kg, from about 0.1 mg / kg to about 30 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 1 mg / kg to about 30 mg / kg, from about 3 mg / kg to about 30 mg / kg, from about 3 mg / kg to about 25 mg / kg, from about 3 mg / kg to about 20 mg / kg, from about 5 mg / kg to about 15 mg / kg, or from about 7 mg / kg to about 12 mg / kg Therefore, for example, the dose of any anti-CD40 antagonist antibody or its antigen binding fragment, for example the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9 or its antigen binding fragment, can be 0.003 mg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg , 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, or another similar dose that is in the range from about 0.003 mg / kg to about 50 mg / kg. The same therapeutically effective dose of an anti-CD40 antagonist antibody or its antigen binding fragment may be administered throughout each week of antibody dosing. Alternatively, different therapeutically effective doses of an anti-CD40 antagonist antibody or its antigen binding fragment can be used during the course of a treatment period.

In some embodiments, the initial therapeutically effective dose of an anti-CD40 antagonist antibody or its antigen-binding fragment as defined elsewhere herein may be in the lower dosage range (i.e., from about 0.003 mg / kg up to about 20 mg / kg) with subsequent doses within the upper dosage range (i.e., from about 20 mg / kg to about 50 mg / kg).

In alternative embodiments, the initial therapeutically effective dose of an antagonistic antiCD40 antibody or its antigen binding fragment as defined elsewhere herein may be in the upper dosage range (i.e., from about 20 mg / kg to approximately 50 mg / kg) with subsequent doses within the lower dosage range (ie, from 0.003 mg / kg to approximately 20 mg / kg). Accordingly, in one embodiment, the initial therapeutically effective dose of the anti-CD40 antagonist antibody or its antigen binding fragment is from about 20 mg / kg to about 35 mg / kg, including about 20 mg / kg, about 25 mg / kg, about 30 mg / kg and about 35 mg / kg, and subsequent therapeutically effective doses of the anti-CD40 antagonist antibody or its antigen binding fragment are from about 5 mg / kg to about 15 mg / kg, including about 5 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg and approximately 15 mg / kg.

In some embodiments of the disclosure, treatment with anti-CD40 antagonist antibody is initiated by administering a "loading dose" of the antibody or its antigen-binding fragment to the subject in need of therapy with anti-CD40 antagonist antibodies. By "loading dose" is meant an initial dose of the anti-CD40 antagonist antibody or its antigen-binding fragment that is administered to the subject, in which the dose of the antibody or its antigen-binding fragment administered is within the range of higher dosage (ie, from about 20 mg / kg to about 50 mg / kg). The "loading dose" can be administered as a single administration, for example, a single infusion in which the antibody or its antigen binding fragment is administered via IV, or as multiple administrations, for example, multiple infusions in which the antibody or its antigen binding fragment is administered IV, provided that the full "loading dose" is administered over a period of approximately 24 hours. After administration of the "loading dose", one or more additional therapeutically effective doses of the anti-CD40 antagonist antibody or its antigen binding fragment are then administered to the subject. Subsequently therapeutically effective doses may be administered, for example, according to a weekly dosing schedule, or once every two weeks, once every three weeks,

or once every four weeks. In such embodiments, subsequent therapeutically effective doses are generally in the lower dosage range (ie, from 0.003 mg / kg to about 20 mg / kg).

Alternatively, in some embodiments, after the "loading dose", subsequent therapeutically effective doses of the antagonist anti-CD40 antibody or its antigen binding fragment are administered according to a "maintenance schedule", in which the therapeutically effective dose of the antibody or its antigen binding fragment is administered once a month, once every 6 weeks, once every two months, once every 10 weeks, once every three months, once every 14 weeks, once every four months, once every 18 weeks, once every five months, once every 22 weeks, once every six months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once every 12 months. In such embodiments, the therapeutically effective doses of the anti-CD40 antagonist antibody or its antigen binding fragment are in the lower dosage range (i.e., from 0.003 mg / kg to about 20 mg / kg), in particular when the doses Thereafter they are administered at more frequent intervals, for example, once every two weeks up to once a month, or within the upper dosage range (i.e., from about 20 mg / kg to about 50 mg / kg), particularly when Subsequent doses are administered at less frequent intervals, for example, when subsequent doses are administered separately for approximately one month to approximately 12 months.

The anti-CD40 antagonist antibodies present in the pharmaceutical compositions described herein for use in the methods disclosed herein may be native or may be obtained by means of recombinant techniques and may be from any source, including sources of mammals such as, for example, mouse, rat, rabbit, primate, pig and human. Preferably such polypeptides are obtained from a human source, and more preferably they are human proteins, recombinant of hybridoma cell lines.

Pharmaceutical compositions useful in the invention may comprise biologically active variants of the anti-CD40 antagonist antibodies of the invention. Such variants should retain the desired biological activity of the native polypeptide such that the pharmaceutical composition comprising the variant polypeptide has the same therapeutic effect as the pharmaceutical composition comprising the native polypeptide when administered to a subject. That is, the variant anti-CD40 antibody will serve as a therapeutically active component in the pharmaceutical composition in a manner similar to that observed for the native antagonist antibody, for example CHIR-5.9 or CHIR-12.12 as expressed by the hybridoma cell line 5.9 or 12.12, respectively. Methods are available in the art to determine if a variant anti-CD40 antibody retains the desired biological activity, and therefore serves as a therapeutically active component in the pharmaceutical composition. The biological activity of the antibody variants can be measured using assays specifically designed to measure the activity of the native antagonist antibody, including the assays described herein.

Any pharmaceutical composition comprising an anti-CD40 antagonist antibody having the binding properties described herein as the therapeutically active component can be used in the methods disclosed herein. Accordingly, liquid, lyophilized or spray dried compositions comprising one or more of the anti-CD40 antagonist antibodies of the invention may be prepared as an aqueous or non-aqueous solution or suspension for subsequent administration to a subject according to the invention. . Each of these compositions will comprise at least one of the anti-CD40 antagonist antibodies of the present invention as a therapeutically or prophylactically active component. By "therapeutically or prophylactically active component" is meant that the antiCD40 antibody is specifically incorporated into the composition to elicit a therapeutic or prophylactic response with respect to the treatment, prevention or diagnosis of a disease or condition in a subject when the composition is administered. Pharmaceutical to that subject. Preferably the pharmaceutical composition comprises stabilizing agents, suitable loading agents, or both to minimize the problems associated with the loss of stability and biological activity of the proteins during preparation and storage.

Formulation compounds may be added to pharmaceutical compositions comprising an anti-CD40 antagonist antibody of the invention. These formulation compounds may include, but are limited to, oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants or fillers. Preferred carbohydrates include sugars or sugar alcohols such as mono-, di- or polysaccharides, or water soluble glucans. Saccharides or glucans may include fructose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulane, dextrin, cyclodextrin α and β, soluble starch, hydroxyethyl starch and carboxymethyl cellulose, or mixtures thereof. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having a hydroxyl group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol and arabitol. These sugars or sugar alcohols can be used individually or in combination. The concentration of sugar or sugar alcohol is between 1.0% and 7% w / v, more preferably between 2.0% and 6.0% w / v. Preferred amino acids include levógira (L) forms of carnitine, arginine, and betaine; however, other amino acids can be added. Preferably the polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. The surfactants that can be added to the formulation are shown in EP documents No. 270,799 and

268,110.

In addition, the antibodies can be chemically modified by covalent conjugation to a polymer to, for example, increase their circulation half-life. Preferred polymers and methods for linking them to peptides are shown in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R (O-CH2 -CH2) n O-R where R can be hydrogen, or a protecting group such as an alkyl or alkanol group. Preferably, the protecting group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG preferably has an average molecular weight between 1,000 and 40,000, more preferably between 2,000 and 20,000, most preferably between 3,000 and 12,000. Preferably, the PEG has at least one hydroxyl group, more preferably it is a terminal hydroxy group. This is the hydroxy group that is preferably activated to react with a free amino group in the inhibitor. However, it will be understood that the type and amount of the reactive groups can be varied to obtain a covalently conjugated PEG / antibody of the present invention.

Water soluble polyoxyethylated polyols are also useful in the present invention. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG) and the like. POG is preferred. One reason is because the glycerol central structure of polyoxyethylated glycerol is the same central structure that occurs in nature in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching will not necessarily be considered as a foreign agent in the body. The POG has a molecular weight of preference in the same range as PEG. The structure for the POG is shown in Knauf et al. (1988) J. Bio. Chem. 263: 15064-15070, and an analysis of POG / IL-2 conjugates can be found in US Patent No. 4,766,106.

Another drug delivery system to increase circulatory half-life is the liposome. Procedures for preparing liposome delivery systems are discussed in Gabizon et al. (1982) Cancer Research 42: 4734; Cafiso (1981) Biochem Biophys Acta 649: 129; and Szoka (1980) Ann. Rev. Bioplsys. Eng. 9: 467. Other drug delivery systems are known in the art and are described, for example, in Poznansky et al. (1980) Drug Delivery Systems (R.L. Juliano, ed., Oxford, N.Y.) pages 253-315; Poznansky (1984) Pharm Revs 36: 277.

The formulation compounds to be incorporated into a pharmaceutical composition should provide stability of the anti-CD40 antagonist antibody or its antigen binding fragment. That is, the anti-CD40 antagonist antibody or its antigen binding fragment should retain its physical and / or chemical stability and should have the desired biological activity, i.e. one or more of the activities defined hereinbefore, including , but not limited to, inhibition of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by cells expressing CD40L or soluble CD40 ligand (sCD40L); inhibition of "survival" intracellular antiapoptotic signals in any cell stimulated by solid phase sCD40L or CD40L; inhibition of CD40 signal transduction in any cell after binding with solid phase sCD40L or CD40L; and inhibition of the proliferation of malignant human B cells as noted elsewhere herein.

Procedures for controlling protein stability are well known in the art. See, for example, Jones (1993) Adv Drug Delivery Rev. 10: 29-90; Lee, ed. (1991) Peptide and Protein Drug Delivery (Marcel Dekker, Inc., New York, New York); and the stability tests disclosed in this document below. In general, protein stability is measured at a chosen temperature for a specified period of time. In preferred embodiments, a stable antibody pharmaceutical formulation provides stability of the anti-CD40 antagonist antibody or its antigen binding fragment when stored at room temperature (approximately 25 ° C) for at least 1 month, at least 3 months, or at least 6 months, and / or is stable at approximately 2-8 ° C for at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months.

A protein such as an antibody, when formulated in a pharmaceutical composition, is considered to maintain its physical stability at a given time if it does not show visual signs (i.e. discoloration or loss of transparency) or signs that can be measured (for example, using size exclusion chromatography (SEC) or UV light scattering) of precipitation, aggregation, and / or denaturation in that pharmaceutical composition. With respect to chemical stability, a protein such as an antibody, when formulated in a pharmaceutical composition, is considered to maintain its chemical stability at a given time point if chemical stability measures are indicative of the protein (i.e., the antibody) maintains its biological activity of interest in that pharmaceutical composition. Procedures for controlling changes in chemical stability are well known in the art and include, but are not limited to, procedures for detecting chemically altered forms of the protein as resulting from the cut, using, for example, SDSPAGE, SEC, and / or matrix-assisted desorption ionization / dispersion mass spectrometry; and degradation associated with changes in molecular charge (for example, associated with deamidation), using, for example, ion exchange chromatography. See, for example, the procedures disclosed in this document below.

It is considered that an anti-CD40 antagonist antibody or its antigen binding fragment, when formulated in a pharmaceutical composition, maintains a desired biological activity at a given time point if the desired biological activity at that time is within 30%, of preference within approximately 20% of the desired biological activity exhibited at the time the pharmaceutical composition was prepared as determined in an assay suitable for the desired biological activity. Assays for measuring the desired biological activity of the anti-CD40 antagonist antibodies disclosed herein, and their fragments that bind to the antigen, can be carried out as described in the Examples herein. See also the tests described in Schultze et al. (1998) Proc. Natl Acad. Sci. USA 92: 8200-8204; Denton et al. (1998) Pediatr. Transplant 2: 6-15; Evans et al. (2000) J. Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13: 12; Kwekkeboom et al. (1993) Immunology 79: 439-444; and U.S. Patent Nos. 5,674,492 and 5,847,082.

In some embodiments of the invention, the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment is formulated in a liquid pharmaceutical formulation. The anti-CD40 antagonist antibody or its antigen binding fragment can be prepared using any method known in the art, including the methods disclosed herein above. In one embodiment, the antagonist anti-CD40 antibody, for example, the CHIR monoclonal antibody

12.12 or CHIR-5.9, or its antigen binding fragment is produced recombinantly in a CHO cell line.

Upon preparation and purification, the anti-CD40 antagonist antibody or its antigen binding fragment can be formulated as a liquid pharmaceutical formulation in the manner set forth herein. Where the anti-CD40 antagonist antibody or its antigen binding fragment must be stored before formulation, they can be frozen, for example, at ≤ -20 ° C, and then thawed at room temperature for further formulation. The liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody or its antigen binding fragment. The amount of antibody or its antigen binding fragment present in the formulation takes into account the route of administration and the desired dose volume.

Thus, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR-5.9 antibody, or its antigen binding fragment in a concentration of about 0.1 mg / ml to about 50 , 0 mg / ml, approximately 0.5 mg / ml to approximately 40.0 mg / ml, approximately 1.0 mg / ml to approximately 30.0 mg / ml, approximately 5.0 mg / ml to approximately 25.0 mg / ml, approximately 5.0 mg / ml to approximately 20.0 mg / ml, or approximately 15.0 mg / ml to approximately 25.0 mg / ml. In some embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or its antigen binding fragment in a concentration of about 0.1 mg / ml to about 5.0 mg / ml, about 5.0 mg / ml to approximately 10.0 mg / ml, approximately 10.0 mg / ml to approximately 15.0 mg / ml, approximately 15.0 mg / ml to approximately 20.0 mg / ml, approximately 20.0 mg / ml to approximately 25 , 0 mg / ml, approximately 25.0 mg / ml to approximately 30.0 mg / ml, approximately 30.0 mg / ml to approximately 35.0 mg / ml, approximately 35.0 mg / ml to approximately 40.0 mg / ml, approximately 40.0 mg / ml to approximately 45.0 mg / ml, or approximately 45.0 mg / ml to approximately 50.0 mg / ml. In other embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or its antigen binding fragment in a concentration of about 15: 0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, approximately 18.0 mg / ml, approximately 19.0 mg / ml, approximately 20.0 mg / ml, approximately 21.0 mg / ml, approximately 22.0 mg / ml, approximately 23.0 mg / ml, approximately 24 , 0 mg / ml, or about 25.0 mg / ml. The liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR-5.9 antibody, or its antigen binding fragment and a buffer that maintains the pH of the formulation in the range of approximately pH 5, 0 to about pH 7.0, including about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 , 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and other such values within from the range of about pH 5.0 to about pH 7.0. In some embodiments, the buffer maintains the pH of the formulation in the range of about pH 5.0 to about pH 6.5, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5, 5, about pH 5.5 to about 7.0, about pH 5.5 to about pH 6.5, or about pH 5.5 to about pH 6.0.

Any suitable buffer that maintains the pH of the liquid anti-CD40 antibody formulation in the range of about pH 5.0 to about pH 7.0 may be used in the formulation, provided that the physicochemical stability and the desired biological activity of the antibody are keep as noted earlier in this document. Buffers include but are not limited to conventional acids and their salts, where the counterion can be, for example, sodium, potassium, ammonium, calcium or magnesium. Examples of conventional acids and their salts that can be used to buffer the liquid pharmaceutical formulation include, but are not limited to, succinic acid or succinate, citric acid or citrate buffers, acetic acid or acetate, tartaric acid or tartrate, phosphoric acid or phosphate, gluconic acid or gluconate, glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate and malic or malate acid. The concentration of the buffer in the formulation can be from about 1 mM to about 50 mM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other similar values within the range of about 1 mM to about 50 mM. In some embodiments, the concentration of the buffer in the formulation is from about 5 mM to about 15 mM, including about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or other similar values within the range of about 5 mM to about 15 mM.

In some embodiments of the invention, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the antagonist anti-CD40 antibody, for example, the CHIR 12,12 or CHIR-5.9 monoclonal antibody, or its antigen-binding fragment and succinate buffer or buffer. Citrate at a concentration that maintains the pH of the formulation in the range of about pH 5.0 to about pH 7.0, preferably about pH 5.0 to about pH 6.5. By "succinate buffer" or "citrate buffer" is meant a buffer comprising a succinic acid salt or a citric acid salt, respectively. In a preferred embodiment, the succinate or citrate counterion is the sodium cation, therefore the buffer is sodium succinate or sodium citrate, respectively. However, any cation is expected to be effective. Other possible succinate or citrate cations include, but are not limited to, potassium, ammonium, calcium and magnesium. As noted above, the concentration of the succinate or citrate buffer in the formulation may be from about 1 mM to about 50 mM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other such values within the range from about 1 mM to about 50 mM. In some embodiments, the concentration of buffer in the formulation is from about 5 mM to about 15 mM, including about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or about 15 mM. In other embodiments, the liquid pharmaceutical formulation comprises the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment at a concentration of about 0.1 mg / ml to about 50.0 mg / ml, or about 5.0 mg / ml to about 25.0 mg / ml, and succinate or citrate buffer, for example, sodium succinate buffer or sodium citrate, at a concentration of about 1 mM to about 20 mM, about 5 mM to about 15 mM, preferably about 10 mM.

Where it is desirable that the liquid pharmaceutical formulation is practically isotonic, the liquid pharmaceutical formulation comprising a therapeutically effective amount of the antagonist anti-CD40 antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR5.9, or its antigen binding fragment , and a buffer to maintain the pH of the formulation within the range of about pH 5.0 to about pH 7.0 may further comprise an amount of an isotonicity agent sufficient to make the formulation practically isotonic. By "practically isotonic" is meant that the aqueous formulation has an osmolarity of about 240 mmol / kg to about 360 mmol / kg, preferably about 240 to about 340 mmol / kg, more preferably about 250 to about 330 mmol / kg, even more preferably about 260 to about 320 mmol / kg, more preferably still about 270 to about 310 mmol / kg. The procedures for determining the isotonicity of a solution are known to those skilled in the art. See, for example, Setnikar et al. (1959) J. Am. Pharm. Assoc. 48: 628.

Those skilled in the art are familiar with a variety of pharmaceutically acceptable solutes useful for providing isotonicity in pharmaceutical compositions. The isotonicity agent can be any reagent capable of adjusting the osmotic pressure of the liquid pharmaceutical formulation of the present invention to a value practically equal to that of a body fluid. It is desirable to use a physiologically acceptable isotonicity agent. Accordingly, the liquid pharmaceutical formulation comprising a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, and a buffer to maintain the pH of The formulation within the range of about pH 5.0 to about pH 7.0 may further comprise components that can be used to provide isotonicity, for example, sodium chloride; amino acids such as alanine, valine and glycine; sugars and sugar alcohols (polyols), including, but not limited to, glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol; acetic acid, other organic acids and their salts, and relatively minor amounts of citrates or phosphates. The person skilled in the art will know other agents that are suitable to provide the optimum tonicity of the liquid formulation.

In some preferred embodiments, the liquid pharmaceutical formulation comprising a therapeutically effective amount of an anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, and a buffer for maintaining the pH of the formulation within the range of about pH 5.0 to about pH 7.0 further comprises sodium chloride as the isotonicity agent. The concentration of sodium chloride in the formulation will depend on the contribution of other components to the tonicity. In some embodiments, the concentration of sodium chloride is about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 50 mM to about 150 mM , approximately 75 mM to approximately 175 mM, approximately 75 mM to approximately 150 mM, approximately 100 mM to approximately 175 mM, approximately 100 mM to approximately 200 mM, approximately 100 mM to approximately 150 mM, approximately 125 mM to approximately 175 mM, approximately 125 mM to approximately 150 mM, approximately 130 mM to approximately 170 mM, approximately 130 mM to approximately 160 mM, approximately 135 mM to approximately 155 mM, approximately 140 mM to approximately 155 mM, or approximately 145 mM to approximately 155 mM. In such an embodiment, the concentration of sodium chloride is approximately 150 mM. In another such embodiment, the concentration of sodium chloride is about 150 mM, the buffer is sodium succinate buffer or sodium citrate in a concentration of about 5 mM to about 15 mM, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment and the formulation has a pH of about pH 5.0 to about pH 7.0, about pH 5, 0 to about pH 6.0, or about pH 5.5 to about pH 6.5. In other embodiments, the liquid pharmaceutical formulation comprises the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, at a concentration of approximately 0.1 mg / ml to approximately 50.0 mg / ml or approximately 5.0 mg / ml to approximately 25.0 mg / ml, approximately 150 mM sodium chloride and approximately 10 mM sodium succinate or sodium citrate, at a pH of approximately pH 5, 5.

Protein degradation by freezing thawing or mechanical shearing during the processing of a liquid pharmaceutical formulation of the present invention can be inhibited by incorporation of surfactants in the formulation to decrease surface tension at the solution-air interface. Accordingly, in some embodiments, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the antagonist anti-CD40 antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR 5.9, or its antigen binding fragment, a buffer to maintain The pH of the formulation within the range of about pH 5.0 to about pH 7.0 and further comprises a surfactant. In other embodiments, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, a buffer to maintain the pH of the formulation within the range of about pH 5.0 to about pH 7.0, an isotonicity agent such as sodium chloride in a concentration of about 50 mM to about 300 mM, and further comprises a surfactant.

Typical surfactants used are nonionic surfactants, including polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); polyoxypropylene-polyoxyethylene esters such as Pluronic F68; polyoxyethylene alcohols such as Brij 35; simethicone; polyethylene glycol such as PEG400; lysophosphatidylcholine; and polyoxyethylene-p-toctylphenol such as Triton X-100. Classical stabilization of pharmaceutical compounds by means of surfactants or emulsifiers is described, for example, in Levine et al. (1991) J. Parenteral Sci. Technol. 45 (3): 160-165. A preferred surfactant used in the practice of the present invention is polysorbate 80. When a surfactant is included, it is typically added in an amount from about 0.001% to about 1.0% (w / v), about 0.001% to about 0.5%, approximately 0.001% to approximately 0.4%, approximately 0.001% to approximately 0.3%, approximately 0.001% to approximately 0.2%, approximately 0.005% to approximately 0.5%, approximately 0.005% to approximately 0.2%, about 0.01% to about 0.5%, about 0.01% to about 0.2%, about 0.03% to about 0.5%, about 0.03% to about 0, 3%, about 0.05% to about 0.5%, or about 0.05% to about 0.2%.

Accordingly, in some embodiments, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the antagonist anti-CD40 antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, the buffer is buffer of sodium succinate or sodium citrate in a concentration of about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 5 mM to about 15 mM; the formulation has a pH of about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, or about pH 5.5 to about pH 6.5 and the formulation further comprises a surfactant, for example, polysorbate 80, in an amount from about 0.001% to about 1.0% or about 0.001% to about 0.5%. Such formulations may optionally comprise an isotonicity agent, such as sodium chloride in a concentration of about 50 mM to about 300 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM. In other embodiments, the liquid pharmaceutical formulation comprises the anti-CD40 antagonist antibody, for example, the CHIR monoclonal antibody.

12.12 or CHIR-5.9, or its antigen binding fragment, in a concentration of about 0.1 mg / ml to about 50.0 mg / ml or about 5.0 mg / ml to about 25.0 mg / ml, including approximately 20.0 mg / ml; about 50 mM sodium chloride to about 200 mM, including about 150 mM sodium chloride; sodium succinate or sodium citrate at a concentration of about 5 mM to about 20 mM; including sodium succinate or sodium citrate at about 10 mM; sodium chloride at a concentration of about 50 mM to about 200 mM, including about 150 mM; and optionally a surfactant, for example, polysorbate 80, in an amount from about 0.001% to about 1.0%, including about 0.001% to about 0.5%; where the liquid pharmaceutical formulation has a pH of about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about pH 6.5, or about pH 5.5 to about pH 6.0.

The liquid pharmaceutical formulation may be essentially free of preservatives and other vehicles, excipients, or stabilizers noted hereinbefore. Alternatively, the formulation may include one or more preservatives, for example, pharmaceutically acceptable antibacterial agents, carriers, excipients or stabilizers described hereinbefore with the proviso that they do not adversely affect the physicochemical stability of the antagonist anti-CD40 antibody. or its antigen binding fragment. Examples of acceptable carriers, excipients or stabilizers include, but are not limited to, additional buffering agents, co-solvents, surfactants, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (eg, Zn-protein complexes) , and biodegradable polymers such as polyesters. A detailed discussion of formulation and selection of pharmaceutically acceptable vehicles, stabilizers and isomolites can be found in Remington’s Pharmaceutical Sciences (18th ed .; Mack Publishing Company, Eaton, Pennsylvania, 1990).

Once the liquid pharmaceutical formulation or other pharmaceutical composition described herein is prepared, it can be lyophilized to prevent degradation. Procedures for lyophilizing liquid compositions are known to those skilled in the art. Just before using, the composition can be reconstituted with a sterile diluent (Ringer's solution, distilled water or saline, for example) that may include additional components. After reconstitution, the composition is preferably administered to the subjects using procedures known to those skilled in the art.

Use of anti-CD40 antagonist antibodies in the manufacture of medicines

The present invention also provides the use of an anti-CD40 antagonist antibody or its antigen binding fragment in the manufacture of a medicament for the treatment of an autoimmune disease and / or an inflammatory disease in a subject, in which the medicament is coordinated with treatment with at least one different therapy. By "coordinated" it is meant that the medication will be used before, during, or after the treatment of the subject with at least one different therapy. Examples of other therapies include, but are not limited to, those described above herein, that is, surgery or surgical procedures (eg, splenectomy, hepatectomy, lymphadenectomy, leukophoresis, cell, tissue or organ transplantation, intestinal procedures , organ perfusion and the like); radiation therapy; therapy such as steroid therapy and non-steroidal therapy, hormone therapy, cytokine therapy, therapy with dermatological agents (for example, topical agents used to treat skin conditions such as allergies, contact dermatitis and psoriasis), immunosuppressive therapy and distinct and similar monoclonal antibody anti-inflammatory therapy, where treatment with additional therapy or with additional therapies takes place before, during, or after the treatment of the subject with the medication to treat an autoimmune disease and / or an inflammatory disease in a subject , in which the medication is coordinated with the treatment with at least one different therapy as noted hereinbefore.

In some embodiments, the medicament comprising the anti-CD40 antagonist antibody, for example the CHIR-12.12 or CHIR-5.9 monoclonal antibody disclosed herein, or its antigen binding fragment, is coordinated with treatment with two other therapies When the medication comprising the anti-CD40 antagonist antibody is coordinated with the treatment with two other therapies, the use of the medication may be before, during, or after the treatment of the subject with one or both of the other therapies.

The present invention also provides the use of an anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9 disclosed herein,

or its antigen-binding fragment in the manufacture of a medicament for treating an autoimmune disease and / or an inflammatory disease in a subject, in which the medicament is used in a subject that has been pretreated with at least one other therapy. By "pretreated" or "pretreatment" is meant that the subject has been treated with one or more

therapies, before receiving the medicine comprising the anti-CD40 antagonist antibody

or its antigen binding fragment. "Pretreated" or "pretreatment" includes subjects who have been treated with the other therapy or other therapies within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks , within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days , or even within 1 day before the start of treatment with the medicament comprising the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 disclosed herein, or its binding fragment to the antigen. It is not necessary that the subject has been a patient who responds to previous treatment with the previous therapy or previous therapies. Therefore, the subject receiving the medicament comprising the anti-CD40 antagonist antibody or its antigen binding fragment could have responded, or could have failed to respond, to pretreatment with the previous cancer therapy, or to one or more therapies. previous where the previous treatment included multiple therapies.

The following examples are offered by way of illustration and not as a limitation.

EXPERIMENTAL

The anti-CD40 antagonist antibodies used in the examples below are CHIR-5.9 and CHIR-12.12. The anti-CD40 antibodies CHIR-5.9 and CHIR-12.12 are human anti-CD40 monoclonal antibodies (mAb) of the human IgG1 subtype generated by immunization of transgenic mice carrying the human IgG1 heavy chain locus and the human K light chain locus (XenoMouse® technology; Abgenix; Fremont, California). SF9 insect cells that express CD40 extracellular domain are used as immunogens.

In summary, splenocytes from mice immunized with murine myeloma cells SP 2/0 or P 3 x 63 Agg 6,653 were condensed in a 10: 1 ratio using 50% polyethylene glycol as previously described by de Boer et al. (1988) J. Immunol. Meth. 113: 143. The condensed cells were resuspended in complete IMDM medium supplemented with hypoxanthine (0.1 mM), aminopterin (0.01 mM), thymidine (0.016 mM), and hIL-6 0.5 ng / ml (Genzyme, Cambridge, Massachusetts). The fused cells were then distributed between the wells of 96-well tissue culture plates, so that each contained 1 growing hybridoma on average.

After 10-14 days the supernatants of the hybridoma populations were analyzed to detect the production of specific antibodies. For the detection of specific antibodies by hybridoma clones, supernatants from each well were combined and tested for specificity of anti-CD 40 activity by ELISA first. They were then used

5 positive for fluorescent staining of cells of B cells transformed with EBV as described for the FACS assay above. Positive hybridoma cells were cloned twice by limiting dilution in IMDM / FBS containing 0.5 ng / ml hIL-6.

A total of 31 splenocytes were condensed with the murine myeloma cells SP 2/0 to

10 generate 895 antibodies that recognize the recombinant CD40 in ELISA. On average about 10% of hybridomas produced using Abgenix XenoMouse® technology may contain the mouse lambda light chain instead of the human kappa chain. Antibodies containing mouse lambda light chain were selected and discarded. A subset of 260 antibodies was selected that also showed binding to the

15 CD40 cell surface for another analysis. Stable hybridomas selected during a series of subcloning procedures were used for additional characterization in binding and functional assays.

Clones of another 7 hybridomas that had antagonistic activity were identified. Based on your

20 relative antagonistic potency and ADCC activities, two hybridoma clones were selected for further evaluation. They are called: 131.2F8.5.9 (5.9) and 153.8E2.D10, D6.12.12 (12.12).

Table 1. Summary of the initial data set with CHIR-5.9 and CHIR anti-CD40 IgG1 antibodies

12.12.

25

Hybridoma to mother

Hybridoma clones cell surface junction Antagonist to ADC C CD C CMCC # DNA sequence of region V

131.2F5

131.2F5.8.5.9 +++ +++ ++ - 12047 Yes

153.8E2

153.8E2D10D6.12 .9 +++ ND ND ND 12063 Yes

The mouse hybridoma line 131.2F8.5.9 (CMCC No. 12047) and the hybridoma line 153.8E2.D10.D6.12.12 (CMCC No. 12056) have been deposited with the American Type Culture

Collection (ATCC; 10801 University Blvd., Manassas, Virginia 20110-22209 (USA)) under Patent Deposit Number PTA-5542 and PTA-5543, respectively.

The cDNAs encoding the variable regions of the candidate antibodies were amplified by PCR, cloned and sequenced. The amino acid sequences for the light chain and heavy chain of the CHIR-12.12 antibody were set forth in Figures 1A and 1B, respectively. See also SEQ ID NO: 2 (light chain for CHIR-12.12 mAb) and SEQ ID NO: 4 (heavy CHIR-12.12 mAb chain). A variant of the CHIR-12.12 mAb heavy chain is shown in Figure 1B (see also SEQ ID NO: 5), which differs from SEQ ID NO: 4 in that it has a serine residue substituted by the alanite residue in position 153 of SEQ ID NO: 4. Nucleotide sequences encoding the light chain and heavy chain of the CHIR-12.12 antibody are set forth in Figures 2 and 2B, respectively. See also SEQ ID NO: 11 (light chain coding sequence for CHIR-12.12 mAb) and SEQ ID NO: 3 (heavy chain coding sequence for CHIR12.12 mAb). The amino acid sequences for the light chain and heavy chain of the CHIR-5.9 antibody are set forth in Figures 3A and 3B, respectively. See also SEQ ID NO: 6 (light chain for CHIR-5-9 mAb) and SEQ ID NO: 7 (heavy CHIR-59 mAb chain). A variant of the CHIR-5-9 mAb heavy chain is shown in Figure 3B (see also SEQ ID NO: 8), which differs from SEQ ID NO: 7 in that it has a serine residue substituted by the alanine residue at position 158 of SEQ ID NO: 7.

As expected for antibodies derived from independent hybridomas, there is substantial variation in nucleotide sequences in the complementary determining regions (CDR). The diversity in the CDR3 region of VH is believed to be what most significantly determines the specificity of the antibody.

As shown by FACS analysis, CHIR-5.9 and CHIR-12.12 specifically bind to human CD40 and can prevent binding of the CD40 ligand. Both mAbs can compete with the prior binding of the CD40 ligand to the CD40 of the cell surface. The binding affinity of CHIR-5.9 to human CD40 is 1.2 x 10-8 M and the binding affinity of CHIR-12.12 to human CD40 is 5 x 10-10 M.

The monoclonal antibodies CHIR-12.12 and CHIR-5.9 are strong antagonists and inhibit CD40 ligand-mediated proliferation of normal B cells in vitro.

Example 1. CHIR-12.12 blocks cell signaling mediated by CD40L

The soluble CD40 ligand (CD40L) activates B cells and induces various aspects of functional responses, including increased survival and proliferation, and activation of the NFκB, ERK / MAPK, PI3K / Akt and p38 signal pathways. . In addition, CD40L-mediated CD40 stimulation provides survival signals by reduction of cleaved PARP and induction of antiapoptotic proteins, XIAP and Mcl-1, in normal B cells. CD40L-mediated CD40 stimulation also recruits TRAF2 and TRAF3 for binding to the cytoplasmic domain of CD40.

The following studies show that CHIR-12.12 directly inhibited all these stimulation effects in normal human B cells. For example, treatment with CHIR

12.12 resulted in the increase in excision of caspase-9, caspase-3 and PARP as well as the reduction of XIAP and Mcl-1 in a time and dose dependent manner, restoring B-cell apoptosis. Treatment with CHIR-12.12 also inhibited the phosphorylation of IκB kinase (IKK) α and β (NFκB pathway), ERK, Akt and p38 in response to CD40L-mediated CD40 stimulation. In addition, it was found that CHIR-12.12 did not cause these apoptotic effects without the initial stimulation of CD40 mediated by CD40L.

CHIR-12.12 inhibited CD40 ligand-mediated survival by inducing PARP cleavage

In these experiments, 0.6 x 106 normal human B cells from healthy donors (percentage purity between 85 and 95%) were stimulated with 1 µg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). Then CHIR-12.12 (10 µg / ml) and control IgG were added. Cells were collected at 0 and 20 minutes, at 2 hours, 6 hours, 18 hours and 26 hours. Excised caspase 9, cleaved caspase 3, cleaved PARP and β actin controls were detected in cell lysates by Western blotting.

Briefly, it was observed that CD40L-mediated CD40 stimulation provided survival signals as it did not lead to increases in cleaved caspase 9, cleaved caspase 3

or PARP cleaved over time, indicating that the cells were not suffering apoptosis. However, treatment with CHIR-12.12 resulted in an increase in these cleavage products, indicating that treatment with CHIR-12.12 nullified the effects of CD40L binding on survival signals in normal B cells stimulated with cCD40L, restoring apoptosis of B cells (data not shown).

CHIR-12.12 inhibited the expression of "survival" antiapoptotic proteins

In these experiments, 0.6 x 10 6 normal human B cells from healthy donors (purity percentage between 85-95%) were stimulated with 1 µg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). Then CHIR-12.12 (10 µg / ml) and control IgG were added. Cells were collected at 0 and 20 minutes, at 2 hours, 6 hours, 18 hours and 26 hours. Mcl-1, XIAP, CD40 and β actin controls were detected in cell lysates by Western blotting.

Briefly, stimulation with sCD40L resulted in sustained expression of Mcl-1 and XIAP over time. However, treatment of sCD40L stimulated cells with CHIR-12.12 resulted in a decrease in the expression of these proteins over time (data not shown). Since Mcl-1 and XIAP are "survival" signals capable of blocking the apoptotic pathway, these results demonstrate that treatment with CHIR-12.12 eliminates blockage against apoptosis in normal B cells stimulated with sCD40L.

Treatment with CHIR-12.12 inhibited the phosphorylation of IKKα (Ser180) and IKKβ (Ser 181) in normal B cells.

In these experiments, 1.0 x 106 normal human B cells from healthy donors (purity percentage between 85-95%) were stimulated with 1 µg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). Then CHIR-12.12 (10 µg / ml) and control IgG were added. Cells were collected at 0 and 20 minutes. IKK α (Ser180) and IKK β (Ser 181) and total IKK β controls were detected in cell lysates by Western blotting.

Briefly, stimulation by means of sCD40L resulted in the phosphorylation of IKKα (Ser180) and IKKβ (Ser 181) over time; however, treatment with CHIR-12.12 nullified this response to stimulation of sCD40L in normal B cells (data not shown).

Treatment with CHIR-12.12 inhibited CD40 ligand-mediated survival in a dose-dependent manner.

In these experiments, 0.6 x 106 normal human B cells from healthy donors (percentage purity between 85 and 95%) were stimulated with 1 µg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). CHIR-12.12 (0.01, 0.1, 0.2, 0.5, 1.0 µg / ml) and control IgG were then added. Cells were collected at 24 hours. The cleaved PARP and β actin controls were detected in cell lysates by Western blotting.

Briefly, treatment with CHIR-12.12 resulted in an increase in PARP cleavage in cells stimulated with sCD40L in a dose-dependent manner and therefore nullified the survival signal pathway in normal B cells stimulated with sCD40L (data not shown).

CHIR-12.12 inhibited the expression of "survival" antiapoptotic proteins in a dose-dependent manner.

In these experiments, 0.6 x 106 normal human B cells from healthy donors (percentage purity between 85 and 95%) were stimulated with 1 µg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). CHIR-12.12 (0.5, 2 and 10 µg / ml) and control IgG were then added. Cells were collected at 22 hours. The controls of Mcl-1, XIAP, cleaved PARP and β actin were detected in cell lysates by Western blotting.

Briefly, treatment with CHIR-12.12 reduced the expression of Mcl-1 and XIAP and increased the expression of PARP in cells stimulated with sCD40L in a dose-dependent manner and therefore nullified these blockages to the apoptotic pathway in B cells. normal stimulated with sCD40L (data not shown).

CHIR-12.12 does not affect the expression of antiapoptotic proteins, cleaved PARP and XIAP, in the absence of soluble CD40L signals.

In these experiments, 1.0 x 10 6 normal human B cells from healthy donors (purity percentage between 85-95%) were stimulated with CHIR-12.12 (10 µg / ml) and control IgG alone (i.e., non-cell were previously stimulated with sCD40L before adding the antibody). Cells were collected at 0, 4, 14 and 16 hours. XIAP, cleaved PARP and β actin controls were detected in cell lysates by Western blotting.

Briefly, the results show that without stimulation of sCD40L, the cells expressed

Increased concentrations of cleaved PARP, while XIAP expression remained constant, both in the cells treated with control IgG and those treated with CHIR-12.12 (data not shown). These data indicate that CHIR-12.12 does not cause apoptosis in normal human B cells without CD40L stimulation.

CHIR-12.12 inhibits the phosphorylation of IKKα (Ser180) and IKKβ (Ser181), Akt, ERK and p38 in normal B cells.

In these experiments, serum was deprived of medium containing 1% FBS 1.0 x 106 normal human B cells from healthy donors (purity percentage between 85 and 95%) and stimulated with sCD40L 1 µg / ml (Alexis Corp ., Bingham, Nottinghamshire, UK). Cultures were treated with CHIR-12.12 (1 and 10 µg / ml) and control IgG. Cells were collected at 0 and 20 minutes. Phospho-IKKα, phospho-IKKβ, total IKKβ, phosfoERK, total ERK, phospho-Akt, total Akt, phospho-p38 and p38 total were detected in cell lysates by Western blotting.

Briefly, stimulation with sCD40L resulted in increases in IKKα / β phosphorylation, ERK phosphorylation, Akt phosphorylation and p38 phosphorylation, thus resulting in cell survival and / or proliferation. Treatment of the cells with CHIR-12.12 nullified the effects of stimulation of sCD40L in these signal pathways in normal B cells (data not shown).

CHIR 12.12 inhibits multiple signal pathways such as PI3K and MEK / ERK in the CD40 signal cascade.

In these experiments, serum was deprived of medium containing 1% FBS 1.0 x 106 normal human B cells from healthy donors (purity percentage between 85 and 95%) and stimulated with sCD40L 1 µg / ml (Alexis Corp ., Bingham, Nottinghamshire, UK). Cultures were also treated with CHIR-12.12 (1 and 10 µg / ml), Wortmanin (a PI3K / Akt inhibitor, 1 and 10 µM), LY 294002 (a PI3K / Akt inhibitor; 10 and 30 µM) and PD 98095 (an inhibitor of MEK, 10 and 30 µg / ml). Cells were collected at 0 and 20 minutes. FosfoERK, phospho-Akt, total Akt, phospho-IKKα / β and total were detected in cell lysates by Western blotting.

Briefly, the results show that CHIR-12.12 annulled the phosphorylation of all these signal transduction molecules, while signal transduction inhibitors showed only specific signal cancellation, indicating that CHIR-12.12 probably inhibited at a stage prior to these signal transduction molecules mediated by CD40L stimulation (data not shown).

CHIR-12.12 inhibits the binding of TRAF2 and TRAF3 signaling molecules to the cytoplasmic domain of CD40 in normal B cells.

In these experiments, serum was deprived for four hours in medium containing 1% FBS 4.0 x 106 normal human B cells from healthy donors (purity percentage between 85 and 95%) and stimulated with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK) for 20 minutes. Cells were collected at 0 and 20 minutes. CD40 was immunoprecipitated using polyclonal anti-CD40 (Santa Cruz Biotechnology, CA), and probed in a Western blot with anti TRAF2 mAb (Santa Cruz Biotechnology, CA), anti TRAF3 mAb (Santa Cruz Biotechnology, CA), and anti-mAb CD40 (Santa Cruz Biotechnology, CA). Briefly, the results show that TRAF2 and TRAF3 co-precipitated with CD40 after stimulation of sCD40L. In contrast, treatment with CHIR-12.12 canceled the formation of the CD40-TRAF2 / 3 signal complex in normal B cells stimulated with sCD40L. There were no changes in the expression of CD40 (data not shown).

Without being bound by theory, the results of these experiments, and the results in the examples summarized above, indicate that the CHIR-12.12 antibody is a dual-acting antagonistic anti-CD40 monoclonal antibody that has a unique combination of attributes. This fully human monoclonal antibody blocks CD40L-mediated CD40 signal pathways for the survival and proliferation of B cells; This antagonism eventually results in cell death. CHIR-12.12 also mediates recognition and binding by effector cells, initiating antibody-dependent cellular cytotoxicity (ADCC). Once CHIR-12.12 is bound to effector cells, cytolytic enzymes are released, resulting in apoptosis and lysis of B cells. CHIR-12.12 is a more potent antitumor antibody than rituximab when compared in preclinical tumor models. .

Example 2: CHIR-5.9 and CHIR-12.12 bind to a different epitope than 15B8 in CD40

The candidate monoclonal antibodies CHIR-5.9 and CHIR-12.12 compete with each other for binding to CD40 but not with 15B8, an anti-CD40 IgG2 mAb (see International Publication Document No. WO 02/28904). Competitive antibody binding studies were designed using Biacore with CM5 biosensor chips with protein A immobilized by means of

Amines coupling, which was used to capture anti-CD40, CHIR-12.12, or 15B8. Normal association / dissociation binding curves were observed with varying concentrations of CD40-his (data not shown). For competition studies, CHIR-12.12 or 15B8 were captured either on the surface of protein A. Subsequently, a CD40-his / Fab complex of CHIR-5.9 was flowed (100 nM CD40: CHIR-5.9 Fab 1 µM), in varying concentrations, across the modified surface. In the case of CHIR-12.12, no association of the observed complex was observed, indicating that CHIR-5.9 blocks the binding of CHIR-12.12 to CD40-his. For 15B8, association of the Fab CHIR-5.9 complex was observed indicating that CHIR-5.9 does not block the binding of 15B8 to the CD40 binding site. However, the

The dissociation rate of the complex increased dramatically (data not shown).

It was also determined that 15B8 and CHIR-12.12 do not compete for the binding of CD40-his. This experiment was carried out by capturing CHIR-12.12 on the protein A biosensor chip, blocking the residual sites of protein A with hIgG1 control, linking CD40-his and a

15 then flowing 15B8 over the modified surface. 15B8 joined under these conditions indicating that CHIR-12.12 does not block the binding of 15B8 to CD40.

Example 3: Binding properties of CHIR-12.12 and CHIR-5.9 mAbs

20 Protein A was immobilized on CM5 biosensor chips through amine coupling. Human anti-CD40 monoclonal antibodies, at a concentration of 1.5 µg / ml, were captured on the modified surface of the biosensor for 1.5 minutes at 10 µl / min. Soluble recombinant CD40-his was flowed over the surface of the biosensor in varying concentrations. The antibody and antigen were diluted in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,

25 Surfactant P 20.005% (HBS-EP). Kinetic and affinity constants were determined using the Biaevaluation software with a model / global interaction setting of

1: 1.

As shown in Table 2 below, there is a 121-fold difference in the rate of dissociation of CHIR-5.9 and CHIR-12.12 that results in a 24-fold affinity for CHIR-12.12.

35

Antibody

ka (M-1 sec-1) kd (sec-1) KD (nM)

Anti-CD40, CHIR-5.9

(12.35 ± 0.64) x 105 (15.0 ± 1.3) x 10-3 12.15 ± 0.35

Anti-CD40, CHIR-12.12

(2.41 ± 0.13) x 105 (1.24 ± 0.06) x 10-4 0.51 ± 0.02

Example 4: Epitope characterization for the CHIR-12.12 and CHIR monoclonal antibodies

5.9

5

To determine the location of the epitope on CD40 recognized by the monoclonal antibodies CHIR-12.12 and CHIR-5.9, SDS-PAGE analysis and Western blotting were performed. Purified CD40 (0.5 µg) was separated on a 4-12% NUPAGE gel under reducing and non-reducing conditions, transferred to PVDF membranes, and probed with antibodies

10 monoclonal in a concentration of 10 µg / ml. Transfers were probed with anti-human IgG conjugated to alkaline phosphatase and developed using the Western® Blue stabilized substrate for alkaline phosphatase (Promega).

The results indicate that the anti-CD40 monoclonal antibody CHIR12.12 recognizes epitopes

15 both in the non-reduced form and in the reduced form of CD40, the non-reduced form of CD40 exhibiting greater intensity than the reduced form of CD40 (Table 3; transfers not shown). The fact that recognition was positive for both forms of CD40 indicates that this antibody interacts with a conformational epitope, part of which is a linear sequence. The monoclonal antibody CHIR-5.9 mainly recognizes the form no

20 reduced CD40 suggesting that this antibody interacts primarily with a conformational epitope (Table 3; transfers not shown).

Table 3. Domain identification.

Domain 1

Domain 2 Domain 3 Domain 4

mAb CHIR-12.12

- + - -

mAb CHIR-5.9

- + - -

mAb 15B8

+ - - -

25 To map the antigenic region on CD40, the four extracellular domains of CD40 were cloned and expressed in insect cells as GST fusion proteins. The secretion of the four domains was secured with a GP67 secretion signal. The supernatant of the insect cells was analyzed by SDS-PAGE analysis and Western blotting to identify the domain that contained the epitope.

The monoclonal antibody CHIR-12.12 recognizes an epitope in Domain 2 under both reducing and non-reducing conditions (Table 4; transfers not shown). In contrast, the CHIR-5.9 monoclonal antibody exhibits very weak recognition for Domain 2 (Table 16; transfers not shown). None of these antibodies recognize Domains 1, 3 or 4 in this analysis.

Table 4. Domain Analysis2.

Reduced

Not reduced

mAb CHIR-12.12

++ +++

mAb CHIR-5.9

+ +

10 To more precisely define the epitope recognized by the CHIR-12.12 mAb, extracellular Domain 2 peptides from CD40 were synthesized, corresponding to the sequence PCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICT (residues 61-104 of the sequence shown in SEQ ID NO: 10 or SEQ ID Nº: 12). Synthesis membranes were generated

SPOT (Sigma) containing thirty-five 10-mer peptides with a displacement of 1 amino acid. Western blot analysis was performed with CHIR-12.12 mAb and anti-human IgG labeled with beta galactosidase as secondary antibody. The transfer was dismantled and re-probed with CHIR-5.9 mAb to determine the region recognized by this antibody.

20 SPOTs analyzes probing with anti-CD40 monoclonal antibody CHIR-12.12 at a concentration of 10 µg / ml gave positive reactions with transfers 18 to 22. The region of the sequence covered by these peptides is shown in Table 5.

25 Table 5. Results of SPOTs analysis probing with anti-CD40 monoclonal antibody CHIR-12.12.

Transfer number

Sequence region

18

HQHKYCDPNL (residues 78-87 of SEQ ID NO: 10 or SEQ ID NO: 12)

19

QHKYCDPNLG (residues 79-88 of SEQ ID NO: 10 or SEQ ID NO: 12)

twenty

HKYCDPNLGL (residues 80-89 of SEQ ID NO: 10 or SEQ ID NO:

12)

twenty-one

KYCDPNLGLR (residues 81-90 of SEQ ID NO: 10 or SEQ ID NO: 12)

22

YCDPNLGLRV (residues 82-91 of SEQ ID NO: 10 or SEQ ID NO: 12)

These results correspond to a linear epitope of: YCDPNL (residues 82-87 of the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12). This epitope contains Y82, D84 and N86, which have been predicted to be involved in the CD40-CD40 ligand interaction.

5

SPOTs with CHIR-5.9 mAbs showed a weak recognition of the peptides represented by the spots 20-22 shown in Table 6, suggesting involvement of the YCDPNLGL region (residues 82-89 of the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12) in its binding to CD40. It should be noted that mAbs CHIR-12.12 and CHR-5.9 compete one

10 with the other by binding to CD40 in the BIACORE analysis.

Table 6. Results of SPOTs analysis probing with anti-CD40 monoclonal antibody CHIR-5.9.

Transfer number

Sequence region

twenty

HKYCDPNLGL (residues 80-89 of SEQ ID NO: 10 or 12)

twenty-one

KYCDPNLGLR (residues 81-90 of SEQ ID NO: 10 or 12)

22

YCDPNLGLRV (residues 82-91 of SEQ ID NO: 10 or 12)

15 Linear epitopes identified by SPOTs are within the B1 module of CD40. The sequence of module B1 of CD40 is:

HKYCDPNLGLRVQQKGTSETDTIC (residues 80-103 of SEQ ID NO: 10 or 12).

20 Within the linear epitope identified for CHIR-12.12 is C83. It is known that this cysteine residue forms a disulfide bond with C103. It is likely that the conformational epitope of the CHIR-12.12 mAb contains this disulfide bond (C83-C103) and / or neighboring amino acids conformationally close to C103.

25

Example 5: Testing in models of autoimmune and inflammatory diseases

Model of systemic lupus erythematosus (SLE).

CHIR-12.12 was tested in a systemic lupus erythematosus (SLE) model in which peripheral blood mononuclear cells (PMBC) of SLE patients are injected into SCID mice. See, for example, the model described in Duchosal et al. (1990) J. Exp. Med. 172: 985

8.

After PBMC transfer of SLE patients in SCID mice, it is determined whether or not treatment with CHIR-12.12 influences T lymphocyte response to autoantigen and autoantibody production and morbid manifestations such as glomerulonephritis. The first group of studies tests CHIR-12.12 as an individual agent followed by testing the effect in a combination with other agents such as CTLA4-Ig.

Multiple sclerosis model.

Experimental autoimmune encephalitis (EAE) of the marmoset monkey is a model for human multiple sclerosis. See, for example, the model described in Raine et al. (1999) Ann. Neurol

46: 144-60 and Hart et al. (2004) Lancet Neurol. 3: 588-97. CHIR-12.12 binds to CD40 marmoset and is tested for efficacy in this model.

Inflammation and atherosclerosis

CHIR-12.12 is tested in vitro for its ability to inhibit CD40L-induced production of matrix degrading enzymes, tissue factor expression, proinflammatory cytokines, and upregulation of adhesion molecules. Subsequent studies test the ability of CHIR-12.12 to show anti-inflammatory activities in vivo using transgenic mice that express the human CD40 molecule. See, for example, the model described in Yasui (2002) Inty. Immunol 14: 319-29.

Transplant.

CHIR-12.12 is tested for its ability to prevent transplant rejection in nonhuman primate models. Renal allograft receptor crayfish acacos are treated with CHIR12.12 antibody. demonstrating the effect of graft acceptance with or without additional immunosuppressive drugs such as cyclosporine, FK506, rapamycin, corticosteroids, CTLA4-Ig molecule and anti-B-cell stimulating antibody and the like. See, for example, the model described in Wee et al. (1992) Transplantation 53: 501-7.

Alzheimer disease.

. CHIR-12.12 is first tested in vitro for its ability to block microglial activation. In vivo efficacy studies with CHIR-12.12 are carried out in doubly transgenic mice that express human CD40 and overproduce beta-amyloid peptide. See, for example, the model described in Tan et al. (2002) Nat. Neurosci. 5: 1288-93.

Example 6: Clinical studies with CHIR-5.9 and CHIR-12.12

Clinical objectives

The general objective is to provide an effective therapy for rheumatoid arthritis (RA) by targeting cells with an anti-CD40 IgG1. The signal for this disease is determined in phase I although some measurement of activity can be obtained in phase I. Initially, the agent is studied as a single agent, but will be combined with other therapeutic agents as development proceeds.

Phase I

•

Evaluate safety and pharmacokinetics - dose increase in subjects with RA.

•

Choose the dose based on safety, tolerability and change in serum markers of CD40. In general, a maximum tolerable dose (DMT) is sought, but other indications of efficacy (depletion of cells carrying CD40 +, etc.) may be appropriate for the dose finding.

•

Consideration of more than one dose, as some phase II dose finding may be necessary.

•

Patients are dosed weekly with real-time pharmacokinetic (Pk) sampling. Initially the maximum allowed dose is a 4 week cycle. Pk can be highly variable depending on the morbid state, density of CD40, etc.

•

This / these assay (s) is open to subjects with RA.

•

The decision to interrupt or continue studies is based on safety, doses and

•

preliminary evidence of therapeutic activity.

•

The activity of the drug as determined by the response rate is determined in Phase II.

•

Identify dose (s) for Phase II.

Phase II

Several trials will be initiated in subjects with RA. More than one dose and more than one program can be tested in a random phase II configuration.

• Target a population of RA that has a current standard of failed care (non-steroidal anti-inflammatory drug therapy (NSAID) and disease-modifying anti-rheumatic drug (DMARD; for example gold and penicillamine))

image 1 The decision to interrupt or continue the study is based on the proof of the therapeutic concept in Phase II

image 1 Determine if a substitute marker can be used as an early indication for clinical efficacy

image 1 Identify the doses for Phase III

Phase III

Phase III will depend on when the signal is detected in phase II and on which competing therapies are considered to be the standard. If the signal is in a phase of the disease where there is no standard of therapy, then a single-arm, well-controlled study may serve as a fundamental trial. If there are competing agents that are considered standards, then direct studies are conducted.

Example 7: Liquid pharmaceutical formulation for anti-CD40 antagonist antibodies

The objective of this study was to investigate the effects of the pH of the solution on the stability of the anti-CD40 antagonist CHIR-12.12 by means of biophysical and biochemical procedures to select the optimal solution environment for this antibody. Differential scanning calorimetry (DSC) results showed that CHIR-12.12 conformation stability is optimal in formulations that have pH 5.5-6.5. Based on a combination of SDS-PAGE analysis, size exclusion HPLC (SEC-HPLC) and cation exchange HPLC (CEX-HPLC), the physicochemical stability of CHIR-12.12 is optimal at a pH of approximately 5.0- 5.5. In view of these results, a pharmaceutical formulation

The recommended liquid comprising this antibody is a formulation comprising CHIR-12.12 at approximately 20 mg / ml formulated in approximately 10 mM sodium succinate, approximately 150 mM sodium chloride and having a pH of approximately pH 5.5.

10 Materials and procedures

The CHIR-12.12 antibody used in formulation studies is a human monoclonal antibody produced by a CHO cell culture process. This mAb has a molecular weight of 150 kDa and consists of two light chains and two heavy chains

15 joined together by disulfide bonds. It is directed against the cell surface CD40 receptor in cells expressing CD40, including normal and malignant B cells, for the treatment of various types of cancer and autoimmune / inflammatory diseases.

The substance of the anti-CD40 drug used for this study was a bulk batch of anti-CD40

20 (CHIR-12.12) purified derived from CHO cells. The composition of the drug substance was CHIR-12.12 antibody 9.7 mg / ml in 10 mM sodium citrate, 150 mM sodium chloride, at pH 6.5. The control sample in the study was the substance of the drug received, followed by freezing at ≤ -60 ° C, thawing at RT and tested together with stability samples at predetermined time points. Stability samples were prepared by dialysis

25 of the drug substance against solutions of different pH and the CHIR concentration

12.12 in each of the samples was determined by UV 280 as presented in the Table

7.

Table 7. CHIR-12.12 formulations.

Buffer composition

pH CHIR-12.12 concentration (mg / ml)

10 mM sodium citrate, 150 mM sodium chloride

4,5 9.0

10 mM sodium succinate, 150 mM sodium chloride

5.0 9.3

Buffer composition

pH CHIR-12.12 concentration (mg / ml)

10 mM sodium succinate, 150 mM sodium chloride

5.5 9.2

10 mM sodium citrate, 150 mM sodium chloride

6.0 9.7

10 mM sodium citrate, 150 mM sodium chloride

6.5 9.4

10 mM sodium phosphate, 150 mM sodium chloride

7.0 9.4

10 mM sodium phosphate, 150 mM sodium chloride

7.5 9.5

10 mM glycine, 150 mM sodium chloride

9.0 9.5

The physicochemical stability of the CHIR-12.12 antibody in the various formulations was tested using the following protocols.

5 Differential scanning calorimetry (DSC)

The conformational stability of different samples of formulations was monitored using a VP-DSC MicroCal after heating from 15 ° C to 90 ° C at a rate of 1 ° C / min.

10 SDS-PAGE

Fragmentation and aggregation were estimated using 4-20% Tris-glycine gel under non-reducing and reducing conditions. Proteins were detected by staining with Coomassie blue.

fifteen

Size exclusion chromatography (SEC-HPLC)

Protein fragmentation and aggregation were also measured by means of an HPLC Water Alliance system with a Tosohaas TSK-GEL 3000SWXL column, with sodium phosphate

20 100 mM, pH 7.0 as mobile phase at a flow rate of 0.7 ml / min.

Cation Exchange Chromatography (CEX-H PLC)

Degradation related to load change was measured using a Waters 600s HPLC system with a Dionex Propac WCX-10 column, with 50 mM HEPES, pH 7.3 as mobile phase A and 50 mM HEPES containing 500 mM NaCl, pH 7.3 as mobile phase B at a flow rate of 0.5 ° C / min.

Results and Discussion

Study of conformational stability.

The thermal deployment of CHIR-12.12 revealed at least two thermal transitions, which probably represent the fusion of deployment of the Fab and Fc domains, respectively. At higher temperatures, the protein was presumably added, resulting in loss of the DSC signal. In order to examine the formulation, the lowest thermal transition temperature was defined as the melting temperature, Tf, in this study. Figure 5 shows the thermal melting temperature as a function of the pH of the formulations. Formulations at pH 5.5-6.5 provided anti-CD40 with superior conformational stability as demonstrated by higher thermal fusion temperatures.

SDS-PAGE analysis.

Samples of CHIR-12.12 formulations at pH 4.5-9.0 were incubated at 40 ° C for 2 months and subjected to SDS-PAGE analysis (data not shown). Under non-reducing conditions, species with molecular weight (MW) of 23 kDa and 2.7 kDa were observed in formulations with pH higher than 5.5, and species with PM of 51 kDa were observed in all formulations, but appeared less at pH 5.0-5.5. A species with PM of 100 kDaa pH 7.5 and pH9.0 could be seen,

Under reducing conditions, CHIR-12.12 was reduced in heavy chains and free light chains with PM of 50 kDa and 24 kDa, respectively. The 100 kDa species appeared not to be fully reducible and increased with the increase in the pH of the solution, suggesting that there could be a different covalent association of disulfide in the molecules. As there were other species with unknown identities in SDS-PAGE, the stability comparison for each formulation is based on the remaining purity of CHIR-12.12. Formulations at pH 5.0-6.0 provided a more stable environment for CHIR-12.12. Few aggregates were detected by means of SDS-PAGE (data not shown).

SEC-HPLC analysis.

5 The SEC-HPLC analysis detected the CHIR-12.12 intact as the main peak species, an aggregation species as a species of anterior peak separated from the main peak species, a large fragment species such as a plateau peak in the back of the main peak species, and small fragment species subsequent to the main peak species were detected. After incubation at 5 ° C and 25 ° C for 3 months, they were detected

10 insignificant amounts of protein fragments and aggregates (<1.0%) in the above formulations and the species of the main peak of CHIR-12.12 remained more than 99% pure (data not shown). However, protein fragments gradually developed after storage at 40 ° C and more fragments were formed at pH 4.5 and pH 6.5-9.0, as shown in Table 8. After incubating the CHIR-12.12 formulations

15 to 40 ° C for 3 months, approximately 2-3% of aggregates at pH 7.5 and pH 9.0 were detected, while less than 1% of aggregates were detected in formulations at other pHs (data not shown). The results of SEC-HPLC indicate that CHIR-12.12 is more stable at a pH of approximately 5.0-6.0,

20 Table 8. SEC-HPLC results of CHIR-12.12 stability samples under real-time and accelerated storage conditions.

Sample

Main peak% Fragments%

t = 0

40 ° C 1 m 40 ° C 2 m 40 ° C 3 m  t = 0 40 ° C 1 m 40 ° C 2 m 40 ° C 3 m

Control

99.4 99.2 99.9 99.5 <1.0 <1.0 <1.0 <1.0

pH 4.5

99.4 93.2 86.0 81.3 <1.0 6.4 13.2 18.1

pH 5.0

99.8 98.7 91.3 89.2 <1.0 <1.0 7.8 10.2

pH 5.5

99.8 98.9 91.4 90.6 <1.0 <1.0 7.6 8.8

pH 6.0

99.6 97.7 90.4 87.3 <1.0 1.9 8.2 11.7

pH 6.5

99.3 93.4 89.0 86.9 <1.0 5.6 9.9 12.4

pH 7.0

99.2 93.9 87.4 85.1 <1.0 5.5 11.1 13.5

pH 7.5

99.1 92.8 84.4 81.9 <1.0 6.4 12.9 16.2

pH 9.0

99.3 82.4 61.6 50.6 <1.0 15.4 36.2 47.6

CEX-FIPLC analysis.

The CEX-HPLC analysis detected the intact CHIR-12.12 as the main peak species, acid variants eluted earlier than the main peak species and lysine addition variants at the C-terminal end eluted after the main peak species . Table 9 shows the dependence of the percentages of the remaining CHIR-12.12 5 species on the main peak and the acid variants on the pH of the solution. The control sample already contained a high degree of acidic species (approximately 33%), probably due to the early stage fermentation and purification processes. CHIR susceptibility

12.12 at higher pH solutions is evidenced by two facts. First, the sample of the initial formulation at pH 9 (t = 0) already generated 12% more acidic species than the control.

10 Second, the percentage of acidic species increased sharply with the increase in pH. The degradation related to the change of charges is probably due to deamidation. The above data indicates that this type of degradation of CHIR-12.12 was minimized at a pH of approximately 5.0-5.5.

15 Table 9. Percentage of peak area by CEX-HPLC for CHIR-12.12 in different pH formulations under real-time and accelerated storage conditions.

Sample

Main peak% Acid variants%

T = 0

5 ° C 25 ° C 40 ° C 40 ° C t = 0 5 ° C 25 ° C 40 ° C 40 ° C

3 m

3 m

1 m 2 m  3 m  3 m 1 m 2 m

Control

49.2 49.8 49.8 49.2 50.3 32.0 33.7 33.7 32.0 33.6

pH 4.5

48.5 49.7 43.7 39.7 30.0 32.5 32.6 38.0 44.2 56.4

pH 5.0

49.6 49.8 48.3 40.6 31.4 32.7 31.8 35.0 44.3 57.1

pH 5.5

50.7 50.3 48.1 40.0 30.2 32.6 31.8 37.8 48.9 63.3

pH 6.0

50.2 49.9 47.9 37.4 23.9 33.1 33.6 38.5 54.9 72.7

pH 6.5

49.4 49.9 42.3 29.7 14.6 33.3 33.6 47.7 65.2 84.6

pH 7.0

49.7 49.9 21.9 - - 34.4 36.4 64.4 - -

pH 7.5

49.3 48.3 12.7 - - 35.5 40.1 79.2 - -

pH 9.0

41.3 31.8 - - - 44.7 59.9 - - -

conclusion

20 pH has a significant effect on the conformational and physicochemical stability of CHIR

12.12. Degradation related to charge change was determined to be the main degradation pathway for CHIR-12.12, which was minimized to pH 5.0-5.5. Based on the general stability data, a recommended liquid pharmaceutical formulation comprising this antibody is a formulation comprising CHIR-12.12 at a concentration of approximately 20 mg / ml formulated in approximately 10 mM sodium succinate, approximately 150 mM sodium chloride , and which has a pH of approximately 5.5.

SEQUENCE LIST

<110> Long, Li Luqman, Mohammad Yabannavar, Asha Zaror, Isabel

<120> USE OF ANTI-CD40 ANTIBODIES ANTAGONISTS FOR THE TREATMENT OF INFLAMMATORY AND AUTOIMMUNE DISEASES AND ORGAN TRANSPLANT REJECTION

<130> PP23725.001 (284072)

<150> 60 / 565,710

<151> 04-27-2004

<150> 60 / 525,579

<151> 11-26-2003

<150> 60 / 517,337

<151> 04-11-2003

<160> 12

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<223> CHIR human anti-CD40 antibody light chain coding sequence

<221> CDS

<222> (1) ... (720)

<400> 1 <210> 2

image2

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> CHIR-12.12 human anti-CD40 antibody light chain

<400> 2

image3

<210> 3

<211> 2016

<212> DNA

<213> Artificial sequence

<220>

<223> CHIR human anti-CD40 antibody heavy chain coding sequence

12.12 (with introns)

<400> 3

image3

<210> 4

<211> 469

<212> PRT

<213> Artificial sequence

<220>

<223> CHIR-12.12 human anti-CD40 antibody heavy chain

<400> 4

image3

<210> 5

<211> 469

<212> PRT

<213> Artificial sequence

5

<220>

<223> CHIR-12.12 human anti-CD40 antibody variant heavy chain

<400> 5

10

fifteen

twenty

25

30

image3

image3

<210> 6

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> CHIR-12.12 human anti-CD40 antibody light chain

<400> 6 <210> 7

image3

<211> 474

<212> PRT

<213> Artificial sequence

5

<220>

<223> CHIR-5.9 human anti-CD40 antibody heavy chain

<400> 7

10

image3

<210> 8

<211> 474

<212> PRT

<213> Artificial sequence

5

<220>

<223> CHIR-5.9 human anti-CD40 antibody variant heavy chain

<400> 8

10

image3

<210> 9

<211> 612

<212> DNA

<213> Homo sapiens

5

<220>

<221> CDS

<222> (1) ... (612)

10 <221> miscellaneous feature

<222> (0) ... (0)

<223> Coding sequence for the short isoform of human CD40

<400> 9

fifteen

image3

<210> 10

<211> 203

<212> PRT

<213> Homo sapiens

<400> 10

image3

<210> 11

<211> 834

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1) ... (834)

<221> miscellaneous feature

<222> (0) ... (0)

<223> Coding sequence for the long isoform of human CD40

<400> 11

image3

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image3

Claims (17)

 Claims 1. An anti-CD40 human monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a human cell expressing CD40, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, for use in a procedure to treat a human subject of an inflammatory disease or an autoimmune disease, the procedure comprising administering to said subject a effective amount of the human anti-CD40 monoclonal antibody, said human anti-CD40 monoclonal antibody being selected from the group constituted by: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen. 2. Use of an effective amount of a human anti-CD40 monoclonal antibody, which is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40 in the preparation of a medicament for treating a human subject of an inflammatory disease or an autoimmune disease, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, said human anti-CD40 monoclonal antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

3.

The monoclonal antibody of claim 1 or the use of claim 2, wherein said inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), discoid lupus, lupus nephritis, sarcoidosis, juvenile arthritis, arthritis rheumatoid, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis, gouty arthritis, rejection of an organ or tissue transplant, graft versus host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory bowel disease Crohn's disease, celiac disease (gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Graves' disease, Hasimoto's thyroiditis, complex immune disease , chronic fatigue syndrome and dysfunction i Immune (SFCDI), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris, interstitial pulmonary fibrosis, sarcoidosis, diabetes mellitus Type I and Type II, delayed type hypersensitivity type 1, 2, 3 and 4, allergy or allergic disorders, asthma, Churg-Strauss syndrome

(allergic granulomatosis), atopic dermatitis, allergic and irritant contact dermatitis, hives, IgE-mediated allergy, atherosclerosis, vasculitis, idiopathic inflammatory myopathies, hemolytic disease, Alzheimer's disease and chronic inflammatory demyelinating polyneuropathy.

Four.

A human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a human cell expressing CD40, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, for use in a procedure to treat a human transplant rejection subject, the method comprising administering to said subject an effective amount of the antibody anti-human CD40 monoclonal, said human anti-CD40 monoclonal antibody being selected from the group constituted by:

a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

5.

Use of an effective amount of a human anti-CD40 monoclonal antibody, which is capable of specifically binding to a human CD40 antigen expressed on the surface of a

cell expressing human CD40 in the preparation of a medicament for treating a human transplant rejection subject, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, said monoclonal antibody being anti -CD40 human selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen.

6.

The monoclonal antibody of claim 4 or the use of claim 5, wherein said treatment further comprises administering an immunosuppressive agent in the pharmaceutically acceptable excipient.

7.

The monoclonal antibody or the use of claim 6, wherein the immunosuppressive agent is selected from the group consisting of cyclosporine, FK506, rapamycin, corticosteroids, CTLA4-Ig and anti-B cell stimulator antibody.

8.

An anti-CD40 human monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40, said monoclonal antibody being free of significant agonist activity when

binds to the CD40 antigen expressed on the surface of said cell, for use in a procedure to treat a human subject of rheumatoid arthritis, the method comprising administering to said subject an effective amount of the antibody anti-human CD40 monoclonal, said human anti-CD40 monoclonal antibody being selected from the group constituted by: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen. 9 Use of an effective amount of human anti-CD40 monoclonal antibody, which is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40 in the manufacture of a medicament for treating a human arthritis subject rheumatoid, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen expressed on the surface of said cell, said human anti-CD40 monoclonal antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12; d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay; and e) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any one of the preceding items a) -d), wherein said fragment maintains the ability to specifically bind to said human CD40 antigen

10.

The monoclonal antibody of claim 8 or the use of claim 9, wherein said treatment further comprises administering an immunosuppressive agent in a pharmaceutically acceptable excipient.

eleven.

The monoclonal antibody or the use of claim 10, wherein the immunosuppressive agent is selected from the group consisting of cyclosporine, FK506, rapamycin, corticosteroids, CTLA4-Ig, an anti-CD20 antibody and anti-lymphocyte stimulator antibody

B. 12. The monoclonal antibody or the use of any preceding claim, wherein said monoclonal antibody is selected from the group consisting of:

(i)

a monoclonal antibody comprising a light chain variable domain containing residues 44-54, 70-76 and 109-117 of SEQ ID NO: 2 or SEQ ID NO: 6;

(ii)

a monoclonal antibody comprising a heavy chain variable domain containing residues 50-54, 69-84 and 114-121 of SEQ ID NO: 4 or SEQ ID NO: 7;

(iii) a monoclonal antibody comprising a light chain variable domain containing residues 46-52, 70-72 and 111-116 of SEQ ID NO: 2 or SEQ ID NO: 6; Y (iv) a monoclonal antibody comprising a heavy chain variable domain containing residues 45-51, 72-74, 115-120 of SEQ ID NO: 4 or SEQ ID NO: 7. 13. The monoclonal antibody or the use of claim 12, wherein said antibody is a monoclonal antibody comprising an amino acid sequence selected from the group consisting of:

(i)

residues 21-132 of SEQ ID NO: 2;

(ii)

residues 21-239 of SEQ ID NO: 2;

(iii) SEQ ID NO: 2;

(iv)

residues 20-139 of SEQ ID NO: 4;

(v)

residues 20-469 of SEQ ID NO: 4;

(saw)

SEQ ID NO: 4;

(vii) residues 20-469 of SEQ ID NO: 5; (viii) SEQ ID NO: 5;

(ix)

waste 21-132 of SEQ ID NO: 2 and waste 20-139 of SEQ ID NO: 4;

(x)

waste 21-239 of SEQ ID NO: 2 and waste 20-469 of SEQ ID NO: 4;

(xi)

waste 21-239 of SEQ ID NO: 2 and waste 20-469 of SEQ ID NO: 5;

(xii) SEQ ID NO: 2ySEC ID NO: 4; Y (xiii) SEQ ID NO: 2 and SEQ ID NO: 5. 14. The monoclonal antibody or the use of claim 12, wherein said monoclonal antibody comprises an amino acid sequence selected from the group consisting of:

(i)

residues 21-132 of SEQ ID NO: 6;

(ii)

residues 21-239 of SEQ ID NO: 6;

(iii) SEQ ID NO: 6;

(iv)

residues 20-144 of SEQ ID NO: 7;

(v)

residues 20-474 of SEQ ID NO: 7;

(saw)

SEQ ID NO: 7;

(vii) residues 20-474 of SEQ ID NO: 8; (viii) SEQ ID NO: 8;

(ix)

waste 21-132 of SEQ ID NO: 6 and waste 20-144 of SEQ ID NO: 7;

(x)

waste 21-239 of SEQ ID NO: 6 and waste 20-474 of SEQ ID NO: 7;

(xi)

waste 21-239 of SEQ ID NO: 6 and waste 20-474 of SEQ ID NO: 8

(xii) SEQ ID NO: 6ySEC ID NO: 7; Y (xiii) SEQ ID NO: 6 and SEQ ID NO: 8.

fifteen.

The monoclonal antibody or the use of any preceding claim, wherein said antibody binds to human CD40 antigen with an affinity (KD) of at least 10-6 M.

16.

The monoclonal antibody or the use of any preceding claim, wherein said antibody binds to human CD40 antigen with an affinity (KD) of at least 10-8 M.

17.

The monoclonal antibody or the use of any preceding claim, wherein said

5 The antibody is selected from the group consisting of the monoclonal antibody CHIR-5.9 which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 which can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543. 18. The monoclonal antibody or use of any preceding claim, wherein said fragment is selected from the group consisting of a Fab fragment, an F (ab ’) 2 fragment, an Fv fragment and a single chain Fv fragment. 19. The monoclonal antibody or use of any preceding claim, wherein said monoclonal antibody is produced in a CHO cell line.