MXPA06004983A - Antagonist anti-cd40 monoclonal antibodies and methods for their use - Google Patents

Abstract

Methods of therapy for treating diseases mediated by stimulation of CD40 signaling on CD40-expressing cells are provided. The methods comprise administering a therapeutically effective amount of an antagonist anti-CD40 antibody or antigen-binding fragment thereof to a patient in need thereof. The antagonist anti-CD40 antibody or antigen-binding fragment thereof is free of significant agonist activity, but exhibits antagonist activity when the antibody binds a CD40 antigen on a human CD40-expressing cell. Antagonist activity of the anti-CD40 antibody or antigen-binding fragment thereof beneficially inhibits proliferation and/or differentiation of human CD40 -expressing cells, such as B cells.

Description

MONOCLONAL ANTIBODIES ANTI-CD40 ANTAGONISTS AND METHODS FOR USE FIELD OF THE INVENTION The invention relates to human antibodies capable of binding the CD40 antigen, to methods for using the antibodies and to methods for the treatment of diseases mediated by the stimulation of the signaling of the CD40 antigen on cells expressing the CD40 antigen. BACKGROUND OF THE INVENTION B cells play an important role during the immune response, normal in vivo. A foreign antigen will bind to the surface immunoglobulins on the specific B cells, initiating a chain of events that include endocytosis, processing, presentation of processed peptides in the MHC class II molecules and upregulation of the B7 antigen on the surface of the B cells. A specific T cell then binds to the B cell via recognition of the T cell receptor (TCR, for its acronym in English) of the processed antigen that is presented in the MHC class II molecule. The stimulation through the TCR activates the T cell and initiates the production of T cell cytosine. A second signal that activates the T cell additionally is an interaction between the T cell.

REF: 172541 CD28 antigen on T cells and B7 antigen on B cells. When the signals mentioned above are received, the CD40 ligand (CD40L or CD154), which is not expressed on latent human T cells, is upregulated on the surface of T cells. Binding of CD40 ligand to CD40 antigen on the surface of B cells stimulates B cells, causing B cells to mature in plasma cells that secrete high levels of soluble immunoglobulin. The CD40 antigen is a 55 kDa cell surface antigen that is present on the surface of both normal and neoplastic human B cells, dendritic cells, antigen-presenting cells (APCs), endothelial cells, cells onocitic and epithelial. Transformed cells from patients with low-grade and high-grade B-cell lymphomas, acute B-cell lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia and Hodgkin's disease express the CD40 antigen. The expression of the CD40 antigen is also detected in two thirds of the cases of acute myeloblastic leukemia and 50% of AIDS-related lymphomas. The malignant B cells of several tumors of the B-cell lineage express a high degree of the CD40 antigen and appear to depend on the signaling of the CD40 antigen for survival and proliferation.

Immunoblastic B-cell lymphomas frequently arise in uncommitted individuals such as allograft recipients and others receiving long-term immunosuppressive therapy, patients with AIDS and patients with primary immunodeficiency syndromes such as X-linked lymphoproliferative syndrome or Wiscott syndrome. Aldrich (Thomas et al. (1991) Adv. Cancer Res. 57: 329; Straus et al. (1993) Ann. Intern. Med. 118: 45). The CD40 antigen is related to the factor receptor. human neuronal growths (NGF), tumor necrosis factor-a receptor (TNT-a) and Fas, suggesting that the CD40 antigen is a receptor for a ligand with functions important in the activation of B cells. The expression of the CD40 antigen in APCs plays an important co-stimulatory role in the activation of both helper T lymphocytes and cytotoxic T lymphocytes. The CD40 antigen receptor is expressed on activated T cells, activated platelets and smooth, vascular, inflamed muscle cells. The CD40 antigen receptors can also be found in eosinophils, synovial membranes in rheumatoid arthritis, dermal fibroblasts and other types of non-lymphoid cells. The binding of CD40L to the CD40 antigen receptor stimulates the proliferation and differentiation of B cells, the production of antibodies, the switching of isotypes and the generation of B cell memory. BRIEF DESCRIPTION OF THE INVENTION Compositions and methods are provided for treating diseases mediated by the stimulation of CD40 antigen signaling in cells expressing the CD40 antigen, including lymphomas, autoimmune diseases and transplant rejections. • The compositions include monoclonal antibodies capable of binding to a human CD40 antigen that is located on the surface of a cell expressing the human CD40 antigen, wherein the binding prevents the growth or differentiation of the cell. The compositions also include monoclonal antibodies capable of specifically binding to a human CD40 antigen that is expressed on the surface of a cell expressing the human CD40 antigen, the monoclonal antibody is free of significant agonist activity, wherein administration of the monoclonal antibody gives resulted in a significantly lower tumor volume than a similar concentration of the chimeric anti-CD20 monoclonal antibody IDEC-C2B8 in a nude mouse xenograft tumor model staged using the human B cell lymphoma cell line Daudi. The compositions also include antigen-binding fragments of these monoclonal antibodies, hybridoma cell lines that produce these antibodies and isolated nucleic acid molecules that encode the amino acid sequences of these monoclonal antibodies. The invention further includes pharmaceutical compositions comprising these anti-CD40 antibodies in a pharmaceutically acceptable carrier. Methods for preventing or treating a disease mediated by stimulation of CD40 antigen signaling are provided, which comprises treating the patient with an anti-CD40 antibody or an antigen-binding fragment thereof that is free of significant agonist activity when binds a CD40 antigen in a cell that expresses the human CD40 antigen. Diseases mediated by stimulation of cells expressing the CD40 antigen include autoimmune diseases, cancers and rejections of organ and tissue grafts. Lymphomas that can be treated or prevented by a method of the present invention include non-Hodgkin lymphomas (high-grade lymphomas, intermediate-grade lymphomas and low-grade lymphomas), Hodgkin's disease, acute lymphoblastic leukemias, myelomas, lymphocytic leukemias Chronicles and myeloblastic leukemias. Particular autoimmune diseases that are contemplated for treatment using the methods of the invention include systemic lupus erythematosus (SLE), rheumatoid arthritis, Crohn's disease, psoriasis, autoimmune thrombocytopenic purpura, multiple sclerosis, ankylosing spondylitis, myasthenia gravis and pemphigus vulgaris. These antibodies could also be used to prevent rejection of organ and tissue grafts by suppressing autoimmune responses to treat lymphomas by suppressing malignant B lymphocytes of the activating signal provided by the CD40 antigen and to deliver toxins to the antigen-bearing cells. CD40 in a specific way. Methods are provided for inhibiting the growth, differentiation and / or proliferation of human B cells and for inhibiting the production of antibodies by B cells in a human patient, as well as providing methods for inhibiting the growth of cancer cells of a cell lineage. B. Methods for identifying antibodies that have antagonist activity towards cells expressing the CD40 antigen are also provided. The monoclonal antibodies described herein have a strong affinity for the CD40 antigen and are characterized by a dissociation equilibrium constant (KD) of at least 10"6 M, preferably at least about 10" 7 M to about 10"8. M, more preferably at least about 10"8 M to about 10" 12 M. The monoclonal antibodies and antigen-binding fragments thereof which are suitable for use in the methods of the invention are capable of specifically binding to an antigen. Human CD40 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 a fragment thereof exhibits antagonistic activity when it binds to the CD40 antigen on normal, human B cells. In another embodiment, the anti-CD40 antibody or a fragment thereof exhibits antagonistic activity when bound to the CD40 antigen in human, malignant B cells. Suitable monoclonal antibodies have human constant regions, preferably also have regions of partially or completely humanized structure; and much more preferably are fully human antibodies or antigen-binding fragments thereof. Examples of these monoclonal antibodies are the antibodies designated herein as CHIR-5.9 and CHIR-12.12; the monoclonal antibodies produced by the hybridoma cell lines designated 131.2F8.5.9 (referred to in this document as cell line 5.9) and 153.8E2.DIO. D6.12.12 (referred to herein as cell line 12.12); a monoclonal antibody comprising a sequence of amino acids 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; a monoclonal antibody comprising an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; and antigen-binding fragments of these monoclonal antibodies that retain the ability to specifically bind to the human CD40 antigen and that are free of significant agonist activity but exhibit antagonist activity when bound to the CD40 antigen in human cells. Examples of these monoclonal antibodies also include a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 12.12; 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; a monoclonal antibody that competes with the monoclonal antibody CHIR-12.12 in a competitive binding assay; and a monoclonal antibody that is an antigen-binding fragment of the monoclonal antibody CHIR-12.12 or any of the above monoclonal antibodies, wherein the fragment retains the ability to specifically bind to the human CD40 antigen. Those skilled in the art recognize that antagonist antibodies and antigen-binding fragments of these antibodies described herein include antibodies and antigen-binding fragments thereof that are produced using recombinantly methods well known in the art. and described later in this document and include, for example, the monoclonal antibodies CHIR-5.9 and CHIR-12.12 c that have been produced recombinantly. In one embodiment of the invention, methods for treatment comprise administering to a patient a therapeutically effective dose of a pharmaceutical composition comprising suitable anti-CD40 antagonist antibodies or antigen-binding fragments thereof. A therapeutically effective dose of the anti-CD40 antibody or a fragment thereof is in the range of about 0.01 mg / kg to about 40 mg / kg, from about 0.01 mg / kg to about 30 mg / kg, of about 0.1 mg / kg at 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, of 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 method of treatment may comprise an individual administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. Anti-CD40 antagonist antibodies which are identified in this document which are suitable for use in the methods of the invention can be modified. Modifications of these anti-CD40 antagonist antibodies include, but are not limited to, immunologically active chimeric anti-CD40 antibodies, humanized anti-CD40 antibodies and immunologically active murine anti-CD40 antibodies. BRIEF DESCRIPTION OF THE FIGURES Figures IA and IB show the binding of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 to the CD40 antigen on the surface of the lymphoma cell line (Ramos).

Figures 2A and 2B illustrate the binding properties of the monoclonal anti-CD40 antibodies CHIR-5.9 and CHIR-12.12 relative to the ligand of the CD40 antigen. Figure 2A shows that the binding of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 to the cell surface antigen CD40 prevents subsequent binding of the CD40 ligand. Figure 2B shows that the monoclonal antibodies CHIR-5.9 and CHIR-12.12 can compete with the CD40 ligand previously bound to the cell surface CD40 antigen. Figures 3A and 3B show the activity of ADCC of the monoclonal antibodies, candidates CHIR-5.9 and CHIR-12.12 against cancer cells of the lymph nodes of patients with non-Hodgkin lymphoma (NHL, for its acronym in English). NK cells enriched from a voluntary donor, either fresh after isolation (Figure 3A) or after overnight culture at 37 ° C (Figure 3B) were used as effector cells in this assay. As the NHL cells also express the CD20 antigen, the target antigen for rituximab (Rituxan ™ 1), the ADCC activity of the candidate mAbs was compared with that of rituximab. Figure 4 demonstrates the in vivo antitumor activity of monoclonal antibodies CHIR-5.9 and CHIR-12.12 compared to that of rituximab using a xenograft B cell lymphoma model in nude mice not graded in steps (Namalwa). Figure 5 demonstrates the in vivo antitumor activity of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 compared to that of rituximab using a xenograft B-cell lymphoma model in nude mice not graded in steps (Daudi). RC, resistance to tumor testing. Figure 6 demonstrates the in vivo antitumor activity of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 compared to that of rituximab using a B-cell lymphoma model of xenograft in nude mice sorted in stages (Daudi). CR, complete regression. Figure 7 shows the protocol used to determine the number of CD20 and CD40 molecules in Namalwa and Daudi cells (whose title is "Number of Molecules of CD20 and CD40 in Namalwa and Daudii cells"). Figure 8 shows the comparative ADCC of mAb CHIR-12.12 and rituximab against Daudi lymphoma cells. Figures 9A and 9B establish the amino acid sequences for the light and heavy chains of mAb CHIR-12.12. The leader 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 9A. The leader 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 they are shown in Figure 9B. The constant, alternative region for the heavy chain of mAb CHIR-12.12 shown in Figure 9B reflects a substitution of a cerin residue for the alanine residue at position 153 of SEQ ID NO: 4. The complete sequence for this variant of the heavy chain of mAb CHIR-12.12 is set forth in SEQ ID NO: 5. Figures 10A and 10B show the coding sequence for the light chain (Figure 10A; SEQ ID NO: 1) and the heavy chain (Figure 10B; SEQ ID NO: 3) for the CHIR-12.12 mAb. Figures HA and 11B establish the amino acid sequences for the light and heavy chains of the CHIR-5.9 mAb. The leader 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 the Figure HA. The leader regions (residues 1-19 of the SEC 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 11B. The constant, alternative region for the heavy chain of mAb CHIR-5.9 shown in Figure 11B reflects a substitution of a serine residue for the alanine residue at position 158 of SEQ ID NO: 7. The complete sequence for this variant of the heavy chain of mAb CHIR-5.9 is set forth in SEQ ID NO: 8.

Figures 12A to 12D show the coding sequence (Figure 12A; SEQ ID NO: 9) for the short isoform of the human CD40 antigen (amino acid sequence shown in Figure 12B; SEQ ID NO: 10) and the coding sequence (FIG. Figure 12C; SEQ ID NO: 11) for the long isoform of the human CD40 antigen (amino acid sequence shown in Figure 12D). Figure 13 shows the thermal fusion temperature of the CHIR-12.12 antibody in different pH formulations measured by means of differential scanning calorimetry (DSC). DETAILED DESCRIPTION OF THE INVENTION The term "tumor", as used herein, refers to all the growth and proliferation of neoplastic cells, either malignant or benign and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammalian animals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, lymphoma and leukemia. The "antibodies" and the "immunoglobulins" (Igs) are glycoproteins that have the same structural characteristics. While antibodies exhibit specificity for binding to an antigen, immunoglobulins include both antibodies and other antibody-like molecules lacking antigen specificity. The polypeptides of the latter class are produced, for example, at low levels by the lymphoid system and at increased levels by myelomas. The term "antibody" is used in the broadest sense and covers completely assembled antibodies, fragments of antibodies that can bind to antigens (eg, Fab ', F' (ab) 2, Fv, single chain antibodies, 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, i.e., the individual antibodies comprising the population are identical except for possible mutations of natural origin that could be present in smaller quantities. The "native antibodies" and the "native immunoglobulins" are usually heterotetrameric glycoproteins of approximately 150,000 daltons, comprised of two identical light chains (L) and two identical heavy (H) chains. 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 heavy and light chain also has intrachain disulfide connections that are regularly separated. Each heavy chain has at one end a variable domain (VH) followed by a variety 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 variable domain of the light chain 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 light and heavy chain. The term "variable" refers to the fact that certain portions of the variable domains differ extensively in their sequence between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed across all the variable domains of the antibodies. This is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable domains of both light chain and heavy chain. The most highly conserved portions of the variable domains are called the structure regions (FR, for its acronym in English) . Each of the variable domains of the heavy and light, native chains comprises four FR regions, which largely adopt a lamina- /? Configuration, connected by three CDRs, which form loops that connect, and in some cases are part of of, the structure of lamina- / ?. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibodies (see Kabat et al. (1991) NIH Publ. 91-3243, Vol. I, pages 647-669). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit several effector functions, such as Fc receptor binding (FcR), involvement of the antibody in cell-dependent toxicity. antibodies, opsonification, initiation of complement-dependent cytoxicity and degranulation of mast cells. The term "hypervariable region" when used herein refers to amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the variable domain of light chain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the variable domain of heavy chain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th ed., Public Health Service, National Institute of Health, Bethesda, MD) and / or those residues of a "hypervariable loop" (ie residues 26-32). (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the variable domain of heavy chain; Clothia and Lesk (1987). "Mol. Biol. 196: 901-917.) The" structure "or" FR "residues are those variable domain residues different from the hypervariable region residues. "antibodies" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody Examples of antibody fragments include the Fab, FAb ', F (ab') 2 and Fv; diabodies; linear (Zapata et al. (1995) Protein Eng. (8 (10): 1057-1062), single-chain antibody molecules, and multisp antibodies Efficient forms of antibody fragments. The papain digestion of the antibodies produces two identical antigen binding fragments, called the "Fab" fragments, each with an individual antigen binding site and a residual "Fc" fragment, whose name reflects its ability to crystallize easily. The pepsin treatment produces an F (ab ') 2 fragment that has two antigen binding sites and is still capable of cross-linking the antigen.

"Fv" is the minimal antibody fragment that contains a complete antigen recognition and binding site. In a double-stranded Fv species, this region consists of a dimer of a variable domain of heavy chain and light chain in close association, non-covalent. In a single chain Fv species, a heavy chain variable domain and a light chain domain can be covalently linked by means of a flexible peptide linker such that light and heavy chains can associate in a "dimeric" structure that is analogous to that in double-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-V__ dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even an individual variable domain (or half of a Fv fragment comprising only three CDRs specific for an antigen) has the ability to recognize and bind an antigen, albeit with a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragments differ from the Fab 'fragments by the addition of some residues to the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the patella region of the antibody. Fab '-SH is the designation in this document for a Fab' fragment in which the cysteine residue (s) of the constant domains carry a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments having patella cysteines therebetween. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) of any vertebrate animal species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (?) 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 major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM and several of these can be further divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The structure of subunits and the three-dimensional configurations of the different classes of immunoglobulins are well known. The different isotypes have different effector functions. For example, human IgGl and IgG3 isotypes mediate antibody dependent cell-mediated cytotoxicity (ADCC) activity. The word "label" when used herein refers to a detectable compound or composition that is directly or indirectly conjugated to the antibody to generate a "labeled" antibody. The label may be detectable by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze the chemical alteration of a substrate compound or a composition that is detectable. Radionuclides that can serve as detectable labels include, for example, I-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, B-212, and Pd. -109. The label could also be a non-detectable entity such as a toxin. The term "antagonist" is used in the broadest sense and includes any molecule that blocks, inhibits or partially or completely neutralizes a biological activity of a native target described herein or the transcription or translation thereof. The "carriers" as used in this document include carriers, pharmaceutically acceptable excipients or stabilizers that are not toxic to the cell or mammal that is exposed to them at the dosages and concentrations employed. Frequently, the physiologically acceptable carrier 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 (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone, 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 nonionic surfactants such as TWEEN ^, polyethylene glycol (PEG) and Pluronics1 ^. The administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. A "host cell", as used herein, refers to a microorganism or a eukaryotic cell or cell line grown as a unicellular entity that can be, or has been, used as a receptor for a recombinant vector or other polynucleotides of transfer and includes the progeny of the original cell that has been transfected. It is understood that the progeny of an individual cell may not necessarily be completely identical in their morphology or in the complement of genomic or total DNA to the original precursor, due to natural, accidental or deliberate mutation. "Human effector cells" are leukocytes that express one or more FcRs and 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 (PBMCs), natural killer (NK) cells, monocytes, macrophages, eosinophils and neutrophils, with PBMC cells being and NK the most preferred. Antibodies having ADCC activity are typically of the IgGI or IgG3 isotype. It should be noted that in addition to the isolation of IgG1 and IgG3 antibodies, these ADCC mediating antibodies can be made by designing a variable region of an antibody different from ADCC or a variable region fragment to a constant region of the IgGI isotype. or IgG3. 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 human FcR of native sequence. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses Fc? RI, Fc? RII and Fc? RII, which include allelic variants and alternately spliced forms of these receptors. Fc? RII receptors include Fc? RIIA (an "activation receptor") and Fc? RIIB (an "inhibition receptor"), which have similar amino acid sequences that differ mainly in the cytoplasmic domains thereof. The Fc? RIIA activation receptor contains an activation configuration based on tyrosine immunoreceptor (ITAM) in its cytoplasmic domain. The Fc? RIIB inhibition receptor contains an immunoreceptor tyrosine-based inhibition (ITIM) configuration in its cytoplasmic domain (see Daeron (1997) Annu., Rev. Immunol., 15: 203-234). The FcRs are reviewed in Ravetch and Kinet (1991) Annu. .Rev. Immunol. 9: 457-492 (1991); Capel and collaborators (1994) I_7iniuno.met.hods 4: 25-34; and from Haas and collaborators (1995) J. "Lab. Clin. Med. 126: 330- 341. Other FcRs, including those to be identified in the future, are comprised 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 a variety of ways to make human antibodies. For example, secretion cells can be immortalized by the infection with the Epstein-Barr virus (EBV, for its acronym in English). However, cells infected with EBV are difficult to clone and usually produce relatively low yields of immunoglobulin only (James and Bell (1987) J. I munol, Methods 100: 5-40). In the future, the immortalization of human B cells could possibly be achieved by introducing a defined combination of transformation genes. This possibility is highlighted by a recent demonstration that the expression of the catalytic subunit of telomerase together with the SV40 large oncoprotein and an oncogenic allele of H-ras resulted in the tumorigenic conversion of the human, normal epithelial cells and fibroblasts (Hahn et al. collaborators (1999) Nature 400: 464-468). It is now possible to produce transgenic animals (e.g., mice) that are capable, with immunization, of producing a repertoire of human antibodies in the absence of the production of endogenous immunoglobulin (Jakobovits et al. (1993) Nature 362: 255-258; Lonberg and Huszar (1995) Jnt. Rev. Immunol 13: 65-96; Fishwild et al. (1996) Nat. Biotechnol., 14: 845-851; Mendez et al. (1997) Nat. Genet., 15: 146-156; 1999) J. Immunol. Methods 231: 11-23; Tomizuka et al. (2000) Proc. Nati, Acad. Sci. USA 97: 722-727; reviewed in Little et al. (2000) Immunol. Today 21: 364-370). For example, it has been described that the deletion of homozygotes from the heavy chain binding region (JH) gene of the antibody in mutant, chimeric and germline mice results in complete inhibition of the production of endogenous antibodies (Jakobovits and collaborators (1993) Proc. Nati, Acad. Sci. USA 90: 2551-2555). The transfer of the genetic ordering of human germline immunoglobulin in these germline mutant mice results in the production of human antibodies with the antigen test (Jacobovits et al. (1993) Nature 362: 255-258). Méndez et al. (1997) (Nature Genetics 15: 146-156) has generated a line of transgenic mice that, when tested with an antigen, generates fully human antibodies of high affinity. This is achieved by integrating germ lines of the megabase human heavy chain and light chain locus (specific positions) in mice with deletion in the endogenous JH segment as described above. These mice (XenoMouseIIMR technology (Abgenix, Fremont, California)) harbor 1,020 kb of the human heavy chain locus containing approximately 66 VH genes, complete DH and J regions and three different constant regions, and also host 800 kb of the human K locus that contains 32 Vsr genes, JK segments and CK genes. The antibodies produced in these mice closely resemble those observed in humans in all aspects, including rearrangement, assembly and gene repertoire. Human antibodies are preferentially expressed on endogenous antibodies due to suppression in the endogenous segment that prevents rearrangement of genes at the murine locus. These mice can be immunized with an antigen of particular interest. The sera of these immunized animals can be examined by the reactivity of antibodies against the initial antigen. The lymphocytes can be isolated from lymph nodes or spleen cells and can be further selected by B cells to select the negative cells in CD138 and positive in CD19. In one aspect, these B cell cultures (BCCs) can be fused to myeloma cells to generate hybridomas as detailed above. In another aspect, these B cell cultures can be further examined for the reactivity against the initial antigen, preferably. This test includes ELISA with the target protein / antigen, a competition assay with known antibodies that binds to the antigen of interest and in vitro binding to transiently transfected CHO cells or other cells that express the target antigen.

The present invention is directed to compositions and methods for treating human patients with diseases mediated by the stimulation of CD40 antigen signaling in cells expressing the CD40 antigen. The methods include treatment with an anti-CD40 antibody of the invention, or an antigen-binding fragment thereof, wherein the administration of the antibody or antigen-binding fragment thereof promotes a positive, therapeutic response within the patient being treated. submit to this method of therapy. Anti-CD40 antibodies that are suitable for use in the methods of the invention bind specifically to a human CD40 antigen expressed on the surface of a human cell or are free of significant agonist activity, but exhibit antagonistic activity when bound to CD40 antigen in a cell expressing the human CD40 antigen. These anti-CD40 antibodies and antigen-binding fragments thereof are referred to herein as anti-CD40 antagonist antibodies. These antibodies include, but are not limited to, the fully human monoclonal antibodies CHIR-5.9 and CHIR-12.12 described below and the monoclonal antibodies that have the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12. Those skilled in the art recognize that antagonist antibodies and antigen-binding fragments of these antibodies described herein include antibodies and antigen-binding fragments thereof that are recombinantly produced using methods well known in the art and described later in this document and include, for example, the monoclonal antibodies CHIR-5.9 and CHIR-12.12 that have been produced recombinantly. Antibodies that have the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 include antibodies that competitively interfere with the binding of the CD40 antigen and / or bind to the same epitopes as the CHIR-5.9 and CHIR antibodies -12.12. An expert could determine if an antibody competitively interferes with antibodies CHIR-5.9 and CHIR-12.12 using standard methods. When those antibodies bind to the CD40 antigen displayed on the surface of human cells, such as human B cells, the antibodies are free of significant agonist activity; in some embodiments, its binding to the CD40 antigen displayed on the surface of human cells results in inhibition of the proliferation and differentiation of those human cells. Thus, anti-CD40 antagonist antibodies that are suitable for use in the methods of the invention include those monoclonal antibodies that can exhibit antagonistic activity towards normal and malignant human cells that express the cell surface CD40 antigen. In some embodiments, the anti-CD40 antibodies of the invention exhibit increased antitumor activity relative to the chimeric anti-CD20 monoclonal antibody IDEC-C2B8, where the antitumor activity is tested with equivalent amounts of those antibodies in a xenograft tumor model. in nude mice using the cell lines' of human lymphoma. The IDEC-C2B8 antibody (IDEC Pharmaceuticals Corp., San Diego, California, commercially available under the trade name Rituxan ™, also referred to as rituximab) is a chimeric anti-CD20 monoclonal antibody containing human IgGl and kappa constant regions with variable regions of murine isolated from a murine anti-CD20 monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood 83: 435-445). Rituximab ™ is approved for the treatment of low-grade non-Hodgkin's lymphoma or follicular B-cell lymphoma (NHL). The discovery of antibodies with superior antitumor activity compared to Rituximab ™ could dramatically improve the methods of cancer therapy for B-cell lymphomas, particularly B-cell non-Hodgkin lymphoma. Suitable models of xenograft tumors in nude mice include those using the human Burkitt lymphoma cell lines known as Namalwa and Daudi. Preferred embodiments test antitumor activity in a nude mouse xenograft tumor model staged using the human lymphoma cell line Daudi as described. described in this document later in Example 17. A nude mouse xenograft tumor model staged using the Daudi lymphoma cell line is more effective in distinguishing the therapeutic efficacy of an antibody than a model that is not classified. by stages, since the dosage of the antibody in the stepped model is initiated only after the tumor has reached a measurable size. In the model that is not staged, antibody dosing generally starts within about 1 day of tumor inoculation and before a palpable tumor is present. The ability of an antibody to function better than Rituxan ™ 1 (ie, to exhibit increased antitumor activity) in a stepped model is a strong indication that the antibody will be therapeutically more effective than Rituxan ™ 1. In addition, in the Daudi model, anti-CD20, the target for Rituxan "11 is expressed on the cell surface at a higher level than the CD40 antigen." "Equivalent amount" of the anti-CD40 antibody of the invention and the Rituxan ™ 1 it is proposed that the same dose in mg be administered on a weight basis.Thus, where the anti-CD40 antibody of the invention is dosed at 0.01 mg / kg of body weight of the mouse used in the tumor model, Rituxan ™ 1 is also dosed at 0.01 mg / kg of mouse body weight Similarly, where the anti-CD40 antibody of the invention is dosed at 0.1, the 10 mg / kg of body weight of the mouse used in the model of tumor, Rituxan ™ 1 is also dosed at 0.1, 1 or 10 mg / kg, respectively, of the mouse body weight When administered in the xenograft tumor model in nude mice, some antibodies of the invention result a tumor volume significantly lower than an equivalent amount of Rituxan ™ 1. Thus, for example, the fully human monoclonal antibody CHIR-12.12 exhibits an increase of at least 20% in antitumor activity relative to that observed with an equivalent dose of Rituxan ™ 1 when tested in the tumor model of xenograft in nude mice sorted in stages using the human lymphoma cell line Daudi in the manner described in the following examples and may exhibit an increase as high as 50% to 60% in antitumor activity in this assay. This increased antitumor activity is reflected in the greater reduction in tumor volume observed with the anti-CD40 antibody of the invention compared to the equivalent dose of Rituxan ™ 1. Thus, for example, depending on the length of time after tumor inoculation, the monoclonal antibody CHIR-12.12 can exhibit a tumor volume that is from about one third to about half that observed for an equivalent dose of Rituxan ™ 1 Another difference in the effectiveness of the antibody is to measure in vi tro the concentration of the antibody necessary to obtain the maximum lysis of the tumor cells in vi tro in the presence of NK cells. For example, the anti-CD40 antibodies of the invention achieve maximum lysis of Daudi cells at an EC50 value of less than 1 and preferably A and much more preferably, 1/10 of the concentration of Rituxan ™ 1. In addition to the monoclonal antibody CHIR-12.12, other anti-CD40 antibodies that would share the characteristics of having a significantly greater efficacy than the equivalent amounts of Rituxan ™ 1 in the assays described above include, but are not limited to: (1) the monoclonal antibody produced by the 12.12 hybridoma cell line; (2) a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence in SEQ ID NO: 2, the sequence in SEQ ID NO: 4, the sequence in SEQ ID NO: 5, the sequence both in SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence in both SEQ ID NO: 2 and SEQ ID NO: 5; (3) 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 in SEQ ID NO: 1, the nucleotide sequence in the SEC ID NO: 3 and the nucleotide sequence both in SEQ ID NO: 1, and in SEQ ID NO: 3; (4) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 12.12; (5) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the amino acid sequence in SEQ ID NO: 10 or SEQ ID NO: 12; (6) a monoclonal antibody that competes with the monoclonal antibody CHIR-12.12 in a competitive binding assay; and (7) a monoclonal antibody that is an antigen-binding fragment of the monoclonal antibody CHIR-12.12 or the above monoclonal antibodies in the preceding articles (1) - (6), wherein the fragment retains the ability to specifically bind to the CD40 antigen human. Anti-CD40 Antibodies Antagonists The monoclonal antibodies CHIR-5.9 and CHIR-12.12 represent anti-CD40 antibodies, antagonists, suitable for use in the methods of the present invention. The CHIR-5.9 and CHIR-12.12 antibodies are fully human anti-CD40 monoclonal antibodies of the IgGI isotype produced from the 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 that contained the heavy chain locus IgG? human and the human K chain locus (XenoMouseMR technology, Abgenix, Fremont, California). Spleen cells were fused with SP2 / 0 mouse myeloma cells (Sierra BioSource). The resulting hybridomas were sub-cloned several times to create stable, monoclonal cell lines 5.9 and 12.12. Other antibodies of the invention can be prepared in a similar manner using mice transgenic for human immunoglobulin loci or by other methods known in the art and / or described herein. The nucleotide and amino acid sequences of the variable regions of the CHIR-12.12 antibody and the amino acid sequences of the variable regions of the CHIR-5.9 antibody are described. More particularly, the amino acid sequences for the leader, variable and constant regions for the light chain and the heavy chain for the CHIR-12.12 mAb are set forth in Figures 9A and 9B, respectively. See also SEQ ID NO: 2 (complete sequence for light chain of mAb CHIR-12.12), SEQ ID NO: 4 (complete sequence for heavy chain for mAb CHIR-12, 12) and SEQ ID NO: 5 ( complete sequence for a heavy chain variant for the mAb CHIR-12 .12 set forth in SEQ ID NO: 4, wherein the variant comprises a substitution of serine by the alanine residue at position 153 of SEQ ID NO: 4 ). The nucleotide sequences encoding the light chain and the heavy chain for the CHIR-12 mAb. 12 are shown in Figures HA and 11B, respectively. See also SEQ ID NO: 1 (coding sequence for light chain for mAb CHIR-12.12) and SEQ ID NO: 3 (coding sequence for heavy chain for mAb CHIR-12.12). The amino acid sequences for the leader, variable and constant regions for the light chain and the heavy chain of the CHIR-5.9 mAb are shown in Figures 10A and 10B, respectively. See also SEQ ID NO: 6 (complete sequence for the light chain of mAb CHIR-5.9), SEQ ID NO: 7 (complete sequence for the heavy chain of mAb CHIR-5, 9) and SEQ ID NO: 8 (complete sequence for a variant of the heavy chain of mAb CHIR-5, 9 set forth in SEQ ID NO. : 7, wherein the variant comprises a serine substitution for the alanine residue at position 158 of SEQ ID NO: 7). In addition, hybridomas that express CHIR-5 antibodies. 9 and CHIR-12. 12 have been deposited with the ATCC with a patent deposit designation of PTA-5542 and PTA-5543, respectively. In addition to the antagonist activity, it is preferable that the anti-CD40 antibodies of this invention have another mechanism of action against a tumor cell. For example, the native antibodies CHIR-5.9 and CHIR-12.12 have ADCC activity. Alternatively, the variable regions of the CHIR-5.9 and CHIR-12.12 antibodies can be expressed in another isotype of antibody having ADCC activity. It is also possible to conjugate the native forms, recombinant forms or antigen-binding fragments of CHIR-5.9 or CHIR-12.12 to a cytotoxin, a therapeutic agent or a radioactive metal ion or radioisotope, as later observed herein. The monoclonal antibodies CHIR-5.9 and CHIR-12.12 bind to the soluble CD40 antigen in the ELISA-type assays, prevent the binding of the CD40 ligand to the CD40 antigen on the cell surface and displace the previously bound CD40 ligand, as determined by means of flow cytometric tests. The antibodies CHIR-5.9 and CHIR-12.12 compete with each other for binding to the CD40 antigen but not to 15B8, the anti-CD40 monoclonal antibody described in the provisional US application Serial No. 60 / 237,556, entitled "Human Anti-CD40 Antibodies ", filed on October 2, 2000 and the international application of PCT No. PCT / US01 / 30857, also entitled" Human Anti-CD40 Antibodies ", filed on October 2, 2001 (Attorney Registration Number PP16092.003), both are incorporated in this document as a reference in their entirety, when they are tested in vi tro for the effects on the proliferation of B antibodies from normal human subjects, CHIR-5.9 and CHIR-12.12 antibodies act as anti-CD40 antagonist antibodies, and CHIR-5.9 and CHIR-12.12 antibodies do not induce a strong proliferation of human lymphocytes from normal subjects. they are able to eliminate the target cells expressing the CD40 antigen by means of antibody-dependent cellular cytotoxicity (ADCC). The binding affinity of the CHIR-5.9 antibody for the human CD40 antigen is 1.2xl0-8 M and the affinity of binding of the CHIR-12.12 antibody is 5x10"10 M, as determined by the Biacore ™ 1 assay. Anti-CD40 antibodies, antagonists, suitable for use in the methods of the present invention exhibit strong affinity for binding to individual sites by the CD40 cell surface antigen. The monoclonal antibodies of the invention exhibit a dissociation equilibrium constant (KD) for the CD40 antigen of at least 10"5 M, at least 3x10" 5 M, preferably at least 10"6 M to 10" 7 M, more preferably from at least 10"8 M to approximately 1Ó ~ 12 M, measured using a standard assay such as Biacore ™ 1. Biacore ™ 1 analysis is known in the field and details are provided in" BIAaplications handbook. "The methods described in WO 01/27160 can be used to modulate the binding affinity.

By "CD40 antigen", "CD40 cell surface antigen", "CD40 receptor" or "CD40" is proposed a transmembrane glycoprotein that belongs to the family of tumor necrosis factor (TNF) receptors (see, for example, U.S. Patent Nos. 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 (2d ed., Academia Press, San. Diego)). Two isoforms of the human CD40 antigen, encoded by alternatively spliced transcript variants of this gene, have been identified. 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 (reported first as GenBank accession No. CAA43045 and identified as isoform 1 in GenBank access No. NP_001241), encoded by SEQ ID NO: 11 (see GenBank accesses Nos. 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 access No. NM_152854)), which also has a signal sequence represented by the first 19 residues. The precursor polypeptides of these two isoforms of the human CD40 antigen share their first 165 residues in common (ie, residues 1-165 of SEQ ID NO: 10 and SEQ ID NO: 12). The precursor polypeptide of the short isoform (shown in SEQ ID NO: 10) is encoded by a transcription variant (SEQ ID NO: 9) lacking a coding segment, which leads to a translation frame change; the resulting CD40 isoform contains a shorter and distinct C-terminus (residues 166-203 of SEQ ID NO: 10) than that contained in the long isoform of CD40 antigen (terminus C shown at residues 166-277 of SEQ ID NO: 10). NO: 12). For purposes of the present invention, the term "CD40 antigen", "CD40 cell surface antigen", "CD40 receptor" or "CD40" includes both short and long isoforms of the CD40 antigen. The anti-CD40 antibodies of the present invention bind to an epitope of the human CD40 antigen that resides in the same location within either the short isoform or the long isoform of this cell surface antigen as later observed herein. The CD40 antigen is displayed on the surface of a variety of cell types, as described elsewhere in this document. By "displayed on the surface" and "expressed on the surface" it is proposed that all or a portion of the CD40 antigen be exposed to the outside of the cell. The CD40 antigen displayed or expressed can be glycosylated in whole or in part. By "agonist activity" it is proposed that the substance function as an agonist. An agonist combines with a receptor on a cell and initiates a reaction or activity that is similar to that or is the same as that initiated by the natural ligand of the receptor. An agonist of the CD40 antigen includes any or all of, but is not limited to, the following responses: proliferation and differentiation of B cells, production of antibodies, intercellular adhesion, generation of B cell memory, isotype switching, upregulation of the Cell surface expression of MHC Class II and CD80 / 86 and secretion of pro-inflammatory cytokines such as IL-8, IL-12 and TNF. By "antagonistic activity" it is proposed that the substance function as an antagonist. An antagonist of the CD40 antigen prevents or reduces the induction of any of the responses induced by the binding of the CD40 receptor to an agonist ligand, particularly CD40L. The antagonist can reduce the induction of one or more of any of the responses to the agonist binding by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50% , 55%, 60%, more preferably 70%, 80%, 85% and much more preferably 90%, 95%, 99% or 100%. Methods for measuring binding specificity and antagonist activity of the anti-CD40 antibody and the CD40 ligand are known to a person skilled in the art and include, but are not limited to, standard competitive binding assays, assays to monitor the immunoglobulin secretion by B cells, B-cell proliferation assays, Banchereau-like B-cell proliferation assays, collaborating T-cell assays for antibody production, co-stimulation of B-cell proliferation assays, and assays for upregulation of markers of B cell activation. See, for example, assays such as those described in WO 00/75348 and U.S. Patent No. 6,087,329, incorporated herein by reference. By "significant" agonist activity we propose an agonist activity of at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% greater than the agonist activity induced by a neutral substance or a negative control measured in an assay of a B cell response. Preferably, a "significant" agonist activity is an agonist activity that is at least 2 times or at least 3 times greater than the agonist activity induced by a neutral substance or a negative control measured in an assay of a B cell response. Thus, for example, where the B cell response of interest is the proliferation of B cells, the activity "significant" agonist would be the induction of a level of B cell proliferation that is at least 2 times greater or at least 3 times higher 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 IgGl, which does not bind to the CD40 antigen serves as the negative control. A substance "free of significant agonist activity" would exhibit an agonist activity no greater than about 25% greater than the agonist activity induced by a neutral substance or a negative control, preferably no more than about 20% higher, 15% higher, 10% higher , 5% higher, 1% higher, 0.5% higher or even no more than approximately 0.1% greater than agonist activity induced by a neutral substance or a negative control measured in a B-cell response assay. Anti-CD40 antibodies Antagonists that are useful in the methods of the present invention are free of a significant agonist activity as will be seen above when they bind to a CD40 antigen in a human cell. In an 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 one response of B cells (for example, proliferation and differentiation or proliferation, differentiation and production of antibodies). As used herein an "anti-CD40 antibody" includes any antibody that specifically recognizes the surface antigen of CD40 B cells, including polyclonal antibodies, monoclonal antibodies, single chain antibodies and fragments thereof such as Fab, F (ab ') 2, Fv and other fragments that retain the antigen binding function of the precursor anti-CD40 antibody. Of particular interest for the present invention are the anti-CD40 antagonist antibodies described herein that share the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described above. These antibodies include but are not limited to the following: (1) the monoclonal antibodies produced by the hybridoma cell lines designated 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.DIO .D6.12.12 ( referred to herein as cell line 12.12), deposited with 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 , the sequences shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequences shown in both 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 , the sequences shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequences shown both 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 FIG. SEQ ID NO: 3 and the sequences shown both in SEQ ID NO: 1 and SEQ ID NO: 3; (5) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the 5.9 hybridoma cell line or the 12.12 hybridoma cell line; (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 an antigen-binding fragment of the monoclonal antibody CHIR-12.12 or CHIR-5.9 or the above monoclonal antibodies in the preceding articles (l) - (7), wherein the fragment retains the ability to specifically bind to the human CD40 antigen. Production of Anti-CD40 Antibodies Antagonists The anti-CD40 antagonist antibodies described herein and for use in the methods of the present invention can be produced using any method of antibody production known to those skilled in the art. In this way, polyclonal sera can be prepared by conventional methods. 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 preferred for the preparation of polyclonal sera due to the volume of serum that can be obtained and the availability of antibodies labeled anti-rabbit and anticabra. Polyclonal sera can be prepared in a transgenic animal, preferably a mouse that carries a human immunoglobulin locus. In a preferred embodiment, Sf9 cells expressing the CD40 antigen are used as the immunogen. The immunization can also be performed by mixing or emulsifying the solution containing the antigen in saline, preferably in an adjuvant such as incomplete Freund's adjuvant and injecting the mixture or emulsion by the parenteral route (generally by the subcutaneous or intramuscular route). A dose of 50-200 μg / injection is typically sufficient. The immunization is generally reinforced 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's complete adjuvant. One can alternatively generate antibodies by means of in vitro immunization using methods known in the art, which for purposes of this invention are considered equivalent to immunization in vivo. Polyclonal antisera are obtained by bleeding 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 means of centrifugation (for example, 1,000 x g for 10 minutes). From rabbits, approximately 20-50 ml can be obtained by bleeding. The production of Sf9 cells. { Spodoptera frugiperda) is described in U.S. Patent No. 6,004,552, incorporated herein by reference. Briefly, the sequences encoding the human CD40 antigen were recombined in a baculovirus using transfer vectors. The plasmids were co-transfected with the baculovirus DNA of the type not cultured in Sf9 cells. Sf9 cells infected with recombinant baculovirus were identified and purified by cloning. Preferably, the antibody is of a monoclonal nature. By "monoclonal antibody" an antibody obtained from a population of substantially homogeneous antibodies is proposed, ie, the individual antibodies comprising the population are identical except for possible mutations of natural origin that may be present in minor amounts. The term is not limited with respect to the species or source of the antibody. The term includes complete immunoglobulins as well as fragments such as Fab, F (ab ') 2, Fv and others that retain the antigen-binding function of the antibody. The monoclonal antibodies are highly specific, being directed against an individual antigenic site, ie, the cell surface antigen CD40 in the present invention. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against an individual determinant in the antigen. The "monoclonal" modifier indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be considered as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495 or can be made by recombinant DNA methods (cf. for example, U.S. 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 U.S. Patent No. 5,514,548. By "epitope" is proposed the part of an antigenic molecule for which an antibody is produced and to which the antibody will bind. The epitopes may comprise linear amino acid residues (ie, the residues within the epitope are sequentially arranged one after the other in a linear fashion), non-linear amino acid residues (referred to herein as "non-linear epitopes"), these epitopes do not sequentially arranged) or amino acid residues, both linear and non-linear. 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 an antigen. The immunization can be carried out by mixing or emulsifying the solution containing the antigen in saline, preferably in an adjuvant such as a complete Freund's adjuvant and injecting the mixture or emulsion by the parenteral route. Any method of immunization known in the art can be used to obtain the monoclonal antibodies of the invention. After immunization of the animal, the spleen (and optionally, several large lymph nodes) are removed and dissociated into individual cells. Spleen cells can be examined by applying a cell suspension to a plate or well coated with the antigen of interest. B cells expressing membrane-bound immunoglobulin that is specific for the antigen bind to the plate and are not removed by rinsing. The resulting B cells, or all of the dissociated cells of the spleen, are then induced to fuse with myeloma cells to form hybridomas and are cultured in a selective medium. The resulting cells are plated by serial dilution and tested for the production of antibodies that specifically bind to the antigen of interest (and that do not bind to unrelated antigens). Hybridomas that secrete the selected monoclonal antibody (mAb) are then cultured either in vi tro (for example, in tissue culture bottles or hollow fiber reactors) or in vivo (as ascites in mice).

Where the anti-CD40 antagonist antibodies of the invention should be prepared using recombinant DNA methods, the DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (eg, by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). The hybridoma cells described herein serve as a preferred source of this 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 cells (CHO) or myeloma cells that do not otherwise produce an immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the host, recombinant cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al. (1993) Curr. Opinion in Immunol, 5: 256 and Phickthun (1992) I munol. Revs. 130: 151. Alternatively, an antibody can be produced in a cell line such as a CHO cell line, as described in U.S. Patent Nos. 5,545,403; 5,545,405 and 5,998,144; incorporated in this document as a reference. 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, eg, the CHIR-12.12 or CHIR-5.9 antibody or an antigen-binding fragment thereof, is produced in CHO cells using the GS ™ 1 gene expression system ( Lonza Biologics, Portsmouth, New Hampshire), which uses glutamine synthetase as a marker. See also U.S. Patent Nos. 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; The contents of which are incorporated in this document as a reference in its entirety. Monoclonal antibodies to the CD40 antigen are known in the art. See, for example, sections devoted to the B cell antigen in McMichael, ed (1987; 1989) Le kocyte Typing III and IV (Oxford University Press, New York); U.S. Patent Nos. 5,674,492; 5,874,082; 5,677,165: 6,056,959: WO 00/63395; International Publications Nos. 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. Immuno 1. 146: 1836; Gasean et al. (1991) J. Immuno 1. 147: 8; Banchereau et al. (1991) Clin. Immunol. Spectrum 3: 8 and Banchereau et al. (1991) Science 251: 70; all of which are incorporated in this document as a reference. Of particular interest for the present invention are the anti-CD40 antagonist antibodies described herein which share the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described above. The term "CD40 antigen epitope" used herein refers to a molecule that has the ability to immunoreact with the anti-CD40 monoclonal antibodies of this invention, excluding the CD40 antigen itself. The epitopes of the CD40 antigen can comprise proteins, protein fragments, peptides, carbohydrates, lipids and other molecules, but for the purposes of the present invention proteins, short oligopeptides, oligopeptide mimics are more commonly (ie, organic compounds that mimic the antibody-binding properties of the CD40 antigen) or combinations thereof. Suitable oligopeptide mimics are described, inter alia, in PCT application US 91/04282.

Additionally, the term "anti-CD40 antibody" used herein includes chimeric anti-CD40 antibodies; these chimeric anti-CD40 antibodies for use in the methods of the invention have the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein. By "chimeric" antibodies are proposed antibodies that are more preferably derived using recombinant deoxyribonucleic acid techniques and which comprise both human (including immunologically "related", eg, chimpanzee) and non-human components. In this way, the constant region of the chimeric antibody is, much more preferably, substantially identical to the constant region of a neutral human antibody; the variable region of the chimeric antibody is much more preferably derived from a non-human source and has the antigenic specificity desired by the CD40 cell surface antigen. The non-human source can be any vertebrate animal source that can be used to generate antibodies to the human CD40 cell surface antigen or a material comprising a human CD40 cell surface antigen. These non-human sources include, but are not limited to, rodents (e.g., rabbits, rats, mice, etc. see, for example, U.S. Patent No. 4,816,567, incorporated herein by reference) and non-human primates. (for example, Old World Monkey, Ape, etc., see, for example, U.S. Patent Nos. 5,750,105 and 5,756,096, incorporated herein by reference). As used herein, the phrase "immunologically active" when used with reference to chimeric anti-CD40 antibodies means a chimeric antibody that binds to the human CD40 antigen. Chimeric and humanized anti-CD40 antibodies are also encompassed by the term anti-CD40 antibody used herein. Chimeric antibodies comprise segments of antibodies derived from different species. Rituxan ™ 1 is an example of a chimeric antibody with a variable region of murine and a constant region of human. By "humanized" we propose 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 the residues of a hypervariable region (also known as the complementarity determining region or CDR) of the receptor are replaced by residues from a hypervariable region of a non-human species. human (donor antibody) such as a mouse, rat, rabbit or non-human primate having the desired specificity, affinity and capacity. The phrase "region of complementarity determination" refers to the amino acid sequences that together define the binding affinity and specificity of the native 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 phrase "constant region" refers to the portion of the antibody molecule that confers effector functions. In a previous work directed towards the production of non-immunogenic antibodies for use in the therapy of a human disease, the constant mouse regions were replaced by constant human regions. The constant regions of the humanized, target antibodies were derived from human immunoglobulins. However, these humanized antibodies still produced an unwanted and potentially dangerous immune response in humans and there was a 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 monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein. Humanization can be carried out essentially following the method 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: 1543-1536), by substituting the CDRs or CDR sequences of rodent or mutant rodent for the corresponding sequences of a human antibody. See also U.S. Patent No. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; incorporated in this document as a reference. In some cases, residues within the framework regions of one or more variable regions of human immunoglobulin are replaced by corresponding non-human residues (see, for example, U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762 and 6,180,370). In addition, the humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further define the performance of the antibody (e.g., to obtain a desired affinity). In general, the humanized antibody will substantially comprise all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the of the framework regions are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional 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; incorporated in this document as a reference. Accordingly, these "humanized" antibodies can include antibodies wherein substantially less than one variable, human, intact domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some structure residues are replaced by residues from analogous sites in rodent antibodies. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089: 5,693,761; 5,693,762; 5,859,205. See also U.S. Patent No. 6,180,370 and International Publication No. WO 01/27160, which disclose humanized antibodies and techniques for producing humanized antibodies that have improved affinity for a predetermined antigen. Also included by the term "anti-CD40 antibodies" are the xenogenetics or modified anti-CD40 antibodies that are produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by endogenous, inactivated immunoglobulin (Ig) loci. In these transgenic animals, the endogenous genes, competent for the expression of light and heavy subunits of the host immunoglobulins, are rendered non-functional and are replaced by the analogous human immunoglobulin 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 Nos. 5,877,397 and 5,939,598, incorporated herein by reference. Preferably, the fully human antibodies for the CD40 antigen are obtained by immunizing transgenic mice. One of these mice is obtained using the XenoMouse ™ 1 technology (Abgenix, Fremont, California) and is described in U.S. Patent Nos. 6,075,181, 6,091,001 and 6,114,598, all of which are incorporated herein by reference. To produce the antibodies described herein, the transgenic mice for the heavy chain locus of human IgGi and the light chain locus Human K were immunized with Sf9 cells expressing the human CD40 antigen. Mice can also be transgenic for other isotypes. The fully human antibodies that are useful in the methods of the present invention are characterized by binding properties similar to those exhibited by the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein. Fragments of 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. In this way, a fragment of an anti-CD40 antibody will retain the ability to bind to the CD40 B cell surface antigen. These fragments are characterized by properties similar to the corresponding full length antagonist anti-CD40 antibody, i.e., the fragments 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 a antagonistic activity when they bind to a CD40 antigen on a cell expressing the human CD40 antigen. These fragments are referred to herein as "antigen binding" fragments. Suitable antigen binding fragments of an antibody comprise a portion of a full length antibody, generally the antigen binding region or variable thereof. Examples of antibody fragments include, but are not limited to, Fab, F (ab ') 2 and Fv fragments and individual chain antibody molecules. By "Fab" is proposed an antigen binding fragment, monovalent of an immunoglobulin which is composed of the light chain and part of the heavy chain. By F (ab ') 2 a fragment of binding to antogenes is proposed, bivalent of an immunoglobulin that contains both light chains and part of both heavy chains. For the "individual chain Fv" or "sFv" antibody fragments, fragments comprising the VH and VL domains of an antibody are proposed, wherein 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, incorporated herein by reference. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which makes it possible for the sFv fragment to form the desired structure for antigen binding. For a review of the sFv fragment see Pluckthun (1994) in The Pharmacology of Monoclonal Antibodies, Vol. 113, ed Rosenburg and Moore (Springer-Verlag, New York), pages 269-369-315. The antigen-binding fragments of the anti-CD40 antagonist antibodies described herein can also be conjugated to a cytotoxin to effect the removal of target cancer cells, as described hereinafter. Antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al. (1990) Nature 348: 552-554 (1990) and U.S. Patent No. 5,514,548. Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent reports describe the production of high affinity human antibodies (range of nM) by chain intermixing (Marks et al. (1992) Bio / Technology 10 - 119-183), as well as combinatorial infection and in vivo recombination. as a strategy to build very large phage libraries (Waterhouse et al. (1993) Nucleic Acids Res. 21: 2265-2266) .Thus, these techniques are viable alternatives to traditional hybridoma monoclonal antibody techniques for isolation of monoclonal antibodies Several techniques have been developed for the production of antibody fragments.Traditionally, these fragments are derived by way of 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 the host, recombinant cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab '-SH fragments can be recovered directly from E. coli and can be 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 the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. Anti-CD40 antagonist antibodies that are useful in the methods of the present invention include the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein as well as antibodies that differ from this antibody but retain the CDRs; and antibodies with one or more amino acid additions, deletions or substitutions, wherein the antagonist activity is measured by the inhibition of B cell proliferation and / or differentiation. The invention also includes anti-CD40, antagonist, deimmunized antibodies, which they can be produced as described in, for example, International Publications Nos. WO 98/52976 and WO 0034317; incorporated in this document as a reference. In this manner, the residues within the antagonist anti-CD40 antibodies of the invention are modified to render the non-immunogenic or less immunogenic antibodies towards humans while retaining their antagonistic activity towards the cells expressing the human CD40 antigen, where activity is measured by trials observed elsewhere in this document. Also included within the scope of the claims are fusion proteins comprising an anti-CD40 antagonist antibody of the invention, or a fragment thereof, fusion proteins that can be synthesized or expressed from corresponding polynucleotide vectors, as is known in the field. These fusion proteins are described with reference to the conjugation of antibodies as seen below. The antibodies of the present invention can have sequence variations produced using the methods described in, for example, patent applications Nos. EP 0 983 303 A1, WO 00/34317 and WO 98/52976, incorporated herein by way of reference. For example, it has been shown that sequences within the CDR can cause an antibody to bind to MHC class II and trigger an undesired response of helper T cells. The conservative substitution may allow the antibody to retain the binding activity even if it loses its ability to trigger an unwanted response of T cells. Any of these conservative or non-conservative substitutions may be made using methods recognized in the art, such as those observed in Another part in this document and the resulting antibodies will be within the scope of the invention. The variant antibodies can be routinely tested for antagonist activity, affinity and specificity using methods described herein. An antibody produced by any of the methods described above or any other method that is not described herein, will be within the scope of the invention if it possesses at least one of the following biological activities: inhibition of immunoglobulin secretion by peripheral, human, normal B cells that are stimulated by T cells; inhibition of survival and / or proliferation of peripheral, human, normal B cells that are stimulated by Jurkat T cells; inhibition of survival and / or proliferation of peripheral, human, normal B cells that are stimulated by cells expressing CD40L or the soluble CD40 ligand (sCD40L); inhibition of intracellular, anti-apoptotic signals "of survival" in any cell stimulated by SCD40L or solid phase CD40L; inhibition of CD40 signal transduction in any cell with ligation with the solid phase SCD40L or CD40L; and inhibition of the proliferation of malignant, human B cells as observed subsequently. These tests can be performed as described in the examples in this document. See also the trials described in Schultze et al. (1998) Proc. Nati Acad. Sci. USA 92: 8200-8204; Dentón 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 and collaborators (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; incorporated in this document as a reference. A representative assay for detecting anti-CD40 antagonist antibodies that are specific for the epitopes of the CD40 antigen identified herein is a "competitive binding assay". Competitive binding assays are serological assays in which unknown elements are detected and quantified by their ability to inhibit the binding of a known ligand, labeled to its specific antibody. This is also referred to as a competitive inhibition assay. In a representative competitive binding assay, the 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 specifically react with an epitope of interest can be identified by examining a series of antibodies prepared against a CD40 protein or a fragment of the protein comprising the particular epitope of the CD40 protein of interest. For example, for the human CD40 antigen, the epitopes of interest include epitopes comprising the linear and / or non-linear amino acid residues of the short isoform of the human CD40 antigen (see GenBank accession No. NP_690593) set forth in Figure 12B (SEQ ID NO: 10), encoded by the sequence set forth in Figure 12A (SEQ ID NO: 9, see also GenBank access No. NM_152854) or the long isoform of the human CD40 antigen (see accesses of GenBank Nos. CAA43045 and NP_001241) exposed in Figure 12D (SEQ ID NO: 12), encoded by the sequence shown in Figure 12C (SEQ ID NO: 11, see accesses of GenBank Nos. X60592 and NM_001250). Alternatively, competitive binding assays with suitable anti-CD40 antagonist antibodies, identified previously, could be used to select monoclonal antibodies comparable with previously identified antibodies. The antibodies used in these immunoassays can be labeled or unlabeled. The unlabeled antibodies can be used in the agglutination; The labeled antibodies can be used in a wide variety of assays, employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an anti-CD40 antibody and an epitope of interest can be facilitated by the binding of a detectable substance to the antibody. Suitable detection means include the use of labels such as radionuclides, enzymes, coenzymes, fluorescent substances, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, complexes of prosthetic groups, 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, dichlorothiazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of a suitable radioactive material include 125 I, 131 I, 35 S or 3 H. These labeled reagents can be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, eg, ELISA, fluorescent immunoassays and the like. See, for example, U.S. Patent Nos. 3,766,162; 3,791,932; 3,817,837 and 4,233,402. Any of the anti-CD40 antibodies, antagonists, previously described or fragments of antibodies thereof can be conjugated before use in the methods of the present invention. Methods for producing conjugated antibodies are known in the art. In this way, the anti-CD40 antibody can be labeled using indirect labeling or an indirect labeling approach. By "indirect labeling" or "indirect labeling approach" it is proposed that a chelating agent be covalently bound to an antibody and at least one radionuclide be inserted into the chelating agent. See, for example, the chelating agents and radionuclides described in Srivagtava and Mease (1991) Nucí. Med. Bio. 18: 589-603, incorporated herein by reference. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 3P and 125μ), dense reagents in electrons, enzymes and ligands that have specific binding partners. Enzymes are typically detected by their activity. For example, aa horseradish peroxidase is usually detected by its ability to convert 3, 3 ', 5, 5' -tetramethylbenzidine (TMB) to a blue pigment, quantifiable by a spectrophotometer. A "specific binding partner" refers to a protein capable of binding to a ligand molecule with high specificity, such as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A and numerous receptor-ligand couplings known in the art. It should be understood that the above description is not intended to categorize the various labels into different classes, since the same label can serve in several different ways. For example, 12? I can serve as a radioactive label or as a dense electron reagent. HRP can serve as an enzyme or as an antigen for a mAb. In addition, one can combine several labels for the desired effect. For example, mAbs and avidin also require labels in the practice of this invention: in this way, one could label a mAb with biotin and detect its presence with avidin labeled with 125 I or with an anti-biotin mAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary experience in the field and are considered as equivalents within the scope of the present invention. Alternatively, the anti-CD40 antibody can be labeled using "direct labeling" or a "direct labeling approach" where a radionuclide is covalently linked directly to an antibody (typically via an amino acid residue). Preferred radionuclides are provided in Srivagtava and Mease (1991) supra. The indirect labeling approach is particularly preferred. See also, for example, International Publications Nos. WO 00/52031 and WO 00/52473, where a linker is used to bind a radioactive label to antibodies; and the labeled forms of the anti-CD40 antibodies described in U.S. Patent No. 6,015,542; incorporated in this document as a reference. In addition, an antibody (or a fragment thereof) can be conjugated to a therapeutic moiety, such as a cytotoxin, a therapeutic agent or a radioactive metal ion or radioisotope. A cytotoxin or cytotoxic agent includes any agent that is harmful to the cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, anti-metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil-decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU ) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine-platinum (II) cisplatin (DDP)), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin) antibiotics (e.g. , dactin icine (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and anti-mitotic agents (eg, vincristine and vinblastine). Radioisotopes include, but are not limited to, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111 and the like. The conjugates of the invention can be used to modify a given biological response; the drug portion should not be considered as limited to therapeutic, chemical, classical agents. For example, the drug portion can be a protein or polypeptide having a desired biological activity. These proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin; a protein such as a tumor necrosis factor, interferon-alpha, interferon-beta, neuronal growth factor, platelet-derived growth factor, tissue plasminogen activator; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony stimulation factor ("GM-CSF"), granulocyte colony stimulation factor ("G-CSF") or other growth factors. The techniques for conjugating this therapeutic portion with antibodies are well known. See, for example, Arnon et al. (1985) "Monoclonal Antibodies for Ir iunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.) pages 243-256; ed Hellstrom et al. (1987) "Antibodies for Drug Delivery", in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pages 623-653; Thorpe (1985) "Antobody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antobodies '84: Biological and Clinical Applications, ed. Pinchera et al., Pages 475-506 (Edi trice Kurtis, Milano, Italy, 1985); "Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy ", in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academia Press, New York, 1985), pages 303-316; and Thorpe et al. (1982) Immunol., Rev. 62: 119- 158. Alternatively, an antibody can be conjugated with a second antibody to form a heterocyte with antibodies played as described in U.S. Pat. No. 4,676,980. In addition, the linkers can be used between the labels and antibodies of the invention (see U.S. Patent No. 4,831,175). Antibodies or antigen-binding fragments thereof can be labeled directly with iodine, indium, radioactive yttrium or other radioactive particle known in the art (U.S. Patent No. 5,595,721). The treatment may consist of a combination of treatment with conjugated and unconjugated antibodies which are administered simultaneously or subsequently (WO 00/52031 and WO 00/52473). Variants of Anti-CD40 Antibody Antagonists Suitable, biologically active variants of the anti-CD40 antagonist antibodies can be used in the methods of the present invention. These variants will retain the desired binding properties of the anti-CD40 antibody, antagonist, precursor. Methods for making antibody variants are generally available in the field. For example, variants of amino acid sequences of an anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-5.9 or CHIR-12.12-described herein, can be prepared by means of mutations in the cloned DNA sequence encoding the antibody of interest. Methods 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. Nati 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 references cited therein, incorporated herein by way of reference. The guide for appropriate substitutions of amino acids that do not affect the biological activity of the polypeptide of interest can be found in the model of Dayhoff et al. (1987) in Atlas of Protein Sequence and Structure (Nat. Biomed. Res. Found., Washington, DC), incorporated herein by reference. Conservative substitutions, such as the exchange of one amino acid for another that has similar properties, may be preferred. Examples of conservative substitutions include, but are not limited to, Gly < = Ala, Val < = > Ile? Leu, Asp = í > Glu, Lys «> Arg, Asn = > Gln and Phe < = > Trp < í? > Tyr. In the construction of variants of the polypeptide of the antagonist anti-CD40 antibody - of interest, modifications are made in such a way that the variants continue to have the desired activity, i.e., a similar binding affinity and are able to bind specifically to a human CD40 antigen that is expressed on the surface of a human cell and that is free from significant agonist activity but exhibiting antagonistic activity when bound to a CD40 antigen on a cell expressing the human CD40 antigen. Obviously, any mutation made in the DNA encoding the variant polypeptide should not place the sequence outside the reading frame and preferably will not create complementary regions that could produce a secondary mRNA structure. See patent application publication EP No. 75,444. In addition, the constant region of an anti-CD40 antagonist antibody can be mutated to alter the effector function in a variety of ways. For example, see U.S. Patent No. 6,737,056B1 and U.S. Patent Application Publication No. 2004 / 0132101A1, which describe Fc mutations that optimize the binding of antibodies to Fc receptors. Preferably, the 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 least 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 monoclonal antibody CHIR-5.9 or CHIR-12.12 described in this document, or with a shorter portion of the reference antibody molecule. More preferably, the molecules share at least 96%, 91%, 98% or 99% sequence identity. For purposes of the present invention, the percentage of sequence identity is determined using the Smith-Waterman homology search algorithm using a matching space search with a space opening penalty of 12 and a space extension penalty of 2, BLOSUM matrix of 62. The Smit-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489. A variant may differ, for example, from the anti-CD40 antibody reference antagonist in an amount as small as 1 to 15 amino acid residues, as small as 1 to 10 amino acid residues, such as 6-10, as small as 5, as small 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 be 30, 40, 50 or more amino acid residues. Corrections can be made for the identity of sequences associated with substitutions or conservative residue spaces (see the Smith-Waterman homology search algorithm). The chemical structure requires a polypeptide capable of specifically binding to the CD40 antigen and of retaining the antagonist activity, particularly when binding to the CD40 antigen on the malignant B cells depends on a variety of factors. Since ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide can be obtained as an acid or basic salt or in neutral form. All these 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 primary amino acid sequence of the polypeptide can be increased by derivatization using sugar portions (glycosylation) or by means of other complementary molecules such as lipids, phosphate, acetyl groups and the like. It can also be increased by conjugation with saccharides. Certain aspects of this increase are made through post-translation processing systems of the producer host; other of these modifications can be introduced in vi tro. In any case, these modifications are included in the definition of an anti-CD40 antibody as used herein as long as the antagonistic properties of the anti-CD40 antibody are not destroyed. It is expected that these modifications may affect the activity quantitatively or qualitatively, either by increasing or decreasing the activity of the polypeptide in the various assays. In addition, the 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. These alterations that do not destroy the antagonist activity do not remove the polypeptide sequence from the definition of anti-CD40 antibodies of interest as used herein. The technique provides substantial guidance with respect to the preparation and use of polypeptide variants. In the preparation of the anti-CD40 antibody variants, a person skilled in the art can easily determine which modifications to the native protein nucleotide or amino acid sequence will result in a variant that is suitable for use as a therapeutically active component of a pharmaceutical composition used in the methods of the present invention. Methods of Therapy Utilizing Anti-CD40 Antibodies Antagonists of the Invention The methods of the invention are directed to the use of anti-CD40 antagonist antibodies to treat patients who have a disease mediated by stimulation of CD40 antigen signaling on expressing cells. the CD40 antigen. By "cell expressing the CD40 antigen" normal and malignant B cells expressing the CD40 antigen are proposed. Methods for detecting CD40 antigen expression in cells are well known in the art and include, but are not limited to, PCR techniques, immunohistochemistry, flow cytometry, Western immunoblotting, ELISA and the like. By a "malignant" B cell any neoplastic B cell is proposed, including but not limited to B cells derived from lymphomas including low, intermediate and high grade B cell lymphomas, immunoblastic lymphomas, non-Hodgkin lymphomas, Hodgkin's disease, lymphomas induced by the Epstein-Barr virus (EBV) and lymphomas related to AIDS, as well as acute lymphoblastic leukemias of B cells, myelomas, chronic lymphocytic leukemias, acute myeloblastic leukemias and the like. "Treatment" is defined herein as the application or administration of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof to a patient or the application or administration of an anti-CD40 antagonist antibody or a fragment thereof to an isolated tissue or cell line of a patient, where the patient has a disease, a symptom of a disease or a predisposition towards a disease, where the purpose is to cure, heal, alleviate, mitigate, alter, remedy, lighten, improve or affect the disease, the symptoms of the disease or the predisposition towards the disease. By "treatment" it is also proposed the application or administration of a pharmaceutical composition comprising the antagonist anti-CD40 antibodies or fragments thereof to a patient or the application or administration of a pharmaceutical composition comprising anti-CD40 antibodies or fragments of the same to an isolated tissue or cell line of a patient who has a disease, a symptom of a disease or a predisposition towards a disease, where the purpose is to cure, heal, alleviate, mitigate, alter, remedy, lighten, improve or affect the disease, the symptoms of the disease or the predisposition towards the disease. By "antitumor activity" we propose a reduction in the proliferation rate or accumulation of malignant cells expressing the CD40 antigen and therefore a decline in the growth rate of an existing tumor or in a tumor arising during therapy and / or the destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells and by thus a decrease in the total size of a tumor during therapy. Therapy with at least one anti-CD40 antibody (or an antigen-binding fragment thereof) causes a physiological response that is beneficial with respect to the treatment of disease states associated with the stimulation of CD40 antigen signaling in expressing cells. the CD40 antigen in a human. The methods of the invention find use in the treatment of non-Hodgkin lymphomas related to proliferation or accumulation, uncontrollable, abnormal B cells. For the purposes of the present invention, these lymphomas will be referred according to the classification scheme of the Work Formulation, that is those B cell lymphomas categorized as low grade, intermediate grade or of high degree (see "The Non-Hodgkin's Lymphoma Pathologic Classification Project", Cancer 49 (1982): 2112-2135). Thus, low-grade B-cell lymphomas include small lymphocytic lymphomas, small, follicular, excised and follicular cell lymphomas of small and large, mixed, follicular cells.; intermediate-grade lymphomas include follicular large cell lymphomas, small, diffuse excised cell lymphomas, small and large cell lymphomas, mixed diffuses, and diffuse large cell lymphomas; and high-grade lymphomas include large cell immunoblastic lymphomas, lymphoblastic lymphomas, and small non-excised Burkitt cell lymphomas or those not of the Burkitt type.

It is recognized that the methods of the invention are useful in the therapeutic treatment of B-cell lymphomas that are classified according to the European and American Lymphoma Classification System, Revised (REAL). These B cell lymphomas include, but are not limited to, lymphomas classified as precursor B cell neoplasms, such as leukemia / B lymphoblastic lymphoma; peripheral B-cell neoplasms, including lymphocytic leukemia, chronic B-cell / small lymphocytic lymphoma, lymphoid / lymphoplasmacytic immunocytosis, mantle cell lymphoma (MCL), follicular central lymphoma (follicular) (including diffuse small cell lymphoma, lymphoma of small and large cells, mixed, diffuse and diffuse large cell lymphomas), marginal zone B-cell lymphoma (including extranodal, nodal and splenic types), hairy cell leukemia, plasmacytoma / myeloma B-cell lymphoma of large, diffuse cells of the primary mediastinal (thymic) subtype, Burkitt's lymphoma, and high-grade B-cell lymphoma similar to Burkitt; acute leukemia; acute lymphocytic leukemia; myeloblastic leukemia; acute myelocytic leukemia; promyelocytic leukemia; myelomonocytic leukemia; monocytic leukemia; erythroleukemia; granulocytic leukemia (chronic myelocytic leukemia); Chronic lymphocytic leukemia; polycythemia vera; multiple myeloma; Waldenstrom macroglobulinemia; heavy chain disease and non-classifiable low-grade or high-grade B-cell lymphomas. It is recognized that the methods of the invention can be useful in the prevention of excessive, additional growths of tumors that arise during therapy. The methods of the invention are particularly useful in the treatment of subjects who have low-grade B-cell lymphomas, particularly those subjects who relapse after standard chemotherapy. Low-grade B-cell lymphomas are more painless than intermediate-grade and high-grade B-cell lymphomas and are characterized by a relapse / remission course. In this way, the treatment of these lymphomas is improved using the methods of the invention, since the episodes of relapses are reduced in number and severity. The anti-CD40 antagonist antibodies described herein may also find use in the treatment of inflammatory diseases and deficiencies or disorders of the immune system including, but not limited to, systemic lupus erythematosus, psoriasis, scleroderma, CREST syndrome, inflammatory myositis, Sjogren's syndrome, mixed connective tissue disease, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, acute respiratory distress syndrome, lung inflammation, idiopathic pulmonary fibrosis, osteoporosis, delayed-type hypersensitivity, asthma, primary biliary cirrhosis, and idiopathic thrombocytopenic purpura . According to the methods of the present invention, at least one anti-CD40 antagonist antibody (or an antigen-binding fragment thereof) defined elsewhere herein is used to promote a positive therapeutic response with respect to a B cell. human, malignant By "positive therapeutic response" with respect to cancer treatment, an improvement in the disease is proposed in association with the antitumor activity of these antibodies or fragments thereof and / or an improvement in the symptoms associated with the disease. That is, one can observe an antiproliferative effect, the prevention of excessive, additional growths of tumors, a reduction in the size of the tumor, a reduction in the number of cancer cells and / or a decrease in one or more symptoms mediated by stimulation. of cells expressing the CD40 antigen. In this way, for example, an improvement in the disease can be characterized as a complete response. By "complete response" the absence of a clinically detectable disease is proposed with the normalization of any previously abnormal radiographic study, bone marrow and cerebrospinal fluid (CSF, for its acronym in English). This response must persist for at least one month after the treatment according to the methods of the invention. Alternatively, an improvement in the disease can be categorized as being a partial response. By "partial response" a decrease of at least about 50% in the entire measurable tumor burden (ie, the number of tumor cells present in the subject) is proposed in the absence of new lesions and persistence for at least one month. This response is applicable for measurable tumors only. The tumor response can be assessed by changes in tumor morphology (ie, total tumor burden, tumor size and the like) using examination techniques such as magnetic resonance imaging (MRI) scan. its acronym in English), x-ray image formation, computed tomographic (CT) scan, bioluminescent imaging, for example, luciferase imaging, bone scan imaging, and sampling of tumor biopsies that include bone marrow aspiration (BMA). In addition to these positive therapeutic responses, the subject who undergoes the therapy with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof may experience the beneficial effect of an improvement in the symptoms associated with the disease. In this way, for B-cell tumors, the subject may experience a decrease in the so-called B symptoms, ie nocturnal perspiration, fever, weight loss and / or urticaria. By "therapeutically effective dose or amount" or "effective amount" an amount of anti-CD40 antagonist antibody or an antigen-binding fragment thereof is proposed which, when administered, produces a positive therapeutic response with respect to the treatment of a patient with a disease comprising the stimulation of cells expressing the CD40 antigen. In some embodiments of the invention, a therapeutically effective dose of the anti-CD40 antibody or a fragment thereof is in the range of about 0.01 mg / kg to about 40 mSf / kg, from about 0.01 mg / kg to about 30 m_-í / 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 method of treatment may comprise an individual administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. A further embodiment of the invention is the use of anti-CD40 antagonist antibodies for monitoring the diagnosis of protein levels in tissue as part of a clinical test procedure, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by the coupling of 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, dichlorotriazilamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of a suitable radioactive material include 125 I, 131 I, 35 S or 3 H.

The anti-CD40 antibodies described herein can be further used to provide reagents, for example, labeled antibodies that can be used, for example, to identify cells expressing the CD40 antigen. This can be very useful in determining the cell type of an unknown sample. The panels of monoclonal antibodies can be used to identify tissue by species and / or by type of organ. Similarly, these anti-CD40 antibodies can be used to screen cells from tissue cultures for contamination (ie, examination for the presence of a mixture of cells expressing the CD40 antigen and not expressing the CD40 antigen in a culture). ). Anti-CD40 antagonist antibodies can be used in combination with known chemotherapeutic agents and cytokines for the treatment of disease states comprising stimulated cells expressing the CD40 antigen. For example, the anti-CD40 antibodies of the invention can be used in combination with cytokines such as interleukin-2. In another embodiment, the anti-CD40 antibodies of the invention can be used in combination with rituximab (IDEC-C2B8, Rituxan ™, IDEC Pharmaceuticals Corp., San Diego, California). Thus, the anti-CD40 antagonist antibodies described herein, or the antigen-binding fragments thereof, are administered in combination with at least one other cancer therapy, including, but not limited to, surgery or surgical procedures (for example, splenectomy, hepatectomy, lymphadenectomy, leukophoresis, bone marrow transplantation and the like); radiation therapy, -chemotherapy; optionally in combination with autologous bone marrow transplantation, where suitable chemotherapeutic agents include, but are not limited to, fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-chlorodeoxyadenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations of them, for example, anthracycline-containing regimens such as CAP (cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincristine, doxorubicin, plus dexamethasone), MP (melphalan plus prednisone) and other cytotoxic and / or therapeutic agents that are used in chemotherapy such as mitoxantrone, daunorubicin, idarubicin, asparaginase and antimetabolites, including but not limited to cytarabine, methotrexate, 5-fluorouracil decarbazine, 6- thioguanine, 6-mercaptopurine and nelarabine; another anticancer therapy of monoclonal antibodies (eg, alemtuzumab (Campath ™ *) and another anti-CD52 antibody that targets the CD52 cell surface glycoprotein on malignant B cells: rituximab (Rituxan ™ 1), the fully human HuMax antibody -CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab / I-131 tositumomab (Bexxar ™ 1), ibritumomab tiuxetan (Zevalin ™) or any other therapeutic anti-CD20 antibody that targets the CD20 antigen on malignant B cells, anti-CD19 antibody (for example, MT103, a bispecific antibody), anti-CD22 antibody (for example, the humanized monoclonal antibody epratuzumab), bevacizumab (Avastin ™ 1) or another anti-cancer antibody that binds target the vascular, human endothelial growth factor, an anti-CD22 antibody that targets the CD22 antigen on malignant B cells (eg, the monoclonal antibody BL-22, an alphaCD22 toxin), an antibody aM-CSF that the macrophage colony stimulation factor be targeted; antibodies that target the activator of nuclear factor-kappaB receptors (RANK) and its ligand (RANKL) _, which are overexpressed in multiple myeloma; an anti-CD23 antibody that targets the CD23 antigen on malignant B cells (e.g., IDEC-152); an anti-CD80 antibody that targets the CD80 antigen (eg, IDEC-114); , an anti-CD38 antibody that targets the CD38 antigen on malignant B cells; antibodies that target the highest class II histocompatibility complex receptors (anti-MHC antibodies) expressed on malignant B cells; other anti-CD40 antibodies (e.g., SGN-40) that target the CD40 antigen on malignant B cells; and antibodies that target the ligand receptor 1 inducer of apoptosis related to tumor necrosis factor (TRAIL-R1) (e.g., the human monoclonal antibody, HGS-ETR1 agonist) and TRAIL-R2 expressed in a variety of solid tumors and tumors of hematopoietic origin); cancer therapy based on small molecules, including, but not limited to, microtubule / topoisomerase inhibitors (e.g., the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin binding agent T900607; XL119; and the inhibitor of aminocamptothecin topoisomerase), SDX-105 (bendamustine hydrochloride), ixabepilone (an epothilone analogue, also referred to as BMS-247550), protein kinase C inhibitors, eg, midostaurin ((PKC-412, CGP 41251, N-benzoylstaurosporine ), pixantrone, eloxatin (an antineoplastic agent), ganita (gallium nitrate), Thalomid ™ 1 (thalidomide), immunomodulatory derivatives of thalidomide (for example, revlimid (formerly revimid)), Affinitak ™ 1 (protein kinase antisense inhibitor) C-alpha), SDX-101 (R-etodolac, which induces apoptosis of malignant lymphocytes), second generation purine nucleoside analogs such as clofarabine, inhibitors of production of Bcl-2 protein by cancer cells (e.g., antisense oblimersen and Genasense ™ 1), proteasome inhibitors (e.g., Velcade ™ * (bortezomib)), small molecule kinase inhibitors (e.g., CHIR- 258), small molecule VEGF inhibitors (eg, ZD-6474), small molecule inhibitors of heat shock protein 90 (HSP, for its acronym in English) (eg, 17-AAG), inhibitors of molecules small histone deacetylases (eg, hybrid / polar citodifferentiation (HPC)) agents such as suberanylohydroxamic acid (SAHA) and FR-901228) and apoptotic agents such as Trisenox ™ (arsenic trioxide) and Xcytrin ™ 1 (motexaphine gadolinium); cancer therapies based on vaccines / immunotherapy that include, but are not limited to, vaccine approaches (eg, Id-KLH, oncology, vitaletine), personalized immunotherapy or active idiotypic immunotherapy (eg, MyVax ™ personalized immunotherapy, formally designated GTOP-99), PromuneMR (CpG 7909, a synthetic agonist for toll-like receptor 9 (TLR9)), interferon-alpha therapy, interleukin-2 (IL-2) therapy, IL-12 therapy, IL-15 and IL-21 therapy; steroid therapy; or another cancer therapy; wherein the additional cancer therapy is administered before, during or subsequent to the anti-CD40 antagonist antibody therapy. Thus, where the combination therapies comprise the administration of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof in combination with the administration of another therapeutic agent, such as with chemotherapy, radiation therapy, other antibody therapy. Anticancer, cancer therapy based on small molecules or cancer therapy based on vaccines / immunotherapy, the methods of the invention include co-administration, using separate formulations or an individual pharmaceutical formulation or and consecutive administration in any order. Where the methods of the present invention comprise combined therapeutic regimens, these therapies can be administered simultaneously, i.e., the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered concurrently or within the same time frame as the other therapy against cancer (ie, therapies are conducted concurrently, but the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is not administered at precisely the same time as the other cancer therapy). Alternatively, the anti-CD40 antagonist antibody of the present invention or an antigen-binding fragment thereof can also be administered before or subsequent to the other cancer therapy. The sequential administration of different cancer therapies can be performed regardless of whether the treated subject responds to the first course of therapy to reduce the possibility of remission or relapse. Where the combination therapies comprise the administration of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof in combination with the administration of a cytotoxic agent, preferably the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered. before administering the cytotoxic agent. In some embodiments of the invention, the anti-CD40 antagonist antibodies described herein or antigen-binding fragments thereof are administered in combination with chemotherapy and optionally in combination with autologous bone marrow transplantation, wherein the antibody and the chemotherapeutic agent (s) can be administered sequentially, in any order, or simultaneously (ie, concurrently or within the same time frame). Examples of suitable chemotherapeutic agents include, but are not limited to, fludarabine or fludarabine phosphate, chloroambucil, vincristine, pentostanin, 2-chlorodeoxyadenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations thereof, eg, regimens containing anthracyclines such as CAP (cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincristine, doxorubicin plus dexamethasone), MP (melphalan plus prednisone) and other cytotoxic and / or therapeutic agents used in chemotherapy such as mitoxanthone, daunorubicin, idarubicin, asparaginase and antimetabolites, which include, but are not limited to cytarabine, methotrexate, decarbazine 5-fluorouracil, 6-thioguanine, 6-mercaptopurine and nelarabine. In some embodiments, the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof is administered prior to treatment with the chemotherapeutic agent. In alternative embodiments, the anti-CD40 antagonist antibody is administered after treatment with the chemotherapeutic agent. In still other embodiments, the chemotherapeutic agent is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. Thus, for example, in some embodiments, the anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof is administered in combination with fludarabine or fludarabine phosphate. . In one of these embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered prior to the administration of fludarabine or fludarabine phosphate. In alternative embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with fludarabine or fludarabine phosphate. In yet other embodiments, fludarabine or fludarabine phosphate is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In other embodiments of the invention, chlorambucil, an alkylating agent, is administered in combination with the anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or a binding fragment to antigens thereof. In another of these modalities, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered prior to the administration of chlorambucil. In alternative embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with chlorambucil. In still other embodiments, chlorambucil is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In still other embodiments, anthracycline-containing regimens such as CAP (cyclophosphamide, doxorubicin plus prednisone) and CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), may be combined with the administration of an antagonist anti-CD40 antibody described herein, example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof. In one of these embodiments, the anti-CD or antagonist antibody or an antigen-binding fragment thereof is administered prior to administration of the anthracycline-containing regimens. In other embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with anthracycline-containing regimens. In yet other embodiments, the anthracycline-containing regimen is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In alternative embodiments, an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof, is administered in combination with alemtuzumab (Campath ™).k. ; distributed by Berlex Laboratories, Richmond, California). Alemtuzumab is a monoclonal, humanized, recombinant antibody (Campath-IH) that targets the CD52 antigen expressed in malignant B cells. In another of these embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered prior to the administration of alemtuzumab. In other embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with alemtuzumab. In still other embodiments, alemtuzumab is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In alternative embodiments, an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, is administered in combination with a therapeutic anti-CD20 antibody. which targets the CD20 antigen on malignant B cells, for example, rituximab (Rituxan ™ 1), the all-human antibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab / l-131 tositumomab (Bexxar ™ 1) or ibritumomab tiuxetan (Zevalin ™ *). In one of these embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered prior to administration of the anti-CD20 antibody. In other embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with the anti-CD20 antibody. In still other embodiments, the anti-CD20 antibody is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In alternative embodiments, an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, is administered in combination with a cancer therapy based in small molecules including, but not limited to, microtubule inhibitors and / or topoisomerase (eg, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin binding agent T900607; XL119; and the topoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustine hydrochloride), ixabepilone (an epothilone analog, also referred to as BMS-247550), protein kinase C inhibitors, for example, midostaurin ((PKC-412, CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (an antineoplastic agent), ganita (gallium nitrate), Thalomid ™ (thalidomide), immunomodulatory derivatives of thalidomide (for example, revlimida (formerly revimid)), Affinitak * (C-alpha protein kinase antisense inhibitor), SDX-101 (R-etodolac, which induces apoptosis of malignant lymphocytes), second generation purine nucleoside analogs such as clofarabine, inhibitors of Bel-2 protein production by cancer cells (e.g., antisense oblimersen and Genasense ™ agents) *), proteasome inhibitors (eg, Velcade ™ * (bortezomib)), small molecule kinase inhibitors (eg, CHIR-258), small molecule VEGF inhibitors (eg, ZD-6474), inhibitors of small molecules of heat shock protein 90 (HSP) (e.g., 17-AAG), small molecule inhibitors of histone deacetylases (e.g., hybrid hybrid / polar HPC) agents such as suberanyl hydroxamic acid (SAHA), and FR- 901228) and apoptotic agents such as Trisenox ™ * (arsenic trioxide) and Xcytrin ™ * (motexafin-gadolinium). In one of these embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered prior to the administration of cancer therapy based on small molecules.

In other embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with cancer therapy based on small molecules. In still other embodiments, cancer therapy based on small molecules is administered simultaneously with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In still other embodiments, an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof, can be used in combination with a cancer therapy. vaccine-based / immunotherapy that includes, but is not limited to, vaccine approaches (eg, Id-KLH, oncology, vitaletine), personalized immunotherapy or active idiotypic immunotherapy (eg, personalized MyVax ™ immunotherapy *, formally designated GTOP- 99), Promune ™ * (CpG 7909, a synthetic toll-like receptor agonist 9 (TLR9)). interferon-alpha therapy, interleukin-2 therapy (IL-2), IL-12 therapy, IL-15 therapy or IL-21 therapy; or steroid therapy. In one of these modalities, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered prior to the administration of the vaccine-based cancer therapy / immunotherapy. In other embodiments, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is administered after treatment with cancer therapy based on vaccines / immunotherapy. In still other embodiments, the vaccine-based cancer therapy / immunotherapy is administered concurrently with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. In one of these embodiments, an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof, can be used in combination with IL-2. IL-2, a known agent for expanding the number of effector, eliminating, natural (NK) cells in treated patients, can be administered before, or concomitantly with, the anti-CD40 antagonist antibody. invention or an antigen binding fragment thereof. This expanded number of NK effector cells can lead to increased ADCC activity of the anti-CD40 antibody, antagonist, administered or an antigen-binding fragment thereof. In other embodiments, IL-21 serves as the immunotherapeutic agent to stimulate NK cell activity when administered in combination with an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 , or a fragment of binding to antigens thereof. In addition, combination therapy with two or more therapeutic agents and an anti-CD40 antagonist antibody described herein may also be used for the treatment of disease states comprising stimulated cells expressing the CD40 antigen, e.g., related cancers. with B cells and autoimmune and / or inflammatory disorders. Without being limiting, examples include a triple combination therapy where two chemotherapeutic agents are administered in combination with an anti-CD40 antagonist antibody described herein and wherein a chemotherapeutic agent and another anti-cancer monoclonal antibody (eg, alemtuzumab, rituximab or anti-CD23 antibody) are administered in combination with an anti-CD40 antagonist antibody described herein. Examples of these combinations include, but are not limited to, combinations of fludarabine, cyclophosphamide and the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof.; and combinations of fludarabine, an anti-CD20 antibody, for example, rituximab (Rituxan ™ *. IDEC Pharmaceuticals Corp., San Diego, California) and the anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9 or a fragment of binding to antigens thereof. Pharmaceutical Formulations and Modes of Administration The anti-CD40 antagonist antibodies of the invention are administered at a concentration that is therapeutically effective to prevent or to treat diseases mediated by cells expressing the CD40 antigen such as SL ?, PBC, ITP, multiple sclerosis, psoriasis, Crohn's disease, graft rejection and B-cell lymphoma. To achieve this goal, antibodies can be formulated using a variety of acceptable excipients that are known in the art. Typically, the antibodies are administered by injection, either intravenously or intraperitoneally. The methods for performing this administration are known to those of ordinary experience in the field. It may also be possible to obtain compositions that can be administered by the topical or oral route or which make possible a transmission through the mucous membranes. Intravenous administration preferably occurs by means of infusion over a period of from about 1 to about 10 hours, more preferably from about 1 to about 8 hours, still more preferably from about 2 to about 100 hours, even more preferably from about 4 to about 6 hours, depending on the anti-CD40 antibody that is administered. The initial infusion with the pharmaceutical composition can be provided over a period of about 4 to about 6 hours with subsequent infusions that are delivered faster. Subsequent infusions may be administered for a period of from about 1 to about 6 hours, including, for example, from about 1 to about 4 hours, from about 1 to about 3 hours or from about 1 to about 2 hours. A pharmaceutical composition of the invention is formulated to be compatible with its proposed route of administration. Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC) or by infusion), oral and pulmonary (e.g., inhalation), nasal, transdermal administration. (topical), transmucosal and rectal. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfide; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetals, citrates or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Anti-CD40 antibodies are typically provided by a standard technique within a pharmaceutically acceptable buffer, eg, sterile saline, sterile buffered water, propylene glycol, combinations of the foregoing, and the like. Methods for preparing parenterally administrable agents are described in Remington's Pharmaceutical Sciences (18th edition, Mack Publishing Company, Eaton, Pa., 1990), incorporated herein by reference. See also, for example WO 98/56418, which describes stabilized pharmaceutical formulations of antibodies that are suitable for use in the methods of the present invention. The amount of at least one anti-CD40 antibody or fragment thereof to be administered is easily determined by a person of ordinary skill in the field without undue experimentation. Factors that influence the mode of administration and the respective amount of at least one anti-CD40 antagonist antibody (or a fragment thereof) include, but are not limited to, the severity of the disease, the history of the disease and the age, height, weight, health and physical condition of the individual who undergoes the therapy. Similarly, the amount of anti-CD40 antagonist antibody or a fragment thereof to be administered will depend on the mode of administration and whether the subject will be subjected to a single dose or multiple doses of this antitumor agent. Generally, a higher dose of the anti-CD40 antibody or a fragment thereof is preferred with an increasing weight of the subject undergoing the therapy. The dose of the anti-CD40 antibody or a fragment thereof 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. Thus, 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. 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 invention, the method comprises administering multiple doses of the anti-CD40 antagonist antibody or a fragment thereof. The method may comprise the administration of 1, 2, 3, 4, 5, 6, 1, 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 a fragment thereof. The frequency and duration of administration of multiple doses of the pharmaceutical compositions comprising the anti-CD40 antibody or a fragment thereof can be readily 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 an individual treatment or, preferably, may include a series of treatments. In a preferred example, a subject is treated with the anti-CD40 antagonist antibody or an antigen-binding fragment thereof in the range of between about 0.1 to 20 mg / kg of body weight, once a week for between about 1 at 10 weeks, preferably between approximately 2 to 8 weeks, more preferably between approximately 3 to 7 weeks and even more preferably for approximately 4, 5 or 6 weeks. Treatment can occur annually to prevent relapse or with the indication of a relapse. It will also be appreciated that the effective dose of antibody or an antigen-binding fragment thereof used for the treatment may increase or decrease through the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic tests as described in this document. Thus, in one embodiment, the dosage regimen includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or a fragment thereof 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 a fragment thereof 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 a fragment thereof on days 1, 3, 5 and 7 of a week in a treatment period; a dosage regimen that includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody or a fragment thereof 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 a fragment thereof on day 1 of a week in a treatment period. The treatment period can include 1 week, 2 weeks, 3 weeks, a month, 3 months, 6 months or a year. The treatment periods may be subsequent or they may be separated from each other by a day, a week, 2 weeks, a month, 3 months, 6 months or a year. In some embodiments, the therapeutically effective doses of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof ranges from about 0.003 9 / ka about 50 tng / kg, from about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 30 mg / kg, from about 0.1 mg / ka 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 kg or from about 7 mg / kg to about 12 mg / kg. Thus, for example, the dose of any anti-CD40 antagonist antibody or an antigen-binding fragment thereof, for example the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof. , it can be 0.003 mg / kd, 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 other of these doses that are within the range of approximately 0.003 mg / kg to approximately 50 mg / kg. The same therapeutically effective dose of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof can be administered during each week of the antibody dosage. Alternatively, different, therapeutically effective doses of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof can be used during the course of a treatment period. In other embodiments, the initial, therapeutically effective dose of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof as defined elsewhere herein may be in the lower dosage range (i.e., approximately 0.003 mg / kg to about 20 mg / kg) with subsequent doses that are within the highest dosage range (i.e. from about 20 mg / kg to about 50 mg / kg). In alternative embodiments, the therapeutically effective initial dose of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof as defined elsewhere herein may be in the higher dosage range (i.e., approximately 20). mg / kg to approximately 50 mg / kg) with subsequent doses that fall within the lower dosage range (ie, from 0.003 mg / kg to approximately 20 mg / kg). Thus, in one embodiment, the therapeutically effective initial dose of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof is from about 20 mg / kg to about 35 ms / k, inclusive of about 20 mg / kg, about 25 mg / kg, about 30 mg / kg and about 35 mg / kg and the subsequent therapeutically effective doses of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof are from about 5 mg / kg to about 15 mg / kg, inclusive of approximately 5 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg and approximately 15 mg / kg. In some embodiments of the invention, antagonist anti-CD40 antibody therapy is initiated by administering a "loading dose" of the antibody or an antigen-binding fragment thereof to the subject in need of antagonist anti-CD40 antibody therapy. By "loading dose" an initial dose of the anti-CD40 antagonist antibody or an antigen binding fragment thereof is proposed which is administered to the subject, where the administered dose of the antibody or an antigen-binding fragment thereof is found within of the highest dosage range (i.e., from about 20 mg / kg to about 50 mg / kg). The "loading dose" can be administered as an individual administration, for example, an individual infusion where the antibody or an antigen-binding fragment thereof is administered IV or as multiple administrations, eg, multiple infusions where the antibody or a Antigen binding fragment thereof is administered IV, as long as the full "loading dose" is administered within a period of about 24 hours. After administration of the "loading dose", the subject is then administered at one or more additional therapeutically effective doses of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. Subsequent 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 these embodiments, subsequent, therapeutically effective doses are generally within the lower dosage range (ie, 0.003 mg / kg to about 20 mg / kg). Alternatively, in some embodiments, after the "loading dose", subsequent therapeutically effective doses of the anti-CD40 antagonist antibody or an antigen binding fragment thereof are administered according to a "maintenance schedule", wherein the Therapeutically effective dose of the antibody or an antigen-binding fragment thereof 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 once every 10 months, once every 11 months or once every 12 months. In these embodiments, the therapeutically effective doses of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof are within the lower dosage range (ie, from 0.003 mg / kg to about 20 mg / kg), particularly when the subsequent doses are administered at more frequent intervals, for example, once every two weeks to once each month or within the highest 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, where subsequent doses are administered for approximately one month to approximately 12 months apart. Anti-CD40 antagonist antibodies that are present in the pharmaceutical compositions described herein for use in the methods of the invention can be native or can be obtained by recombinant techniques and can be from any source, including mammalian animal sources such as, for example, mice, rats, rabbits, primates, pigs and humans. Preferably, these polypeptides are derived from a human source and more preferably are human, recombinant proteins from hybridoma cell lines. The pharmaceutical compositions that are useful in the methods of the invention may comprise biologically active variants of the anti-CD40 antagonist antibodies of the invention. These variations 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. This 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 expressed by the hybridoma cell line 5.9 or 12.12, respectively . Methods for determining whether a variant anti-CD40 antibody retains the desired biological activity and therefore serves as a therapeutically active component in the pharmaceutical composition are available in the field. The biological activity of the antibody variants can be measured using assays specifically designed to measure the activity of the native antagonist antibody, including assays described in the present invention. 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 of the invention. Thus, liquid, lyophilized or spray-dried compositions comprising one or more of the antagonist anti-CD40 antibodies of the invention can be prepared as an aqueous or nonaqueous solution or a suspension for subsequent administration to a subject according to the invention. with the methods of the invention. Each of these compositions will comprise at least one of the anti-CD40 antagonist antibodies of the present invention as a therapeutic or prophylactically active component. By "therapeutically or prophylactically active component" is proposed the anti-CD40 antibody that is specifically incorporated into the composition to produce a therapeutic or prophylactic response, desired with respect to the treatment, prevention or diagnosis of a disease or condition within a subject when the Pharmaceutical composition is administered to that subject. Preferably, the pharmaceutical compositions comprise appropriate stabilizing agents, bulking agents or both to minimize the problems associated with the loss of stability and biological activity of the proteins during preparation and storage. Formulation elements can be added to pharmaceutical compositions comprising an anti-CD40 antagonist antibody of the invention. These formulation elements may include, but are not limited to, oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants or bulking agents. Preferably, the carbohydrates include sugars and sugar alcohols such as mono-, di- or polysaccharides or water-soluble glycans. The saccharides or glucans can include fructose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, ce and β-cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose or mixtures thereof. The "sugar alcohol" is defined as a hydrocarbon of 4 to 8 carbon atoms 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% and 7% w / v, more preferably between 2.0% and 6.0% w / v. Preferably, amino acids include levogy (L) forms of carnitine, arginine and betaine; however, other amino acids can be added. Preferred 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 Nos. 270,799 and 268,110. Additionally, the antibodies can be chemically modified by means of covalent conjugation to a polymer to increase their average lifetime in circulation, for example. Preferred polymers and methods for binding them to peptides are shown in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546; all of which are incorporated in this document as a reference in its entirety. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). The PEG is soluble in water at room temperature and has the general formula: R (0-CH2-CH2) nO-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 carbon atoms, 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 has a preferred 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 hydroxy group, more preferably it is a terminal hydroxy group. This hydroxy group 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 active groups can be varied to achieve a PEG / covalent conjugated antibody conjugate 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. The POG is preferred. One reason is because the glycerol backbone of the polyoxyethylated glycerol is the same backbone that occurs naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be a foreign agent in the body. POG has a preferred molecular weight in the same range as PEG. The structure of the POG is shown in Knauf et al. (1988) J. Bio. Chem. 263: 15064-15070 and a discussion on POG / IL-2 conjugates is found in U.S. Patent No. 4,766,106, both incorporated by this act by way of reference in its entirety. Another drug delivery system for increasing the average lifespan in circulation is the liposome. Methods for preparing liposome delivery systems are described in Gabizon et al. (1982) Cancer Research 42: 4734; Cafiso (1981) Biochem Biophys Acta 649: 129; and Szoka (1980) Ann. Rev. Biophys. Eng. 9: 467. Other drug delivery systems are known in the art and are described in, for example, Poznansky et al. (1980) Drug Delivery Systems (R.L. Juliano, ed. Oxford, N.Y.) pages 253-315; Poznansky (1984) Pharm Revs 36: 211. The formulation elements to be incorporated into a pharmaceutical composition should provide the stability of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. That is, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof must retain its physical and / or chemical stability and must have the desired biological activity, i.e. one or more of the antagonist activities defined herein above. which include, but are not limited to, the inhibition of immunoglobulin secretion by peripheral, human, normal B cells that are stimulated by T cells; the inhibition of the survival and / or proliferation of peripheral, human, normal B cells stimulated by Jurkat T cells; inhibiting the survival and / or proliferation of peripheral, human, normal B cells that are stimulated by cells expressing CD40L or a soluble CD40 ligand (sCD40L); the inhibition of intracellular, anti-apoptotic signals of "survival" in any cell stimulated by sCD40L or solid phase CD40L; the inhibition of CD40 signal transduction in any cell with the ligation with the solid phase SCD40L or CD40L; and the inhibition of the proliferation of malignant, human B cells as observed elsewhere in this document. Methods for monitoring the stability of proteins 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 described in this document later. Generally, the stability of the proteins is measured at a selected temperature for a specific period of time. In preferred embodiments, a pharmaceutical formulation of stable antibody provides stability for the anti-CD40 antagonist antibody or an antigen-binding fragment thereof when stored at room temperature (approximately 25 ° C) for at least 1 month, at least 3 months. 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 retain its physical stability at a given point in time if it does not show visual signs (i.e., discoloration or loss of clarity) or measurable signs (e.g. , 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 retain its chemical stability at a given point in time if the chemical stability measurements are indicative that the protein (i.e. an antibody) retains the biological activity of interest in that pharmaceutical composition. Methods for monitoring changes in chemical stability are well known in the art and include, but are not limited to, methods for detecting chemically altered forms of the protein such as the result of trimming, using, for example, SDS-PAGE , SEC and / or matrix-assisted laser / flight time desorption mass spectrometry; and the degradation associated with changes in molecular charge (eg, associated with deamidation), using, for example, ion exchange chromatography. See, for example, the methods described in this document later. An anti-CD40 antagonist antibody or an antigen-binding fragment thereof, when formulated into a pharmaceutical composition, is considered to retain a desired biological activity at a given point in time if the biological activity desired at that time is within about 30%, preferably within about 20% of the desired biological activity that is exhibited at the time the pharmaceutical composition was prepared as determined in a suitable assay to measure the desired biological activity. Assays for measuring the desired biological activity of the anti-CD40 antagonist antibodies described herein and the antigen binding fragments thereof can be performed as described in the examples described herein. See also the assays described in Schultze et al. (1998) Proc. Nati Acad. Sci. USA 92: 8200-8204; Dentón and collaborators (1998) Pedia tr. 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 I munology 13:12; Kwekkeboom et al. (1993) Immunology 79: 439-444; and U.S. Patent Nos. 5,674,492 and 5,847,082; incorporated in this document as a reference. In some embodiments of the invention, the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 or an antigen-binding fragment thereof is formulated in a liquid pharmaceutical formulation. The anti-CD40 antagonist antibody or an antigen-binding fragment thereof can be prepared using any method known in the art., including those methods described in this document above. In one embodiment, the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof is produced recombinantly in a CHO cell line. After its preparation and purification, the anti-CD40 antagonist antibody or an antigen-binding fragment thereof can be formulated as a pharmaceutical, liquid formulation in the manner set forth herein. Where the anti-CD40 antagonist antibody or an antigen-binding fragment thereof must be stored prior to formulation, it can be frozen, for example, at = -20 ° C and then thawed at room temperature for the additional formulation. The liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. The amount of antibody or antigen-binding fragment thereof that is present in the formulation takes into consideration the route of administration and the desired dose volume. In this manner, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody, for example, the antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof at a concentration of about 0.1 mg / ml to about 50.0 mg / ml, from about 0.5 mg / ml to about 40.0 mg / ml, from about 1.0 mg / ml to about 30.0 mg / ml, from about 5.0 mg / ml to about 25.0 mg / ml, of about 5.0 mg / ml at about 20.0 mg / ml or about 15.0 mg / ml to about 25.0 mg / ml. In some embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or an antigen-binding fragment thereof at a concentration of about 0.1 mg / ml to about 5.0 mg / ml, from about 5.0 mg / ml to about 10.0. mg / ml, from about 10.0 mg / ml to about 15.0 mg / ml, from about 15.0 mg / ml to about 20.0 mg / ml, from about 20.0 mg / ml to about 25.0 mg / ml, of about 25.0 mg / ml at about 30.0 mg / ml, from about 30.0 mg / ml to about 35.0 mg / ml, from about 35.0 mg / ml to about 40.0 mg / ml, from about 40.0 mg / ml to about 45.0 mg / ml or about 45.0 mg / ml at approximately 50.0 mg / ml. In other embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or an antigen-binding fragment thereof at a concentration of about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 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 approximately 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 an antigen-binding fragment thereof and a buffer that maintains the pH of the formulation in the range of approximately pH 5.0 to about pH 7.0, inclusive 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 of these values within 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 from about pH 5.0 to about pH 6.5, from about pH 5.0 to about pH 6.0, from about pH 5.0 to about pH 5.5, from about pH 5.5 to about 7.0, from about pH 5.5 to about pH 6.5 or from 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 can be used in the formulation, provided that the physico-chemical stability and the desired biological activity of the antibody are retained as previously observed in this document. Suitable buffers include, but are not limited to, conventional acids and salts thereof, where the counter-ion may be, for example, sodium, potassium, ammonium, calcium or magnesium. Examples of conventional acids and salts thereof that may be used to buffer the liquid, pharmaceutical formulation include, but are not limited to, succinic acid or succinate buffers, citric or citrate acid, 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 acid or malate. The concentration of buffer within the formulation can be from about 1 mM to about 50 mM, inclusive of about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM. 15 mM, 20 mM, 25 mM, 30 M, 35 mM, 40 mM, 45 mM, 50 mM or other of these values within the range of about 1 mM to about 50 mM. In some embodiments, the buffer concentration within the formulations is from about 5 mM to about 15 mM, inclusive of 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 of these 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 monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof and buffer. succinate or citrate buffer at a concentration that maintains the pH of the formulation in the range from about pH 5.0 to about pH 7.0, preferably from about pH 5.0 to about pH 6.5. By "succinate buffer" or "citrate buffer" is proposed a buffer comprising a salt of succinic acid or a salt of citric acid, respectively.

In a preferred embodiment, the counter-ion of succinate or citrate is the sodium cation and thus 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 succinate or citrate buffer within the formulation may be from about 1 mM to about 50 mM, inclusive of about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM or other of these values within the range of about 1 mM to about 50 M. In some embodiments, the concentration of the buffer within the formulation is about 5 mM to about 15 mM, inclusive of 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 an antigen-binding fragment thereof at a concentration of about 0.1 mg / ml at about 50.0 mg / ml or about 5.0 mg / ml to about 25.0 mg / ml and the succinate or citrate buffer, for example, sodium succinate or sodium citrate buffer, at a concentration of about 1 mM to about 20 mM , from about 5 mM to about 15 mM, preferably about 10 mM. Where it is desirable for the liquid pharmaceutical formulation to be quasi-isotonic, 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 a binding fragment antigens thereof 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 render the formulation almost isotonic. By "quasi isotonic" it is proposed that the aqueous formulation have an osmolarity of about 240 mmol / kg to about 360 mmol / kg, preferably from about 240 to about 340 mmol / kg, more preferably from about 250 to about 330 mmol / kg, even more preferably from about 260 to about 320 mmol / kg, even more preferably from about 270 to about 310 mmol / kg. Methods 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 that are 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 almost equal to that of a body fluid. It is desirable to use a physiologically acceptable isotonicity agent. In this manner, 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 an antigen-binding fragment thereof and a buffer for maintaining the pH of the formulation within the range of about pH 5.0 to about pH 7.0, can further comprise components that can be used to provide isotonicity, eg, 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 or their salts and relatively minor amounts of citrates or phosphates. The person of ordinary experience would know of additional agents that are suitable to provide optimum tonicity of the liquid formulation.

In some preferred embodiments, 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 an antigen-binding fragment thereof and a buffer for maintaining the pH of the formulation within the range of about pH 5.0 to about pH 7.0, further comprising sodium chloride as an 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 from about 50 mM to about 300 mM, from about 50 mM to about 250 mM, from about 50 mM to about 200 mM, from about 50 mM to about 175 mM, of about 50 mM at about 150 mM, from about 75 mM to about 175 mM, from about 75 mM to about 150 mM, from about 100 mM to about 175 mM, from about 100 mM to -about 200 mM, from about 100 mM to about 150 mM, from about 125 mM, to about 175 mM, from about 125 mM to about 150 mM, from about 130 mM to about 170 mM, from about 130 mM to about 160 mM , from about 135 mM to about 155 mM, from about 140 mM to about 155 mM or from about 145 mM to about 155 mM. In one of these embodiments, the concentration of sodium chloride is about 150 mM. In another of these embodiments, the concentration of sodium chloride is about 150 mM, the buffer is a buffer of sodium succinate or sodium citrate at a concentration of about 5 mM to about 15 mM, the pharmaceutical formulation, liquid comprises an amount Therapeutically effective anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof and the formulation has a pH from about pH 5.0 to about pH 7.0, of about pH 5.0 to about pH 6.0 or from 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 an antigen-binding fragment thereof, at a concentration of about 0.1 mg / ml at about 50.0 mg / ml or about 5.0 mg / ml to about 25.0 mg / ml, sodium chloride at about 150 mM and sodium succinate or sodium citrate at about 10 mM, at a pH of about pH 5.5.

Protein degradation due to defrosting or mechanical cutting during the processing of liquid pharmaceutical formulations of the present invention can be inhibited by the incorporation of surfactants into the formulation in order to decrease the surface tension at the solution-air interface. Thus, in some 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 an antigen binding fragment thereof, a buffer to maintain the pH of the formulation within the range of about pH 5.0 to about pH 7.0 and also 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 an antigen-binding fragment thereof, a buffer that maintains 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 at a concentration of about 50 mM to about 300 mM and further comprising a surfactant. Typical surfactants used are non-ionic surfactants, which include 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-t-octylf enol such as Triton X-100. Classical stabilization of pharmaceutical formulations by surfactants or emulsifiers is described, for example, in Levine et al. (1991) J. Parenteral Sci. Technol. 45 (39: 160-165, incorporated herein by reference.) A preferred surfactant that is employed in the practice of the present invention is polysorbate 80. Where a surfactant is included, it is typically added in an amount of about 0.001% at about 1.0% (w / v), from about 0.001% to about 0.5%, from about 0.001% to about 0.4%, from about 0.001% to about 0.3%, from about 0.001% to about 0.2%, of about 0.005% to about 0.5%, from about 0.005% to about 0.2%, from about 0.01% to about 0.5%, from about 0.01% to about 0.2%, from about 0.03% to about 0.5%, from about 0.03% to about 0.3%, from about 0.05% to about 0.5%, or from about 0.05% to about 0.2% .Thus, in some embodiments, the liquid pharmaceutical formulation comprises a quantity t For example, the pharmaceutically effective anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, the buffer is a buffer of sodium succinate or sodium citrate at a concentration of about 1 mM to about 50 mM, from about 5 mM to about 25 mM or from about 5 mM to about 15 mM; the formulation has a pH from about pH 5.0 to about pH 7.0, from about pH 5.0 to about pH 6.0 or from 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 from about 0.001% to about 0.5%. These formulations may optionally comprise an isotonicity agent, such as sodium chloride at a concentration of about 50 mM to about 300 mM, from about 50 mM to about 200 mM or from about 50 mM to about 150 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 an antigen-binding fragment thereof, at a concentration of about 0.1 mg / ml at about 50.0 mg / ml or about 5.0 mg / ml to about 25.0 mg / ml, inclusive about 20.0 mg / ml; sodium chloride from about 50 mM to about 20 mM, including sodium chloride at about 150 mM; sodium succinate or sodium citrate from 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, inclusive about 50 mM; and optionally a surfactant, for example, polysorbate 80, in an amount from about 0.001% to about 1.0%, inclusive from about 0.001% to about 0.5%; wherein the liquid pharmaceutical formulation has a pH from about pH 5.0 to about pH 7.0, from about pH 5.0 to about pH 6.0, from about pH 5.0 to about pH 5.5, from about pH 5.5 to about pH 6.5 or from about pH 5.5 to approximately pH 6.0. The liquid pharmaceutical formulation can be essentially free of any, conservative or other carrier, excipient or stabilizer as noted hereinabove. Alternatively, the formulation may include one or more preservatives, for example, antibacterial agents, the pharmaceutically acceptable carriers, excipients or stabilizers described hereinabove when provided do not adversely affect the physicochemical stability of the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. Examples of acceptable carriers, excipients and 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 complexes). of Zn-protein) and biodegradable polymers such as polyesters. A full description of the formulation and selection of pharmaceutically acceptable carriers, stabilizers and isomolytes can be found in Remington's Pharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton, Pennsylvania, 1990), incorporated herein by reference. After the pharmaceutical, liquid formulation or other pharmaceutical composition described herein is prepared, it can be lyophilized to prevent degradation. The methods for lyophilizing liquid compositions are known to those of ordinary experience in the field. Just prior to use, the composition can be reconstituted with a sterile diluent (Ringer's solution, distilled water or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art. Use of Anti-CD40 Antibodies Antagonists in the Preparation of Medications The present invention also provides the use of an anti-CD40 antagonist antibody or an antigen-binding fragment thereof in the preparation of a medicament for the treatment of a subject for a cancer. characterized by the growth of neoplastic B cells, wherein the medicament is coordinated with treatment with at least one other cancer therapy. Cancers characterized by growth of neoplastic B cells include, but are not limited to, the cancers related to B cells described hereinbefore, for example, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, B-cell lymphoma. , high-grade B-cell lymphoma, intermediate-grade B-cell lymphoma, low-grade B-cell lymphoma, acute B-cell lymphoblastic leukemia, myeloblastic leukemia, Hodgkin's disease, plasmacytoma, follicular lymphoma, small follicular excised lymphoma, lymphoma of large follicular cells, small follicular mixed excised lymphoma, small diffuse excised cell lymphoma, diffuse small lymphocytic lymphoma, prolymphocytic leukemia, lymphoplasmacytic lymphoma, marginal zone lymphoma, mucosal-associated lymphoid tissue lymphoma, monocytoid B-cell lymphoma, lymphoma splenic, hairy cell leukemia, c-cell lymphoma diffuse large cells, B cell lymphoma large mediastinal, lymphomatoid granulomatosis, intravascular lymphomatosis, diffuse mixed cell lymphoma, diffuse large cell lymphoma, immunoblastic lymphoma, Burkitt's lymphoma, AIDS-related lymphoma, and mantle cell lymphoma. By "coordinated" it is proposed that the medicament comprising the anti-CD40 antagonist antibody or an antigen-binding fragment thereof should be used either before, during or after treatment of the subject with at least one other cancer therapy. Examples of other cancer therapies include, but are not limited to, surgery; radiation therapy; chemotherapy; optionally in combination with autologous bone marrow transplantation, where suitable chemotherapeutic agents include, but are not limited to, fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-chlorodeoxyadenosine (cladribine), cyclophosphamide, doxorubicin, prednisone and combinations of the same, for example, anthracycline-containing regimens such as CAP (cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincristine, doxorubicin plus dexamethasone), MP (melphalan plus prednisone) and other cytotoxic and / or therapeutic agents used in chemotherapy such as mitoxantrone, daunorubicin, idarubicin, asparaginase and antimetabolites including, but not limited to, cytarabine, methotrexate, 5-fluorouracil decarbazine, 6 -thioguanine, 6-mercaptopurine and nelarabine; another anti-cancer monoclonal antibody therapy (eg, alemtuzumab) (Campath ™ *) or another anti-CD52 antibody that targets the CD52 cell surface glycoprotein on cells B malignant; rituximab (Rituxan ™ *), the fully human antibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab / l-131 tositumomab (Bexxar ™ *), ibritumomab tiuxetan (Zevalin ™ *) or any other therapeutic anti-CD20 antibody that targets the CD20 antigen on malignant B cells; an anti-CD19 antibody (for example MT103, a bispecific antibody); an anti-CD22 antibody (for example the humanized monoclonal antibody epratuzumab); bevacizumab (Avastin ™ *) or another anti-cancer antibody that targets the endothelial, vascular, human growth factor; an anti-CD22 antibody that targets the CD22 antigen on malignant B cells (eg, the monoclonal antibody BL-22, an alphaCD22 toxin); an α-M-CSF antibody that targets the stimulation factor of macrophage colonies; antibodies that target the activator of nuclear factor-kappaB receptors (RANK) and its ligand (RANKL), which are overexpressed in multiple myeloma; an anti-CD23 antibody that targets the CD23 antigen on malignant B cells (e.g., IDEC-152); an anti-CD38 antibody that targets the CD38 antigen on malignant B cells; antibodies that target the highest class II histocompatibility complex receptors (anti-MHC antibodies) expressed on malignant B cells; other anti-CD40 antibodies (e.g., SGN-40) that target the CD40 antigen on malignant B cells; and antibodies that target the ligand receptor that induces apoptosis related to tumor necrosis factor 1 (TRAIL-R1) (eg, the monoclonal antibody, human, HGS-ETR1 agonist) expressed in a variety of solid tumors and tumors of hematopoietic origin); cancer therapy based on small molecules including, but not limited to, microtubule and / or topoisomerase inhibitors (eg, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin binding agent T900607; XL119; and the inhibitor aminocamptothecin topoisomerase), SDX-105 (bendamustine hydrochloride), ixabepilone (an epothilone analog, also referred to as BMS-247550), protein kinase C inhibitors, for example midostaurin ((PKC-412, CGP-41251, N-benzoylstaurosporine ), pixantrone, eloxatin (an antineoplastic agent), granite (gallium nitrate), Thalomid ™ * (thalidomide), immunomodulatory derivatives of thalidomide (for example, revlimid (formerly revimid)), Affinitak ™ * (antisense inhibitor of protein kinase C-alpha), SDX-101 (R-etodolac, which induces apoptosis of malignant lymphocytes), second generation purine nucleoside analogs such as clofarabine, inhibitors of Bcl-2 protein production by cancer cells (e.g., antisense oblimersen and Genasense ™ agents), proteasome inhibitors (e.g., Velcade ™ * (bortezo ib)), small molecule kinase inhibitors ( for example, CHIR-258), small molecule VEGF inhibitors (e.g., ZD-6474), small molecule inhibitors of heat shock protein 90 (HSP) (e.g., 17-AAG), small molecule inhibitors of histone deacetylases (e.g., hybrid hybrid / polar HPC) agents such as suberanylohydroxamic acid (SAHA) and FR-901228) and apoptotic agents such as Trisenox ™ * (arsenic trioxide) and Xcytrin ™ * (motex gadolinium) refine); cancer therapies based on vaccines / immunotherapy that include but are not limited to, vaccine approaches (eg, Id-KLH, oncology, vitaletine), personalized immunotherapy or active ideotypic immunotherapy (eg, personalized MyVax ™ immunotherapy *, formally designated GTOP-99), Promune ™ * (CpG 7909, a synthetic agonist for the toll-like receptor 9 (TLR9)), interferon-alpha therapy, interleukin-2 (IL-2) therapy, IL- therapy 12; IL-15 therapy and IL-21 therapy; steroid therapy; or another cancer therapy; wherein the treatment with the additional cancer therapy or additional cancer therapies occurs before, during or subsequent to treatment of the subject with the medicament comprising the anti-CD40 antagonist antibody or an antigen-binding fragment thereof, as Observe earlier in this document. In some embodiments, the present invention provides the use of the anti-CD40 antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof in the preparation of a medicament for the treatment of a B cell lymphoma, eg, a non-Hodgkin lymphoma, in a subject, wherein the drug is coordinated with treatment with at least one other cancer therapy selected from the group consisting of chemotherapy, anti-cancer antibody therapy, anti-cancer therapy, cancer based on small molecules and cancer therapy based on vaccines / immunotherapy, where the drug should be used either before, during or after the treatment of the subject with the other cancer therapy or, in the case of combination therapies multiple, either before, during or after treatment of the subject with the other cancer therapies. Thus, for example, in some embodiments, the invention provides for the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, in the preparation of a medicament for treating a B-cell lymphoma. , for example, non-Hodgkin lymphoma, in a subject, wherein the medicament is coordinated with chemotherapy treatment, wherein the chemotherapeutic agent is selected from the group consisting of cytoxan, doxorubicin, vincristine, prednisone and combinations thereof, for example CHOP. In other embodiments, the invention provides for the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, in the preparation of a medicament for the treatment of a B-cell lymphoma, eg, lymphoma. non-Hodgkin's, in a subject, wherein the medicament is coordinated with treatment with at least one other anti-cancer antibody selected from the group consisting of alemtuzumab (Campath ™ *) and another anti-CD52 antibody that targets the CD52 cell surface glycoprotein on malignant B cells; rituximab (Rituxan ™ *), the fully human antibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab / l-131 tositumomab (Bexxar ™ *), ibritumomab tiuxetan (Zevalin ™ *) or any other therapeutic anti-CD20 antibody that targets the CD20 antigen on malignant B cells; an anti-CD19 antibody (e.g., MT103, a bispecific antibody); an anti-CD22 antibody (for example, the monoclonal antibody, humanized epratuzumab); bevacizumab (Avastin ™ *) or another anti-cancer antibody that targets the endothelial, vascular, human growth factor; and any combination thereof; wherein the medicament is to be used either before, during or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during or after the treatment of the subject with the others therapies against cancer. In still other embodiments, the present invention provides the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, in the preparation of a medicament for the treatment of a B-cell lymphoma, for example. Non-Hodgkin lymphoma, in a subject, wherein the medicament is coordinated with treatment with at least one other small molecule-based cancer therapy selected from the group consisting of microtubule inhibitors and / or topoisomerase (eg, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin binding agent T900607; XL119; and the topoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustine hydrochloride), ixabepilone (an epothilone analog, also referred to as BMS-247550), protein inhibitors kinase C, eg, midostaurin ((PKC-412, CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (an antineoplastic agent), ganita (nitrate g) alio), Thalomid ™ (thalidomide), an apoptotic agent such as Xcytrin ™ * (motexaphine gadolinium), inhibitors of Bcl-2 protein production by cancer cells (e.g., antisense oblimersen and Genasense ™ agents) nelarabine and any combination thereof; wherein the medicament is to be used either before, during or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during or after the treatment of the subject with the other therapies against cancer. In still other embodiments, the present invention provides the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof, in the preparation of a medicament for the treatment of a B-cell lymphoma, for example. , non-Hodgkin lymphoma, in a subject, wherein the drug is coordinated with treatment with at least one other cancer therapy based on vaccines / immunotherapy selected from the group consisting of vaccine approaches (eg, Id-KLH, oncophagous, vitaletine), personalized immunotherapy or active ideotypic immunotherapy (eg, MyVax ™ * customized immunotherapy, formally designated GTOP-99), Promune ™ * (CpG 7909, a synthetic agonist for toll-like receptor 9 (TLR9)), therapy of interleukin-2 (IL-2) IL-12 therapy, IL-15 therapy and IL-21 therapy and any combination thereof; wherein the medicament is to be used either before, during or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during or after the treatment of the subject with the other therapies against cancer. In some embodiments, the present invention provides for the use of the anti-CD40 antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment thereof in the preparation of a medicament for the treatment of a B cell-related leukemia, for example, acute B-cell lymphocytic leukemia (B-ALL), in a subject, wherein the medicament is coordinated with treatment with at least one other cancer therapy selected from the group consisting of chemotherapy and anti-metabolite therapy, where the drug should be used before, during or after treatment of the drug with the other cancer therapy or, in the case of multiple combination therapies, either before, during or after the treatment of the subtlety with the other cancer therapies. Examples of these modalities include, but are not limited to, those instances where the medicament comprising the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or an antigen-binding fragment of the it is coordinated with the treatment with a chemotherapeutic agent or anti-metabolites, selected from the group consisting of cytoxan, doxorubicin, vincristine, prednisone, cytarabine, mitoxantrone, idarubicin, asparaginase, methotrexate, 6-thioguananine, 6-mercaptopurine and combinations of the same; wherein the medicament is to be used before, during or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during or after the treatment of the subject with the other anti-cancer therapies. Cancer. In one of these examples, the drug is coordinated with the treatment with cytarabine plus daunorubicin, cytarabine plus mitoxantrone and / or cytarabine plus idarubicin; wherein the medicament is to be used before, during or after treatment of the subject with B-ALL with the other cancer therapy or, in the case of multiple combination therapies, either before, during or after treatment of the subject with the other therapies against cancer. The invention also provides the use of the antagonist anti-CD40 antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 described herein or an antigen-binding fragment thereof in the preparation of a medicament for the treatment of a subject for a cancer characterized by the growth of neoplastic B cells, which include the B cell-related cancers described hereinabove, wherein the medicament is used in a subject that has previously been treated with at least one other cancer therapy. By "pre-treated" or "pre-treatment" it is proposed that the subject have received one or more other cancer therapies (ie, have been treated - with at least one other cancer therapy) before receiving the medication comprises the anti-CD40 antagonist antibody or an antigen-binding fragment thereof. "Previously treated" or "pre-treatment" includes subjects who have been treated with at least one other cancer therapy within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within of 6 months, 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 of 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 described herein or an antigen binding fragment. of the same. It is not necessary that the subject has responded to pre-treatment with previous cancer therapy, or previous cancer therapies. In this way, the subject receiving the medicament comprising the anti-CD40 antagonist antibody or an antigen-binding fragment thereof could have responded or could not have responded (ie, the cancer was refractory), upon pre-treatment with previous cancer therapy or one or more of the previous cancer therapies where the pre-treatment comprised multiple cancer therapies. Examples of other cancer therapies for which the subject may have received the pre-treatment prior to receiving the medicament comprising the anti-CD40 antagonist antibody or an antigen-binding fragment thereof include, but are not limited to, Surgery; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplantation, where suitable chemotherapeutic agents include, but are not limited to, those listed above in this document; another anti-cancer monoclonal antibody therapy that includes, but is not limited to, those anti-cancer antibodies listed above in this document; cancer therapy based on small molecules that includes, but is not limited to, the small molecules listed above in this document; cancer therapies based on vaccines / immunotherapy that include, but are not limited to, those listed above in this document; steroid therapy; another cancer therapy; or any combination thereof. The "treatment" in the context of the coordinated use of a medicament described herein with one or more other cancer therapies is defined herein as the application or administration of the medicament or other cancer therapy to a subject or the application or administration of the drug or other cancer therapy to an isolated tissue or cell line of a subject, where the subject has a cancer characterized by the growth of neoplastic B cells, a symptom associated with a cancer of this type or a predisposition towards the development of this cancer, where the purpose is to cure, heal, alleviate, mitigate, alter, remedy , lighten, improve or affect cancer, any associated symptoms of cancer or predisposition to the development of cancer. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL SECTION The anti-CD40 antagonist antibodies used in the following examples 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 of the human IgGx subtype (mAbs) generated by the immunization of transgenic mice carrying the heavy chain locus of IgG? human and the human K light chain locus (XenoMouse ™ technology *; Abgenix; Fremont, California). As shown by the FACS analysis, the antibodies CHIR-5.9 and CHIR-12.12 bind specifically to the human CD40 antigen and can prevent the binding of the CD40 ligand. Both mAbs can compete with the CD40 ligand previously bound to the CD40 antigen on the cell surface. Both antibodies are strong antagonists and inhibit proliferation mediated by the CD40 ligand in normal B cells, as well as cancer cells from patients with NHL and CLL. In vi tro, both antibodies eliminate cancer cell lines as well as the primary cancer cells of patients with NHL through ADCC. The dose-dependent antitumor activity is observed in a human xenograft lymphoma model. The binding affinity of the CHIR-5.9 antibody to the human CD40 antigen is 1.2 x 10 ~ 8 M and the binding affinity of the CHIR-12.12 antibody to the human CD40 antigen is 5 x 10"10 M. The Mouse Hybridoma Line 131.2F8 .5.9 (CMCC # 12047) and the hybridoma line 153.8E2.DIO .D6.12.12 (CMCC # 12056) have been deposited at the American Type Culture Collection [ATCC; 10801 Unersity Blvd., Manassas, Virginia 20110-2209 (USA) )] under patent deposit number PTA-5542 and PTA-5543, respectively The following protocols have been used in the examples described below: ELISA Assay for Quantitation of Immunoglobulin Human IgM and IgG concentrations were estimated by means of the assay ELISA 96-well ELISA plates were coated with 2 μg / ml goat anti-human IgG MAb (The Jackson Laboratory, Bar Harbor, Maine) or with 2 μg / ml goat anti-human IgM MAb 4102 (BioSource International, California) in 0.05 M carbonate buffer (pH 9.6) by incubation for 16 hours at 4 ° C. Plates were washed 3 times with PBS-Tween-20 ™ * 0.05% (PBS-Tween) and saturated with BSA for 1 hour. After 2 washes, the plates were incubated for 2 hours at 37 ° C with different dilutions of the test samples. After 3 washes the bound Ig was detected by incubation for 2 hours at 37 ° C with 1 μg / ml of goat anti-human IgG labeled with peroxidase or goat anti-human IgM Mab. The plates were washed 4 times and the bound peroxidase activity was revealed by the addition of O-phenylenediamine as a substrate. The human IgG or IgM standards (Caltaq, Burlingame, California) were used to establish a standard curve for each assay.

Isolation of Peripheral Blood Mononuclear Cells (PBMC) from Human Peripheral Blood 20 ml of Ficoll-Paque ™ * solution (low endotoxin, Pharmacia) were added to a 50 ml polystyrene tube, in 3 tubes, 30 minutes before adding the blood. The Ficoll-Paque ™ * solution was heated to room temperature. 3 L of bleach was prepared in 1:10 dilution and used to wash all the tubes and pipettes containing the blood. The blood was stratified on top of the Ficoll-Paque ™ * solution without altering the Ficoll ™ * layer, in 1.5 ml of blood / 1 ml of Ficoll-Paque ™. The tubes were centrifuged at 1700 rpm for 30 minutes at room temperature with the centrifuge brake deactivated. As much of the top layer (plasma) was removed as possible, minimizing the vacuum in order to avoid the removal of the second layer of solution. The second layer, which contains the B and T lymphocytes, was collected using a sterile Pasteur pipette and placed in two 50 ml polystyrene tubes. The collection was diluted with 3 x the volume of cold RPMI without additives and the tubes were centrifuged at 1000 RPM for 10 minutes. The media was removed by aspiration and the cells of both 50 ml tubes were resuspended in a total of 10 ml of cold RPMI (with additives) and transferred to a 15 ml tube. Cells were counted using the hemacytometer, then centrifuged at 1000 RPM for 10 minutes. The media was removed and the cells resuspended in 4 ml of RPMI. This fraction contained the PBMC. Isolation of PBMC B Cells 100 μl of Dynabeads ™ * (anti-CD19 h) were placed in a 5 ml plastic tube. 3 ml of sterile PBS was added to the beads and mixed and placed on a magnetic support, then allowed to settle for 2 minutes. The solution was removed using a Pasteur pipette. 3 ml of sterile PBS were added, mixed and placed on a magnetic support, then allowed to settle for 2 minutes. This procedure with sterile PBS was repeated once more for a total of 3 washes. The PBMC were added to the beads and incubated, while mixing, for 30 minutes at 40 ° C. The tube containing the PBMC and the beads was placed on the magnetic support for 2 minutes, then the solution was transferred to a new 5 ml tube in the magnetic support. After 2 minutes, the solution was transferred to a new 15 ml tube. This step was repeated four more times and the solutions of the first four times were collected in the 15 ml tube, then centrifuged at 1000 RPM for 5 minutes. This step produced the pellet for T-cell separation. 100 μl of RPMI (with additives) was added to collect the beads and the solution was transferred into a 0.7 ml tube. 10 μl of Dynal Detacha Beads ™ * beads were added to the suspension at room temperature and allowed to rotate for 45 minutes. The suspension was transferred to a new 5 ml tube and 3 ml of RPMI (with additives) were added. The tube was placed on the magnetic support for 2 minutes. The solution was transferred to a new 5 ml tube in the support for 2 minutes, then to a 15 ml tube. The previous step was repeated three more times, collecting the solution in the 15 ml tube. The 15 ml tube was centrifuged at 1000 RPM for 10 minutes and the cells were resuspended in 10 ml of RPMI. The washing step was repeated 2 more times for a total of 3 washes. The cells were counted before the last centrifugation. This step completed the purification of the B cells. The cells were stored in FCS 90% and DMSO 10% and frozen at -800 ° C. T cell isolation The human T Cell Enrichment Column ™ * column (R & D systems, anti-CD3 h column kit) was prepared using 20 ml of 1X column wash buffer when mixing 2 ml of wash buffer. column 10 X and 18 ml distilled water, sterile. The column was cloned with 70% ethanol and placed on top of a 15 ml tube. The top cap of the column was removed first to prevent air extraction at the bottom of the column. After, the bottom lid was removed and the tip was cleaned with 70% ethanol. The fluid within the column was allowed to drain into the 15 ml tube. A new sterile 15 ml tube was placed under the column after the column cushion had drained to the level of the white filter. The PBMC fraction depleted in B cells was suspended in 1 ml of buffer and added to the top of the column. The cells were allowed to incubate with the column at room temperature for 10 minutes. The T cells were eluted from the column with 4 aliquots of 2 ml each of 1 X column wash buffer. The harvested T cells were centrifuged at 1000 RPM for 5 minutes. The supernatant was removed and the cells were resuspended in 10 ml of RPMI. The cells were counted and centrifuged once more. The supernatant was removed, completing the purification of the T cells. The cells were stored in FCS 90% and DMSO 10% and frozen at -80 ° C. For the above procedures, the RPMI composition contained FCS 10% (inactivated at 56 ° C for 45 minutes), Pen / Strep 1% (100 u / ml Penicillin, 0.1 μg / ml Streptomycin), glutamate 1%, purevate sodium 1%, 2-ME 50 μm. Flow Cytofluorometric Assay Ramos cells (106 cells / sample) were incubated in 100 μl of primary antibody (10 μg / ml in PBS-BSA) for 20 minutes at 4 ° C. After 3 washes with PBS-BSA or HBSS-BSA, the cells were incubated in 100 μl of FITC F (ab ') 2 fragments of goat anti- (human IgG) antibodies (Caltaq) for 20 minutes at 4 °. C. After 3 washes with PBS-BSA and 1 wash with PBS, the cells were resuspended in 0.5 ml of PBS. The analyzes were performed with a FACSCAN V device (Becton Dickinson, San José, California). Generation of Hybridoma Clones Splenocytes from immunized mice were fused with SP 2/0 or P3 x 63Ag8.653 murine myeloma cells at a ratio of 10: 1 using 50% polyethylene glycol as previously described by de Boer et al. (1988). J. Immunol. Meth. 113: 143. The fused cells were resuspended in complete IMDM medium which was supplemented with hypoxanthine (0.1 mM), aminopterin (0.01 mM), thymidine (0.016 mM) and 0.5 ng / ml of hIL-6 (Genzyme, Cambridge, Massachusetts). The fused cells were then distributed between the wells of the 96-well tissue culture plates, so that each well contained 1 hybridoma growing on average. After 10-14 days, the supernatants of the hybridoma populations were examined for the production of specific antibodies. For the examination of the production of specific antibodies by the hybridoma clones, the supernatants of each well were accumulated and tested for the specificity of anti-CD40 activity by means of the ELISA assay first. The positive supernatants were then used for staining with fluorescent cells of the EBV transformed B cells as described for the previous FACS assay. The positive hybridoma cells were cloned twice by limiting the dilution in IMDM / FBS containing 0.5 ng / ml of hIL-6. Example 1: Production of Anti-CD40 Antibodies Several anti-CD40, antagonistic, fully human monoclonal antibodies of the IgGl isotype were generated. Transgenic mice carrying the human IgGl heavy chain locus and the human K chain locus (Abgenix? -1 XenoMouse ™ * technology (Abgenix; Fremont, California)) were used to generate these studies. The SF9 insect cells expressing the CD40 extracellular domain were used as immunogen. A total of 31 spleens of mice were fused with mouse myeloma SP2 / 0 cells to generate 895 antibodies that recognize the recombinant CD40 antigen in the ELISA assay (Tables 1A and IB). On average, approximately 10% of the hybridomas produced using the Abgenix XenoMouse ™ 1 technology can contain the mouse lambda light chain instead of the human kappa chain. Antibodies containing the light lambda mouse chain were selected. A subset of 260 antibodies that also showed binding to the cell surface CD40 antigen was selected for further analysis. The stable hybridomas selected during a series of subcloning procedures were used for further characterization. in the union- and functional tests.

Table 1A. A Typical Fusion Table 1 B. Summary of Four Merger Sets It was identified that the clones of 7 mother hybridomas have antagonistic activity. Based on their relative antagonistic potency and ADCC activities, two hybridoma clones were selected. Their names are: 131.2F8.5.9 (5.9) and 153.8E2.D10.D6.12.12 (12.12). It was identified that the clones from 7 different hybridomas had antagonistic activity (Table 2 above). Based on their relative antagonistic potencies and ADCC activities, two hybridoma clones were selected for further evaluation. These were called 131.2F8.5.9 (5.9) and 153.8E2.DIO.D6.12.12 (12.12). The binding profile of these two antibodies to the CD40 + lymphoma cell line is shown as a flow cytometric histogram in Figure 1. Example 2: Polynucleotide and Amino Acid Sequences of the Human Anti-CD40 Antibodies 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 the heavy chain of the CHIR-12.12 antibody are set forth in Figures 9A and 9B, respectively. See also SEQ ID NO: 2 (light chain for mAb CHIR-12.12) and SEQ ID NO: 4 (heavy chain for mAb CHIR-12.12). A variant of the heavy chain for mAb CHIR-12.12 is shown in Figure 9B (see also SEQ ID NO: 5), which differs from SEQ ID NO: 4 in that it has a serine residue substituted by the residue of alanine at position 153 of SEQ ID NO: 4. The nucleotide sequences encoding the light chain and heavy chain of the CHIR-12.12 antibody are set forth in Figures 10A and 10B, respectively. See also SEQ ID NO: l (coding sequence for light chain for mAb CHIR-12.12) and SEQ ID NO: 3 (coding sequence for heavy chain for mAb CHIR-12.12). The amino acid sequences for the light chain and the heavy chain of the CHIR-5.9 antibody are set forth in Figures HA and 11B, respectively. See also SEQ ID NO: 6 (light chain for mAb CHIR-5.9) and SEQ ID NO: 7 (heavy chain for mAb CHIR-5.9). A variant of the heavy chain for mAb CHIR-5.9 is shown in Figure 11B (see also SEQ ID NO: 8), which differs from SEQ ID NO: 7 in that it has a serine residue substituted by the residue of alanine at position 158 of SEQ ID NO: 7. As expected for antibodies derived from independent hybridomas, there is substantial variation in the nucleotide sequences in the complementarity determining regions (CDRs). It is believed that the diversity in the CDR3 region of VH determines more significantly the specificity of the antibodies. Example 3: Effect of Antibodies CHIR-5.9 and CHIR-12.12 on the Interaction of CD40 / CD40L In Vi tro The candidate antibodies CHIR-5.9 or CHIR-12.12 prevent the binding of the CD40 ligand to the CD40 antigen on the cell surface and displace the ligand of CD40 previously bound. The antibodies CHIR-5.9 and CHIR-12.12 were tested for their ability to prevent binding of the CD40 ligand to the CD40 antigen on the surface of a lymphoma cell line (Ramos). The binding of both antibodies (unlabeled) prevented the subsequent binding of the PE-CD40 ligand as measured by flow cytometric assays (Figure 2A). In a second set of assays, the two antibodies were tested for their ability to replace the CD40 ligand previously bound to the cell surface CD40 antigen. Both antibodies were effective to compete for the previously bound CD40 ligand, with the CHIR-5.9 antibody which was slightly more effective than the CHIR-12.12 antibody (Figure 2B). Example 4: Antibodies CHIR-5.9 and CHIR-12.12 bind to a Different Epitope on the CD40 Antigen 15B8 The monoclonal antibodies, candidates CHIR-5.9 and CHIR-12.12 compete with each other for binding to the CD40 antigen but not to 15B8, an anti-CD40 mAb of IgG2 (see international publication No. WO 02/28904). Antibody competition binding studies using Biacore ™ * were designed using CM5 biosensor microcircuits with protein A immobilized via the amine coupling, which was used to capture either anti-CD40, CHIR-12.12 or 15B8. Normal association / dissociation binding curves were observed with varying concentrations of CD40-his (data not shown). For the competition studies, either of the CHIR-12.12 or 15B8 antibodies were captured on the surface of protein A. Subsequently, a complex of CD40-his / Fab of CHIR-5.9 (CD40 100 nM: Fab of CHIR-5.9 1 μM), in varying concentrations, was flowed through the modified surface. In the case of the CHIR-12.12 antibody, no association of the complex was observed, indicating that the CHIR-5.9 antibody blocks the binding of the CHIR-12.12 antibody to the CD40-his. For antibody 15B8, the association of the observed CHIR-5.9 Fab complex indicating that the CHIR-5.9 antibody does not block the binding of antibody 15B8 to the CD40 binding site. However, the proportion of the complex increased dramatically (data not shown). It has also been determined that antibodies 15B8 and CHIR-12.12 do not compete for binding to CD40-his. This experiment was established by capturing the CHIR-12.12 antibody in the biosensor microcircuit of protein A, blocking the residual protein A sites with control hIgG__, binding CD40-his and then flowing the 15B8 antibody on the modified surface. The 15B8 antibody did not bind under these conditions indicating that the CHIR-12.12 antibody does not block the binding of the 15B8 antibody to the CD40 antigen. Example 5: Binding Properties of Selected Hybridomas. Protein A was immobilized on CM5 biosensing microcircuits via the amine coupling. The human anti-CD40 monoclonal antibodies, at 1.5 μg / ml, were captured on the surface of the modified biosensor for 1.5 minutes at 10 μl / minute. The soluble, recombinant CD40-his was flowed on the surface of the biosensor in varying concentrations. Antibody and antigen were diluted with 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 surfactant (HBS-EP). The kinetic and affinity constants were determined using the Biaevaluation computer program with a 1: 1 interaction / global model fit. As shown in Table 3 below, there is a 121-fold difference in the ratio of CHIR-5.9 or CHIR-12.12 resulting from a 24-fold higher affinity for the CHIR-12.12 antibody.

Example 6: Antibodies CHIR-5.9 and CHIR-12.12 are Potent Antagonists for CD40-Mediated Proliferation of Human Lymphocytes of Normal Subjects The coupling of the CD40 antigen by the CD40 ligand induces the proliferation of human B cells. It is expected that an anti-CD40 antagonist antibody inhibits this proliferation. Two candidate antibodies (CHIR-5.9 and CHIR-12.12) were tested for their ability to inhibit proliferation of PBMC induced by CD40 ligands of normal human subjects. CHO cells fixed in transfectant formaldehyde expressing the CD40 ligand (CD40L) were used as a source of the CD40 ligand. Human PBMC were cultured for 4 days with the CHO cells fixed in formaldehyde expressing the CD40 ligand in the presence of varying concentrations of the anti-CD40 mAb CHIR-5.9 or CHIR-12.12. Proliferation was measured by the incorporation of tritiated thymidine. The cells were induced with thymidine labeled with tritium at 37 ° C for 14-18 hours. It was found that both antibodies were very effective in inhibiting the proliferation of human PBMC induced by CD40 ligands (Table 4A, mAb CHIR-5.9, Table 4B, mAb CHIR-12.12). The experiment was performed with multiple PBMC donors (n = 12 for CHIR-5.9 and n = 2 for CHIR-12.12) to ensure that the inhibition observed was not a peculiarity of the cells of an individual donor. The following evaluations with 4 additional PBMC donors were carried out for mAb CHIR-12.12 with similar trends observed. A wide range of antibody concentrations (0.01 μg / ml to 100 μg / ml) were used in these trials. The almost complete inhibition of proliferation induced by CD40 ligands could be achieved at a concentration of 0.1 μg / ml of antibodies in most cases. The concentration of antibodies (pM) to inhibit 50% of lymphocyte proliferation induced by CD40 ligands (IC50) for the lymphocytes of 6 donors produced an average IC50 value (pM) of 47 (SD = 21, donor 1.24; donor 2, 66, donor 3, 45, donor 4, 84, donor 5, 30, donor 6, 35), which compares favorably with the average IC50 value (pM) of 49.65 shown in Table 4B. Based on the current data set, both candidate antibodies appear similar in their potency for the inhibition of normal PBMC proliferation induced by CD40 ligands.

Table 4A. Effect of mAb CHIR-5.9 on proliferation of PBMC induced by CD40L CHO- PBMC + Conc. Of HulgGI CHIR-5.9 PBMC CD40L CHO- Abs Exp. # Alone only CD40L% of (μg / ml) CPM inhibition CPM inhibition PBMC-010 1851 121 4436 1 5080 -26 2622 74 1851 121 4436 0.25 5498 -43 2907 62 1851 121 4436 0.0625 6029 -65 2619 74 1851 121 4436 0.0156 5814 -56 1199 131 PBMC-011 Donated? # 1 2162 178 8222 10 13137 -84 2252 101 2162 178 8222 1 11785 -61 1438 115 2162 178 8222 0.1 10758 -43 1249 119 2162 178 8222 0.01 11322 -53 4705 60 Donor # 2 2216 294 7873 10 16679 -164 2362 103 2216 294 7873 1 014148 -117 1202 124 2216 294 7873 0.1 12422 -85 756 133 2216 294 7873 0.01 13870 -112 6606 24 Donor # 3 2396 241 11021 10 11641 -7 2631 100 2396 241 11021 1 13528 - 30 1450 114 2396 241 11021 0.1 12176 -14 990 120 2396 241 11021 0.01 11895 -10 5357 68 Donor # 4 4552 133 15301 10 22098 -64 3768 109 4552 133 15301 1 19448 -39 2040 125 4552 133 15301 0.1 18398 -29 1728 128 4552 133 15301 0.01 22767 -70 9481 55 PBMC-012 777 117 6041 10 7327 -25 2150 76 777 117 6041 1 6212 -3 1550 87 777 117 6041 0.1 7006 -19 828 101 777 117 6041 0.01 7524 -29 1213 94 PB C-014 1857 73 7889 100 9399 -25 3379 76 1857 73- 7889 20 8120 -4 3870 67 1857 73 7889 4 8368 -8 2552 90 1857 73 7889 0.8 9564 -28 1725 103 PBMC-015 Donor # 1 3202 127 10485 100 15425 -69 1497 126 3202 127 10485 20 11497 -14 1611 124 3202 127 10485 4 11641 -16 1359 128 3202 127 10485 0.8 12807 -32 1490 126 Donor # 2 3680 175 15145 100 21432 -56 1792 118 3680 175 15145 20 16998 -16 1779 118 3680 175 15145 4 17729 -23 1965 117 3680 175 15145 0.8 17245 -19 2217 115 Donor # 3 2734 152 19775 100 22967 -19 1664 107 2734 152 19775 20 21224 -9 1848 106 2734 152 19775 4 20658 -5 1534 108 2734 152 19775 0.8 18923 5 1262 110 PBMC-016 1118 36 13531 0.1 10928 21 745 103 1118 36 13531 0.05 11467 17 962 102 1118 36 13531 0.01 11942 13 3013 85 PBMC-017 962 75 12510 1 13597 -9 258 107 % of average inhibition of human PBMC at 100 μglml -42 107 % inhibition average of human PBMC at 10 μglgl -69 98 % inhibition of average human PBMC at 1 μg / ml -41 % of average inhibition of human PBMC at 0.1 μglml -28 117 % average inhibition of human PBMC at 0.01 μg / ml -44 64 % average inhibition of human PBMC -35 101 % inhibition: 100- (CPM with Abs-PBMC alone-CHO-CD40L alone) / (CPM of PBMC + CHO-CD40L- PBMC alone-CHO-CD40L alone) * 100% Table 4B. Effect of mAb CHIR-12.12 on proliferation of PBMC induced by CD40L CHO-PBMC + Conc. HulgGI CH1R-12.12 PBMC CD40L CHO- of Abs% IC50% Exp only CD40L (μg / ml) CPM inhibition CPM inhibition (nM) PBMC-025 Donor # 1 4051 32 42292 0.1 33354 23 440 110 4051 32 42292 0.01 37129 14 8696 88 4051 32 42292 0.001 40271 5 32875 25 4051 32 42292 0.0001 40034 6 37261 13 24.22 Donor # 2 2260 31 14987 0.1 15767 -6 365 115 2260 31 14987 0.01 17134 -17 6734 65 2260 31 14987 0.001 20142 -41 16183 -9 2260 31 14987 0.0001 17847 -23 16187 -9 65.96 PBMC-025 Donor # 1 2039 35 19071 0.1 17136 11 624 109 2039 35 19071 0.01 16445 15 6455 74 2039 35 19071 0.001 16195 17 17833 7 2039 35 19071 0.0001 18192 5 17924 7 45 Dor? Ador # 2 2016 64 17834 0.1 17181 4 2078 100 2016 64 17834 0.01 16757 7 10946 44 2016 64 17834 0.001 18613 -5 17924 -1 2016 64 17834 0.0001 17169 4 18569 -5 84 PB C-028 Donor # 1 4288 45 22547 1 18204 24 2098 112 4288 45 22547 0.1 20679 10 1827 114 4288 45 22547 0.01 22799 -1 6520 88 4288 45 22547 0.001 23547 -5 22327 1 4288 45 22547 0.0001 24778 -12 24124 - 9 30.07 Donor # 2 2148 58 54894 1 48545 12 5199 94 2148 58 54894 0.1 45708 17 5091 95 2148 58 54894 0.01 51741 6 18890 68 2148 58 54894 0.001 52421 5 50978 7 2148 58 54894 0.0001 54778 0 52581 4 34.68 PBMC-029 Donor # 1 609 69 10054 0.1 11027 -10 2098 85 609 69 10054 0.01 10037 0 1827 88 609 69 10054 0.001 10222 -2 6520 38 609 69 10054 0.0001 11267 -13 22327 -131 28.06 Donor # 2 7737 57 23132 0.1 21254 12 2536 134 7737 57 23132 0.01 21726 9 10249 84 7737 57 23132 0.001 22579 4 23380 -2 7737 57 23132 0.0001 22491 4 23183 0 55.35 PB C-030 Donor # 1 2739 47 53426 0.1 60116 -13 2132 101 2739 47 53426 0.01 56411 -6 14297 77 2739 47 53426 0.001 59167 -11 55868 -5 2739 47 53426 0.0001 59290 -12 60865 -15 35.52 Donor # 2 4310 50 53781 0.1 52881 2 3208 102 4310 50 53781 0.01 52741 4 30716 47 4310 50 53781 0.001 53072 1 53628 0 4310 50 53781 0.0001 58045 -9 54343 -1 102.88 PBMC-032 Donor # 1 2458 42 14058 0.1 16579 -22 636 116 40.36 2458 42 14058 0.01 19250 -45 3358 93 2458 42 14058 0.001 19852 -50 20639 -57 2458 42 14058 0.0001 19161 -44 18907 -42% average inhibition of Human PBMC at 0.1 μglml 3 107% average inhibition of human PBMC at 0.01 μglml -1 74% average inhibition of human PBMC at 0.001 μg / ml -7 0% average inhibition of human PBMC at 0.0001 μg / ml -8 -17 49.65 % inhibition: 100- (CPM with Abs-PBMC alone-CHO-CD40L alone) / (PBMC CPM + CHO-CD40L-PBMC alone-CHO-CD40L only) * 100% In addition to B cells, human PBMCs also contain natural killer cells that can mediate antibody-dependent cytotoxicity (ADCC). To clarify the mechanism of antibody-mediated inhibition of proliferation, assays were performed with purified B cells of human PBMC. Similar to the results obtained with the PBMC, both antibodies potently inhibited the proliferation of purified B cells induced by CD40 ligands (Table 5, n = 3). These data demonstrate that the antagonist activity of the candidate antibodies and not the ADCC mechanism is the cause of inhibition of proliferation in these assays. Table 5. Effect of anti-CD40 antibodies on the proliferation of human, purified B cells induced by CD40 ligands CPM HulgGI CHIR-5.9 CHIR-12.12 Cells Conc. Donor Cells CHO- B + CHO- Abs%%% Exp. # # B CD40L CD40L (μg / ml) CPM inhibition CPM inhibition CPM inhibition Cell B-004 1 418 89 3132 100 429 103 271 109 152 114 418 89 3132 20 3193 -2 316 107 222 111 418 89 3132 4 3175 -2 144 114 235 110 418 89 3132 0.8 6334 -122 245 110 63 117 2 81 73 27240 100 28311 -4 85 100 77 100 81 73 27240 20 24707 9 65 100 94 100 81 73 27240 4 23081 15 108 100 68 100 81 73 27240 0.8 26252 4 87 100 77 100 Cell B-005 3 267 75 24552 1 25910 -6 291 100 102 101 267 75 24552 0.1 28447 -16 259 100 108 101 267 75 24552 0.01 26706 -9 2957 89 4922 81 % inhibition averaged 0 -3 103 103 % inhibition: 100- (CPM with Abs B cells alone-CHO-CD40L alone) / (CPM of B cells with CHO-CD40L-B cells alone-CHO-CD40L alone) * 100% Example 7: CHIR-Antibodies 5.9 and CHIR-12.12 do not Induce a Strong Proliferation of Human B Cells of Normal Subjects The CD40 ligand activates the proliferation of normal B cells and B-cell lymphoma cells. The binding of some anti-CD40 antibodies (agonists) can provide a similar stimulatory signal for the proliferation of normal and cancerous B cells. Antibodies with strong B cell stimulatory activity are not suitable candidates for the therapeutic treatment of B-cell lymphomas. The two candidate antibodies were tested for their ability to induce proliferation of B cells from normal voluntary donors. B cells purified by the Ficoll-Hypaque Plus ™ * gradient centrifugation of PBMC from normal donors were cultured in 96-well plates with varying concentrations of candidate antibodies (range 0.001 to 100 μg / ml) for a total of 4 days. In the positive control group, the PBMCs were cultured with CHO cells fixed with formaldehyde expressing the CD40 ligand. B cell proliferation was measured by incorporation of tritium labeled thymidine at 37 ° C for 14-18 hours. While the CD40 ligand presented on the CHO cells induced vigorous proliferation of B cells resulting in an average stimulation index (SI) of 145, the candidate antibodies induced only a weak proliferation with a stimulation index of 2. 89 and 5 08 for CHIR-12 antibodies. 12 and CHIR-5. 9, respectively (n = 3) (Table 6). Table 6. Proliferation of purified B cells from human subjects, normal in response to candidate anti-CD40 mAbs Cells B + CHO-Conc Cells Cells B cells CD40L of Abs B + hulgG1 B + CHIR-5.9 B + CHIR-12.12 Exp. # Donor # CPM CPM YES (1) (μg / ml) CPM YES (2) CPM YES (2) CPM YES (2) Cells 1 418 3132 7.49 100 498 1.19 401 0.96 458 1.10 B-004 418 3132 20 245 0.59 232 0.56 370 0.89 Frozen 418 3132 4 241 0.58 232 0.56 211 0.50 418 3132 0.8 376 0.90 298 0.71 230 0.55 Frozen 2 81 27240 336.30 100 34 0.42 454 5.60 122 1.51 81 27240 20 48 0.59 706 8.72 255 3.15 81 27240 4 41 0.51 567 7.00 367 4.53 81 27240 0.8 34 0.42 736 9.09 408 5.04 Cells 3 267 24552 91.96 1 691 2.59 2101 7.87 1223 4.58 B-005 267 24552 0.1 686 2.57 2267 8.49 1557 5.83 267 24552 0.01 808 3.03 2203 8.25 1027 3.85 267 24552 0.001 567 2.12 846 3.17 826 3.09 Stimulation index SI) Prom (? Dio 145.25 1.29 5.08 2.88 index of E. (1) : = CPM (cells B + C, HO-CD40L) / CP (cells B alone) index of E. (2): = CPM (cells B + A bs) / CP (PBMC so) In addition to the cells B, human PBMCs contain cell types that carry the Fc receptors (FcR) for the IgG1 molecules that can provide the cross-linking of anti-CD40 antibodies bound to the CD40 antigen in B cells.This cross-linking could potentially increase the activity stimulating anti-CD40 antibodies To confirm the lack of B-cell stimulating activity of CHIR-5.9 and CHIR-12.12 antibodies in the presence of cross-linking cells, proliferation experiments were performed with total PBMC containing B cells as well as cells FcR + The data of these experiments (Table 7 A, mAb CHIR-5.9, Table 7B, mAb CHIR-12.12) confirm that these candidate antibodies even in the presence of cells carrying FcR do not stimulate in general the proliferation of B cells on the background proliferation induced by the Human control IgGl (n = 10). The CD40 ligand induced an average SI stimulation index (SI) of 7.41. The values - t_ of average SI with the candidate antibodies were 0.55 and 1.05 for antibodies CHIR-12.12 and CHIR-5.9, respectively. Only one of the 10 donor PBMCs that were tested showed some stimulatory response to the CHIR-5.9 antibody (donor # 2 in Table 7). The lack of stimulatory activity by the candidate mAbs was further confirmed by measuring the proliferation of PBMCs in response to the candidate anti-CD40 antibodies that are immobilized on the plastic surface of Table 7A. Proliferation of PBMC from human, normal subjects in response to mAb CHIR-5.9 Conc. PBMC + CHO-CD40L from Abs PBMC + hulgG1 PBMC + CHIR-5.9 Exp. # PBMC CPM index (1) (μg / ml) CPM index (2) CPM index (2) PBMC- 597 010 1417 5279 3.73 1 1218 0.86 3 4.22 481 1417 5279 0.25 1712 1.21 5 3.40 0.062 364 1471 5279 5 1449 1.02 2 2.57 0.015 324 1417 5279 6 1194 0.84 2 2.29 PBMC- 317 011 Donator *! 2138 8247 3.86 10 3047 1.43 7 1.49 361 2138 8247 2726 1.28 7 1.69 201 2138 8247 0.1 2026 0.95 1 0.94 186 2138 8247 0.01 2424 1.13 0 0.87 1156 452 Donor # 2 2374 1 4.87 10 4966 2.09 3 1.91 1156 244 2374 1 1 2544 1.07 5 1.03 1156 146 2374 1 0.1 2177 0.92 0.62 1156 189 2374 1 0.01 4672 1.97 0.80 211 Donor # 3 3229 7956 2.46 10 5035 1.56 9 0.66 109 3229 7956 2826 0.88 9 0.34 105 3229 7956 0.1 2277 0.71 2 0.33 189 3229 7956 0.01 3078 0.95 9 0.59 1431 517 Donor *! 4198 4 3.41 10 5012 1.19 6 1.23 1431 470 4198 4 3592 0.86 1.12 1431 431 4198 4 0.1 5298 1.26 9 1.03 1431 540 4198 4 0.01 5758 1.37 0 1.29 PBMC- 247 014 2350 8787 3.74 100 2722 1.16 1 1.05 244 2350 8787 20 2315 0.99 7 1.04 165 2350 8787 4 2160 0.92 9 0.71 167 2350 8787 0.8 2328 0.99 1 0.71 PBMC- 1293 168 015 Donor # 1 3284 6 3.94 100 3598 1.10 2 0.51 1293 156 3284 6 20 2751 0.84 2 0.84 1293 110 3284 6 4 3135 0.95 5 0.34 1293 141 3284 6 0.8 4027 1.23 9 0.43 1912 510 Donor # 2 6099 1 3.14 100 2999 0.49 4 0.84 1912 391 6099 1 20 4025 0.66 7 0.64 1912 334 6099 1 4 4496 0.74 1 0.55 1912 413 6099 1 0.8 3834 0.63 9 0.68 1982 120 Donor # 3 2479 6 8.00 100 3564 1.44 4 0.49 1982 2479 6 20 1874 0.76 782 0.32 1982 2479 6 4 1779 0.72 634 0.26 1982 2479 6 0.8 2274 0.92 937 0.38 PBMC- 1578 103 016 1148 9 13.75 0.1 1255 1.09 6 0.90 1578 1148 9 0.05 1284 1.12 871 0.76 1578 1148 9 0.01 1446 1.26 952 0.83 YES PBMC Average 5.09 1.06 1.03 Index (1): = (PBMC + CHO-CD40L) / PBMC Index (2): = (PBMC + Abs) / PBMC Table 7B. Proliferation of PBMC from normal human subjects in response to mAb CHIR-12. 2 Exp. # PBMC + CHO-CD40L Copc. PBMC + hulgG1 PBMC + CH1R-12.12 Abs PBMC CPM index (1) (μg / ml) CPM index (2) CPM index (2) PBMC-025 Donor # 1 4051 42292 10.44 0.1 2909 0.72 2451 0.61 4051 42292 0.01 4725 1.17 8924 2.20 4051 42292 0.001 8080 1.99 8782 2.17 4051 42292 0.0001 4351 1.07 4342 1.07 Donor # 2 2260 14987 6.63 0.1 2538 1.12 6741 2.98 2260 14987 0.01 3524 1.56 8921 3.95 2260 14987 0.001 3159 1.40 4484 1.98 2260 14987 0.0001 2801 1.24 2533 1.12 PBMC-026 Donor # 1 2085 18313 8.78 0.1 1386 0.66 2761 1.32 2085 18313 0.01 2871 1.38 3162 1.52 2085 18313 0.001 2602 1.25 3233 1.55 2085 18313 0.0001 1709 0.82 1766 0.85 Donor # 2 676 18054 26.71 0.1 660 0.98 2229 3.30 676 18054 0.01 2864 4.24 1238 1.83 676 18054 0.001 693 1.03 1507 2.23 676 18054 0.0001 984 1.46 811 1.20 PBMC-027 Donor # 1 2742 13028 4.75 0.1 4725 1.72 2795 1.02 2742 13028 0.01 4575 1.67 5353 1.95 2742 13028 0.001 3218 1.17 3501 1.28 2742 13028 0.0001 5107 1.86 4272 1.56 Donor # 2 1338 11901 8.89 0.1 1633 1.22 1943 1.45 1338 11901 0.01 1520 1.14 5132 3.84 1338 11901 0.001 1517 1.13 2067 1.54 1338 11901 0.0001 1047 0.78 2076 1.55 PBMC-028 Donor # 1 4288 22547 5.26 0.1 3686 0.86 2525 0.59 4288 25547 0.01 3113 0.73 2047 0.48 4288 22547 0.001 4414 1.03 3515 0.82 4288 22547 0.0001 2452 0.57 4189 0.98 Donor # 2 2148 54894 25.56 0.1 9127 4.25 5574 2.59 2148 54894 0.01 4566 2.13 6515 3.03 2148 54894 0.001 5285 2.46 5919 2.76 2148 54894 0.0001 4667 2.17 4298 2.00 PBMC-029 Donor # 1 609 10054 16.51 0.1 359 0.59 363 0.60 609 10054 0.01 473 0.78 956 1.57 609 10054 0.001 461 0.76 1159 1.90 609 10054 0.0001 625 1.03 558 0.92 Donor # 2 7737 23132 2.99 0.1 4940 0.64 3493 0.45 7737 23132 0.01 6041 0.78 3644 0.47 7737 23132 0.001 5098 0.66 5232 0.68 7737 23132 0.0001 5135 0.66 5241 0.68 PB C-030 Dopper # 1 4164 57205 13.74 10 2713 0.65 1046 0.25 4164 57205 1 3627 0.87 1576 0.38 4164 57205 0.1 4590 1.10 1512 0.36 4164 57205 '0.01 4384 1.05 2711 0.65 Donor # 2 3324 53865 16.20 10 6376 1.92 4731 1.42 3324 53865 1 4720 1.42 5219 1.57 3324 53865 0.1 3880 1.17 5869 1.77 3324 53865 0.01 3863 1.16 5657 1.70 PBMC-032 Donor # 1 1808 15271 8.45 10 2349 1.30 4790 2.65 1808 15271 1 3820 2.11 5203 2.88 1808 15271 0.1 2098 1.16 6332 3.50 1808 15271 0.01 1789 0.99 5005 2.77 YES Average of PBMC 11.92 1.30 1.62 lndlce (1): = CPM of (PBMC + CHO-CD40L) / CPM of PBMC Index (2): = CPM of (PBMC + Abs) / CPM of PBMC of the culture wells (n = 2). The mean SI values with the CD40 ligand in the stimulation of CHIR-12.12 and CHIR-5.9 were 22, 0.67 and 1.2, respectively (Table 8). These data taken together show that the candidate anti-CD40 antibodies do not possess strong, stimulating properties of B cells.

Table 8: Proliferation of PBMC from human subjects, normal in response to immobilized anti-CD40 antibodies Conc. PBMC PBMC + CHO-CD40L from Abs PBMC + hulgG1 PBMC + CHIR-5.9 PBMC + CHIR-12.12 Exp. # CPM CPM Sl (1) (ug / ml) CPM SI (2) CPM YES (2) CPM YES (2) PB C-012 225 6808 30.26 10 279 1.24 734 3.26 200 0.89 225 6808 1 175 0.78 178 0.79 161 0.72 225 6808 0.1 156 0.69 226 1.00 249 1.11 225 6808 0.01 293 1.30 232 1.03 254 1.13 Fixed assets- 857 11701 13.65 1000 479 0.56 1428 1.67 384 0.45 004 857 11701 100 543 0.63 839 0.98 265 0.31 857 11701 10 487 0.57 411 0.48 262 0.31 857 11701 1 632 0.74 372 0.43 376 0.44 Estir nulation index 1 'remedy 21.96 0.81 1.21 0.67 index of E. (1): = CPM (PBMC + CHO-CD40L) / CPM (PBMC) index of E. (2): = CPM (PBMC + mAbs) / CPM (PBMC) Example 8: Antibodies CHIR-5.9 and CHIR-12.12 are Able to Remove Target Cells carrying the CD40 Antigen by means of ADCC Candidate antibodies can eliminate cells target carrying the CD40 antigen (lymphoma lines) by means of the ADCC mechanism. Both antibodies CHIR-5.9 and CHIR-12.12 are fully human antibodies of the IgGI isotype and are expected to have the ability to induce the elimination of the target cells by means of the ADCC mechanism. They were tested for their ability to eliminate cancer cell lines in in vitro trials. Two human lymphoma cell lines (Ramos and Daudi) were selected as target cells for these assays. PBMC or NK-enriched cells from 8 normal, volunteer donors were used as effector cells in these assays. The most potent ADCC response was observed with CHIR-12.12 compared to CHIR-5.9 against both target cells of the lymphoma cancer cell line. The lymphoma cell lines also express the antigen CD20, the target antigen for rituximab (Rituxan ™ *), which allowed comparison of the ADCC activity of these two candidate mAbs with the ADCC activity of rituximab. For the purpose of the lymphoma cell line, a specific lysis was observed, average of 35%, 59% and 47% for CHIR-5.9, CHIR-12.12 and rituximab respectively when used respectively at a concentration of 1 μg / ml ( Table 9). The two antibodies did not show much activity in the complement-dependent cytotoxicity assays (CDC).

Example 9. CD40 Antigen is Present on the Surface of NHL Cells of Patients with Lymph Node Biopsy NHL cells were isolated from biopsies of lymph nodes of patients and kept in liquid nitrogen until use. The viability of the cells at the time of the analysis exceeded 90%. The cells of two patients sensitive to rituximab and three patients resistant to rituximab (five patients in total) were stained with a direct labeling of either 15B8-FITC or 15B8 plus anti-huIgG2-FITC and analyzed by means of flow cytometry . It was discovered that the NHL cells of all patients express the CD40 antigen.

Table 10 shows that an average of 76% of NHL cells express the CD40 antigen (a range of 60-91%). Table 10 % Positive average to patients with NHL treated with anti-CD20 mAb b Response of patients to anti-CD20 mAb; CR = complete responder; NR = non-responder c% of cells at the entrance of lymphocytes that stained positively MS81, anti-CD40 mAb agonist '75S8, anti-CD40 antagonist mAb' n.d. it was not done Example 10: Antibodies CHIR-5.9 and CHIR-12.12 Do Not Stimulate Proliferation of Cancer Cells from Lymphatic Nodules of Patients with NHL It is known that the CD40 ligand provides a stimulatory signal for the survival and proliferation of lymphoma cells of patients with NHL. The binding of some anti-CD40 (agonist) antibodies can provide a stimulatory signal, similar to the proliferation of cancer cells in patients. Antibodies with strong B-cell stimulatory activity are not suitable candidates for the therapeutic treatment of B-cell lymphomas. The two candidate antibodies were tested for their ability to induce the proliferation of NHL cells from 3 patients. Cells isolated from lymph node biopsies (LN) were cultured with varying concentrations of candidate antibodies (range 0.01 to 300 μg / ml) for a total of 3 days. Cell proliferation was measured by the incorporation of tritiated thymidine. None of the two candidate mAbs induced any proliferation of cancer cells at any concentration tested (Table 11). Antibodies even in the presence of exogenously added IL-4, a B cell growth factor, did not induce the proliferation of NHL cells (tested in one of the three patients). These results indicate that CHIR-5 antibodies. and CHIR-12.12 are not anti-CD40 agonist antibodies and do not stimulate the in vitro proliferation of NHL cells from patients. Table 11. Proliferation of LN cancer cells from patients with NHL in response to candidate anti-CD40 mAbs Example 11. Antibodies CHIR-5. 9 and CHIR-12. 12 Can Block Proliferation of Cancer Cells Mediated by CD40 Ligands from Patients with Non-Hodgkin's Lymphoma The coupling of the CD40 antigen by the CD40 ligand induces the proliferation of cancer cells from patients with NHL. It is expected that an anti-CD40 antagonist antibody inhibits this proliferation. The two candidate anti-CD40 antibodies were tested at varying concentrations (0.01 μg / ml at 100 μg / ml) for their ability to inhibit the proliferation of NHL cells induced by CD40 ligands of the patients. NHL cells from the patients were cultured in suspension on a feeder expressing CD40L in the presence of IL-4. The proliferation of NHL cells was measured by the incorporation of 3H-thymidine. It was found that both antibodies were very effective in inhibiting the proliferation of NHL cells induced by CD40 ligands.

(Table 12, n = 2). The almost complete inhibition of proliferation induced by CD40 ligands could be achieved at an antibody concentration of 1.0 to 10 μg / ml.

Table 12. Inhibition of the proliferation of cancer cells induced by CD40 ligands of the LN of patients with NHL% inhibition: 100- (CPM with Abs of test / CPM with control mAb) * 100% Example 12: Effect of CHIR-5.9 Antibody on a Variety of Viable NHL Cells when Cultivated with Cells Carrying CD40 Ligand The effects of CHIR-5.9 antibody on numbers of viable NHL cells when cultured with cells bearing ligands of CD40 for an extended period of time (day 7, 10 and 14) were investigated. The CD40 ligand-mediated signaling through the CD40 antigen is important for the survival of B cells. This set of experiments evaluates the effect of the anti-CD40 antibodies on the numbers of NHL cells on day 7, 10 and 14. NHL cells from five patients were cultured in suspension on irradiated feeder cells expressing CD40L in the presence of IL-4. Control human IgG and CHIR-5.9 antibodies were added at concentrations of 10 g / ml on day 0 and day 7. Viable cells under each condition were counted on the specific day. The numbers of cells in the control group (IgG) increased with time as expected. Reduced numbers of cells were recovered from the cultures treated with CHIR-5.9 compared to the control group. The largest levels of reduction in cell numbers by the CHIR-5.9 antibody were observed on day 14 and were on average 80.5% (a range of 49-94%) compared to the isotype control (n = 5) . These data are summarized in Table 13.

Table 13. Effect of anti-CD40 antibody (CHIR-5.9 / 5.11) on the cell numbers of patients with NHL during a prolonged culture period (day 7, 10 and 14)% reduction compared to control Abs = 100 - (Test Abs / Control Abs) * 100 Example 13. Antibodies CHIR-5.9 and CHIR-12.12 are Able to Remove Cancer Cells from Lymphatic Nodules of Patients with NHL by means of ADCC Both antibodies CHIR-5.9 and CHIR-12.12 are completely human antibodies of isotype I G_ and were shown that induce the elimination of lymphoma cell lines in vitro by means of the ADCC mechanism (Table 9). They were tested for their ability to kill cancer cells from an individual patient with NHL in in vitro assays. NK cells enriched from voluntary, normal donors either fresh after isolation or after overnight culture at 37 ° C were used as effector cells in this assay. Similar results were obtained with both recently isolated NK cells and NK cells used after overnight culture. The highest level of ADCC was observed with the CHIR-12.12 antibody compared to the CHIR-5.9 antibody against the patient's HL cells. The NHL cells also express the antigen CD20, the target antigen for rituximab (Rituxan ™ *), which allowed the comparison of the ADCC activity of these two candidate mAbs with rituximab. The CHIR-12.12 antibody and rituximab show a similar level of ADCC activity with the lowest CHIR-5.9 record in this assay. These data are shown in Figures 3A and 3B.

Example 14. Antibodies CHIR-5.9 and CHIR-12.12 can Block the Survival and Proliferation Mediated by CD40 of Cancer Cells of Patients with CLL. Candidate antibodies can block the survival and proliferation mediated by CD40 of cancer cells of patients with CLL. The patients' CLL cells were cultured in suspension on CHO cells fixed with formaldehyde expressing CD40L under two different conditions: the addition of human isotype antibody IgG (control); and the addition of a monoclonal antibody to either CHIR-5.9 or CHIR-12.12. All antibodies were added at concentrations of 1, 10 and 100 // g / mL in the absence of IL-4. Cell counts were performed in 24 and 48 hours by means of the MTS assay. Reduced numbers of cells were recovered from the cultures treated with CHIR-5.9 (n = 6) and CHIR-12.12 (n = 2) compared to the control group. The largest differences in cell numbers between the cultures treated with anti-CD40 mAb and the cultures treated with control antibodies were observed at the time point of 48 hours. These data are summarized in Table 14.

Table 14. The effect of the candidate antibodies on the survival and proliferation induced by the CD40 antigen of cancer cells of patients with CLL measured at 48 hours after the start of the culture. *% reduction compared to control Abs = 100- (Test Abs / Control Abs) * 100 Example 15: Antibodies CHIR-5.9 and CHIR-12.12 Show Antitumor Activity in Animal Models Pharmacology / efficacy in vivo Candidate mAbs are expected to produce the desired pharmacological effects to reduce tumor burden either by / both by the two antitumor mechanisms , blockade of proliferation / survival signal and induction of ADCC. Currently available human lymphoma xenograft models utilize long-term lymphoma cell lines that, in contrast to primary cancer cells, do not depend on stimulation of the CD40 antigen for growth and survival. Therefore, the component of the antitumor activity of these mAbs based on the blocking of tumor proliferation / survival signal is not expected to contribute to antitumor efficacy in these models. The effectiveness in these models is dependent on the ADCC, the second antitumor mechanism associated with mAbs CHIR-5.9 and CHIR-12.12. Two models of human xenograft lymphoma based on the Namalwa and Daudi cell lines were evaluated by the antitumor activities of the candidate mAbs. To further demonstrate their therapeutic activity, these candidate mAbs were evaluated in a human lymphoma model of xenograft classified by stages and not classified by stages that is based on the Daudi cell line. Brief description of in vivo efficacy data When administered by the intraperitoneal route (ip) once a week for a total of 3 doses, the CHIR-12.12 antibody, one of the two candidate mAbs, significantly inhibited the growth of non-stage B-cell lymphoma, aggressive (Namalwa) in a dependent manner of the dose (Figure 4). The second candidate mAb, CHIR-5.9, was tested only in a single dose in this study and was less effective than the CHIR-12.12 antibody at the same dose. Interestingly, it was found that the CHIR-12.12 antibody was more effective in this model than rituximab. It is possible that the lower efficacy of rituximab could be due to the low expression of CD20 in the Namalwa lymphoma cell line. The efficacy observed with the candidate mAbs is of greater importance because only one of the two mechanisms of cancer cell elimination (ADCC) is operative in the current xenograft lymphoma model. It is expected that two mechanisms of elimination, ADCC and blocking of the survival signal contribute to antitumor activities in patients with human lymphoma. This is likely to increase the 'change of efficacy achievement in patients with human lymphoma. The candidate anti-CD40 mAbs also showed a tendency towards inhibition of tumor growth in a second B-cell lymphoma model (Daudi model not validated, data not shown). In the following studies, it was shown that the two candidate antibodies have a dose-dependent antitumor efficacy in the Daudi lymphoma models both non-staged and staged (Figures 5 and 6, respectively). In the Daudi model staged, mAb CHIR-12.12 was more effective in reducing tumor volume than a similar dose of Rituxan ™ *. Models of Human B-cell Lymphoma of xenograft To ensure consistent growth of tumors, nude mice deficient in T cells were irradiated throughout the body at 3 Gy to further suppress the immune system one day before tumor inoculation. The tumor cells were inoculated subcutaneously in the right flank at 5 x 106 cells per mouse. Treatment was started either one day after tumor implantation (human B-cell lymphoma models of xenograft, subcutaneous, non-graded, Namalwa and Daudi) or when the tumor volume reached 200-400 mm3 (Daudi model sorted in stages, usually 15 days after tumor inoculation). Mice carrying the tumor were injected intraperitoneally (i.p.) with the anti-CD40 mAbs once a week at the indicated doses. Tumor volumes were recorded twice a week. When the volume of the tumor in any group reached 2500 mm3, the study was completed. It should be noted that in the Daudi model classified by stages, the tumor volume data were analyzed until day 36 due to the death of some mice after that day. Complete regression (CR) was counted until the end of the study. The data were analyzed using the ANOVA or Kruskal-Walli test and a subsequent test, corresponding to the comparison of multiple groups. In the Namalwa staged model, the anti-CD40 mAb CHIR-12.12, but not the Rituxan ™ * (rituximab), significantly inhibited (p = < 0.01) the growth of Namalwa tumors (tumor volume reduction of 60 % against 25% for rituxumab, n = 10 mice / group) (Figure 4). Thus, in this model, the anti-CD40 mAb CHIR-12.12 was more potent than rituximab. It is noteworthy that the second candidate mAb, CHIR-5.9, was at least as effective as rituximab at a dose of 1/10 than rituximab. Both anti-CD40 mAbs CHIR-12.12 and rituxan significantly prevented the development of tumors in the non-staged Daudi tumor model (14/15 resistance to tumor testing) (Figure 5). When these anti-CD40 monoclonal antibodies were further compared in the Daudi model of stage-graded xenograft, in which the treatment started when the subcutaneous tumor was palpable, the anti-CD40 mAb CHIR-12.12 at 1 mg / kg caused a significant reduction of the tumor (p = 0.003) with 60% complete regression (6/10), while rituximab at the same dose did not significantly inhibit tumor growth or cause complete regression (0/10). See Figure 6. In summary, the anti-CD40 mAb CHIR-12.12 significantly inhibited tumor growth in experimental lymphoma models. At the same dose and the same regimen, mAb CHIR-12.12 showed better anti-cancer activity than Rituxan ™ * (rituximab). In addition, no clinical signs of toxicity were observed at this dose and this regimen. These data suggest that the anti-CD40 mAb CHIR-12.12 has a potent anti-human lymphoma activity in vitro and in xenograft models and could be clinically effective for the treatment of lymphoma. Example 16: Pharmacokinetics of Antibodies CHIR-5.9 and CHIR-12.12 The pharmacokinetics of anti-CD40 mAb in mice was studied after IV and IP administration of individual doses. The anti-CD40 mAb exhibited a high systemic bioavailability after IP administration and a terminal, prolonged average lifespan (>;5 days) (data not revealed) . This pilot study was conducted to assist in the design of pharmacology studies; however, it is of little or no importance for the development activity of this Ab since this fully human mAb does not cross-react with the mouse CD40 antigen. Example 17: Characterization of the Epitope for the Monoclonal Antibodies CHIR-12.12 and CHIR-5.9 To determine the location of the epitope on the CD40 antigen that is recognized by the monoclonal antibodies CHIR-12.12 and CHIR-5.9, the SDS-PAGE analysis was carried out. Western immunoblotting. The purified CD40 antigen (0.5 μg) was separated on a 4-12% NUPAGE gel under reducing and non-reducing conditions, transferred to PVDF membranes and probed with monoclonal antibodies at a concentration of 10 μg / ml. The spots were probed with anti-human IgG conjugated with alkaline phosphatase and were developed using the substrate stabilized with Western BlueTM for alkaline phosphatase (Promega). The results indicate that the anti-CD40 monoclonal antibody CHIR-12.12 recognizes epitopes in both reduced and unreduced forms of the CD40 antigen, with the non-reduced form of the CD40 antigen exhibiting greater intensity than the reduced form of the CD40 antigen (Table 15; spots not shown). The fact that the recognition was positive for both forms of the CD40 antigen indicates that this antibody interacts with a part of the conformational epitope of which is a linear sequence. The monoclonal antibody CHIR-5.9 mainly recognizes the non-reduced form of the CD40 antigen, which suggests that this antibody interacts with a primary conformational epitope (Table 15-, spots not shown). Table 15. Domain identification To analyze the antigenic region in the CD40 antigen, the four extracellular domains of the CD40 antigen were cloned and expressed in insect cells as GST fusion proteins. The secretion of the four domains was ensured with a GP67 secretion signal. The supernatant of insect cells was analyzed by SDS-PAGE analysis and Western immunoblotting to identify the domain containing the epitope. The monoclonal antibody CHIR-12.12 recognizes an epitope in domain 2 under both reducing and non-reducing conditions (Table 16, spots not shown). In contrast, the monoclonal antibody CHIR-5.9 exhibits a very weak recognition for domain 2 (Table 16, spots not shown). None of these antibodies recognizes domains 1, 3 or 4 in this analysis. Table 16. Analysis of domain 2 To more precisely define the epitope recognized by the mAb CHIR-12.12, the peptides were synthesized from the extracellular domain 2 of the CD40 antigen, which corresponds to the sequence PCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICT (residues 61-104 of the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12). The SPOTsMR (Sigma) membranes containing thirty-five 10-er peptides were generated with a deviation of 1 amino acid. Western blot analysis was performed with the CHIR-12.12 mAb and the anti-human IgG beta-galactocidase as the secondary antibody. The spot was removed and probed again with mAb CHIR-5.9 to determine the region recognized by this antibody. SPOTsMR analyzes probing with the anti-CD40 monoclonal antibody CHIR-12.12 at 10 μg / ml yielded positive reactions with spots 18 to 22. The sequence region covered by these peptides is shown in Table 17. Table 17. Analysis results SPOTsM probing with the anti-CD40 monoclonal antibody CHIR-12.12 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 interaction of the CD40 antigen-CD40 ligand. SPOTsMR analysis with mAb CHIR-5.9 showed weak recognition of the peptides represented by spots 20-22 shown in Table 18, suggesting an involvement of the YCDPNLGL region (residues 82-89 of the sequence shown in SEC ID NO: 10 or SEQ ID NO: 12) in its binding to the CD40 antigen. It should be noted that mAbs CHIR-12.12 and CHIR-5.9 compete with each other for binding to the CD40 antigen in the BIACORE assay. Table 18. SPOTsMR analysis results probing with the anti-CD40 monoclonal antibody CHIR-5.9 The linear epitopes identified by the SPOTsMR analyzes are within the CD40 Bl module. The sequence of the CD40 Bl module is: HKYCDPNLGLRVQQKGTSETDTIC (residues 80-103 of SEQ ID NO: 10 or SEQ ID NO: 12). Within the linear epitope identified by the CHIR-12.12 antibody is C83. It is known that this cysteine residue forms a disulfide bond with C103. It is likely that the conformational epitope of mAb CHIR-12.12 contains this disulfide bond (C83-C103) and / or surrounding amino acids that conforming C103 conformationally. Example 18. Number of CD20 and CD40 Molecules in Namalwa and Daudi Cells The number of CD20 and CD40 molecules in Namalwa and Daudi cells was determined as summarized in Figure 7, using antibody concentrations of 0.01, 0.1, 1, 10 and 100 μg / ml. As can be seen in Figure 7, the average number of CD20 molecules (target for rituximab) is higher in both Namalwa and Daudi cell lines than the number of CD40 molecules (target for mAb CHIR-12.12). Example 19: ADCC of CHIR-12.12 mAb and Rituximab Against Daudi Lymphoma Cells Rituximab y. the candidate mAb CHIR-12.12 were tested in vi tro by ADCC activity at varying concentrations against the Daudi lymphoma cell line as target (T) cells and purified NK cells from healthy human volunteers as effector cells (E). Freshly isolated human NK cells were mixed with calcein-labeled Daudi lymphoma cells at an E: T ratio of 10. The cell mixture was incubated for 4 hours at 37 ° C in the presence of established concentrations of either the mAb CHIR-12.12 or rituximab. The level of calcein released from the target cells lysed in the supernatant was measured as Arbitrary Fluorescence Units (AFU). The percentage of specific lysis was calculated as: 100 x (AFU test - AFU spontaneous release) / (maximum release of AFU - spontaneous release of AFU), where the spontaneous release of AFU is the calcein released by target cells in the absence of antibody or NK cells and the maximum release of AFU is the calcein released by target cells with lysis by the detergent. The lysis of the Daudi cells was observed depending on the concentration of the antibody (Figure 8, Table 19 below). The specific, maximal lysis of the target lymphoma cells induced by the anti-CD40 mAb was higher compared to the lysis induced by rituximab (63.6% vs. 45.9%, n = 6, paired t test of the CHIR-12.12 mAb against rituximab, p = 0.0002). In addition, the ED50 value for rituximab was on average (n = 6) 51.6 pM, 13 times higher than the ED50 value for the anti-CD40 mAb CHIR-12.12 for this activity.

Table 19. Comparative ADCC of the CHIR-12.12 mAb and rituximab against Daudi lymphoma cells.

Example 20: CHIR-12.12 Antibody Blocks CD40L-Mediated Surveillance and Signaling of CD40L Antigen in Human, Normal B Cells The soluble CD40 ligand (CD40L) activates B cells and induces various aspects of functional responses, including increased survival and proliferation, and activation of signaling pathways NFKB, ERK / MAPK, Pl3K / Akt and 938. In addition, stimulation of the CD40L-mediated CD40L antigen provides survival signals by reducing cleaved PARP and inducing the anti-apoptotic proteins, XIAP and Mcl-1, in normal B cells. CD40L-mediated stimulation of the CD40L antigen also recruits TRAF2 and TRAF3 to bind to the cytoplasmic domain of the CD40 antigen. The following studies demonstrate that the CHIR-12.12 antibody directly inhibited all of these stimulation effects in normal human B cells. For example, treatment with the CHIR-12.12 antibody resulted in an increased cleavage of caspase-9, caspase-3 and PARP as well as a reduction of XIAP and Mcl-1 in a time and dose dependent manner., restoring B cell apoptosis. Treatment with CHIR-12.12 antibody also inhibited phosphorylation of kinase and I? B (IKK) and? (via NFKB), ERK, Akt and p38 in response to stimulation of CD40 antigen mediated by CD40L. Furthermore, it was discovered that the CHIR-12.12 antibody did not trigger these apoptotic effects without an initial stimulation of the CD40 antigen mediated by CD40L. Survival inhibited by the CHIR-12.12 antibody mediated by the CD40 ligand by inducing PARP excision 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 of SCD40L (Alexis Corp., Bingham, No tinghamshire, UK). The CHIR-12.12 antibody (10 μg / ml) and the control IgG were then added. Cells were harvested at 0, 20 minutes, 2 hours, 6 hours, 18 hours and 26 hours. The controls for cleaved caspase-9, cleaved caspase-3, cleaved PARP and β-actin were detected in the cell lysates by means of Western blotting. In summary, it was observed that CD40L-mediated stimulation of the CD40L antigen provided survival signals since it did not result in increases of cleaved caspase-9, cleaved caspase-3 or cleaved PARP over time, indicating that the cells were not they underwent apoptosis. However, treatment with the CHIR-12 .12 antibody resulted in an increase in these cleavage products, indicating that treatment with the CHIR-12 .12 antibody abrogated the effects of CD40L binding on survival signaling in normal B cells stimulated with SCD40L, restoring B cell apoptosis (data shown). The CHIR-12 antibody. 12 inhibited the expression of anti-apoptotic proteins of "survival" In these experiments, 0. 6 x 10s normal human B cells from healthy donors (purity percentage between 85-95%) were stimulated with 1 μg / ml of SCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). The CHIR-12 .12 antibody (10 μg / ml) and the control IgG were then added. Cells were harvested at 0, 20 minutes, 2 hours, 6 hours, 18 hours and 26 hours. The controls of Mcl-1, XIAP, CD40 and β-actin were detected in cell lysates by Western immunoblotting.

In summary, stimulation with sCD40L resulted in sustained expression of Mcl-1 and XIAP over time. However, the treatment of cells stimulated with sCD40L with CHIR-12. 12 resulted in a decrease in the expression of those proteins over time (data not shown). Since Mcl-1 and XIAP are "survival" signals capable of blocking the apoptotic pathway, these results show that treatment with the CHIR-12 antibody. 12 removes the blocking against apoptosis in normal B cells stimulated with SCD40L. Treatment with the CHIR-12.12 antibody inhibited the phosphorylation of IKK (Ser 180) and IKK β (Ser 181) in normal B cells. In these experiments, 1.0 x 10 6 human, normal healthy donor cells (percent purity between 85-95%) were stimulated with 1 μg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). The CHIR-12.12 antibody (10 μg / mL) and the control IgG were then added. The cells were harvested at 0, and 20 minutes. The IKKa (Ser 180) and the IKK β (Ser 181) phosphorylated and the total IKK β controls were detected in cell lysates by means of the Western Blot assay. In summary, stimulation by means of sCD40L resulted in the phosphorylation of IKKa (Ser 180) and IKK β (Ser 181) over time; However, treatment with the CHIR-12 antibody. 12 canceled this response to stimulation with sCD40L in normal B cells (data not shown). Treatment with the CHIR-12.12 antibody inhibited survival mediated by the CD40 ligand in a dose-dependent manner. In these experiments, 0.6 x 10 6 normal, healthy human donor B cells (percent purity between 85-95%) stimulated with 1 μg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). The CHIR-12.12 antibody (0.01, 0.1, 0.2, 0.5, 1.0 μg / ml) and the control IgG were then added. The cells were harvested in 24 hours. The cleaved PARP and β-actin controls were detected in cell lysates by means of Western immunoblotting. In summary, treatment with the CHIR-12.12 antibody resulted in increased cleavage of PARP in cells stimulated with sCD40L in a dose-dependent manner and thus nullified the survival signaling pathway in normal B cells stimulated with sCD40L (data not shown). The CHIR-12.12 antibody inhibited the expression of "survival" anti-apoptotic proteins in a dose-dependent manner In these experiments, 0.6 x 10e human B cells, normal from healthy donors (purity percentage between 85-95% ) were stimulated with 1 μg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). The CHIR-12.12 antibody (0.5, 2 and 10 μg / ml) and the control IgG were then added. The cells were harvested in 22 hours. The controls of Mcl-1, XIAP, cleaved PARP and β-actin were detected in cell lysates by means of Western immunoblotting. In summary, the treatment with the CHIR-12.12 antibody reduced the expression of Mcl-1 and XIAP and the increased expression of cleaved PARP in cells stimulated with the sCD40L in a dose-dependent manner and thus nullified these blockages to the pathway apoptotic in normal B cells stimulated with sCD40L (data not shown). The CHIR-12.12 antibody did not affect the expression of anti-apoptotic proteins, cleaved PARP and XIAP, in the absence of soluble CD40L signaling. In these experiments, 1.0 x 10 6 normal human B cells from healthy donors (percentage of purity between 85-95%) were treated with the CHIR-12.12 antibody (10 μg / ml) and the control IgG only (i.e., the cells were not previously stimulated with the sCD40L before the addition of the antibody). The cells were harvested at 0, 4, 14 and 16 hours. The controls of XIAP, cleaved PARP and β-actin were detected in cell lysates by Western immunoblotting. In summary, the results show that without the stimulation with the SCD40L, the cells expressed increased concentrations of cleaved PARP, while the expression of XIAP remained constant, both in control cells treated with IgG and in cells treated with the CHIR-12.12 antibody (data not shown). These data indicate that the CHIR-12.12 antibody did not trigger apoptosis in human, normal B cells without stimulation with CD40L. The CHIR-12.12 antibody inhibited the phosphorylation of IKK (Ser 180) and IKKβ (Ser 181), Akt, ERKf and p38 in normal B cells. In these experiments, 1.0 x 10s human B cells, normal from healthy donors (percent purity between 85-95%) were collected from the serum in media containing 1% FBS and stimulated with 1 μg / ml SCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). The cultures were treated with the CHIR-12.12 antibody (1 and 10 μg / ml) and the control IgG. Cells were harvested at 0 and 20 minutes. Phospho-IKKa, phospho-IKKβ, total IKKβ, phospho-ERK, total ERK, phospho-Akt, total Akt, phospho-p38 and total p38 were detected in cell lysates by Western immunoblotting. Briefly, stimulation with sCD40L resulted in increases in IKK / β phosphorylation, ERK phosphorylation, Akt phosphorylation and p38 phosphorylation, thus leading to survival and / or proliferating cells. Treatment of the cells with the CHIR-12.12 antibody abrogated the effects of stimulation with SCD40L on these signaling pathways in normal B cells (data not shown). The CHIR-12.12 antibody inhibits multiple signaling pathways such as P13K and MEK / ERK in the CD40 antigen signaling cascade. In these experiments, 1.0 x 10 6 human B cells, normal from healthy donors (purity percentage between 85-95% ) were collected from serum in media containing 1% FBS and stimulated with 1 μg / ml sCD40L (Alexis Corp. Bingham, Nottinghamshire, UK). The cultures were also treated with the CHIR-12.12 antibody (1 and 10 μg / ml), Wortmanin (a PI3K / Akt inhibitor).; 1 and 10 μM), LY 294002 (an inhibitor of PI3K / Akt; 10 and 30 μM) PD 98095 (an inhibitor of MEK, 10 and 30 μg / ml). Cells were harvested at 0 and 20 minutes. Phospho-ERK, phospho-Akt, total Akt, phospho-IKKa / β and total were detected in cell lysates by means of Western immunoblotting. In summary, the results show that the CHIR-12.12 antibody abolished the phosphorylation of all these signal transduction molecules, whereas the signal transduction inhibitors showed only a specific signal cancellation, indicating that the CHIR-12.12 antibody probably inhibits upstream these signal transduction molecules mediated by stimulation with CD40L (data not shown). The CHIR-12 antibody. 12 inhibits the binding of the signaling molecules TRAF2 and TRAF3 to the toplasmic cy domain of the CD40 antigen in normal B cells. In these experiments, 4.0 x 10 6 human B cells, normal from healthy donors (purity percentage between 85-95%) were collected. of serum for four hours in media containing 1% FBS and stimulated with 1 μg / ml sCD40L (Alexis Corp. Bingham, Nottinghamshire, UK) for 20 minutes. Cells were harvested at 0 and 20 minutes. The CD40 antigen was immunoprecipitated using a polyclonal anti-CD40 antibody (Santa Cruz Biotechnology, CA) and probed on a Western blot with an anti-TRAF2 mAb (Santa Cruz Biotechnology, CA), an anti-TRAF3 mAb (Santa Cruz Biotechnology, CA) and an anti-CD40 mAb (Santa Cruz Biotechnology, CA). In summary, the results show that the TRAF2 and TRAF3 molecules were co-precipitated with the CD40 antigen after stimulation with SCD40L. In contrast, treatment with the CHIR-12.12 antibody abolished the formation of the CD40-TRAF2 / 3 signaling complex in normal B cells stimulated with sCD40L. There were no changes in the expression of the CD40 antigen (data not shown). Without being limited by theory, the results of these experiments and the results in the examples summarized above indicate that the CHIR-12.12 antibody is a monoclonal antibody, anti-CD40, double-acting antagonist having a unique combination of attributes. This fully human monoclonal antibody blocks the CD40L antigen signaling pathways mediated by CD40L for the survival and proliferation of B cells; this antagonism leads to the death of fundamental cells. The CHIR-12.12 antibody also mediates recognition and binding by effector cells, initiating antibody-dependent cellular cytotoxicity (ADCC). Once CHIR-12.12 antibody binds effector cells, cytolytic enzymes are released leading to apoptosis and B-cell lysis. CHIR-12.12 antibody is a more potent antitumor antibody than rituximab when compared in tumor models pre-clinical Example 21. Agonist and Antagonist Activity Against Primary Cancer Cells of Patients with NHL, CLL and NM In collaboration with clinical investigators, candidate mAbs are tested for a variety of activities (listed below) against cells of primary cancers of patients with NHL and CLL and multiple myeloma. • Agonist effect in proliferation assays (8 patients with NHL, 8 patients with CLL and 8 patients with MM) • Antagonistic effect in proliferation assays (8 patients with NHL, 8 patients with CLL and 8 patients with MM) • Apoptotic effect by means of the Annexin V assay (3 -4 patients with NHL, 4 patients with CLL and 4 patients with MM) • Reversion of the survival signal through the Annexin V assay (3 patients with NHL, 3 patients with CLL and 3 patients with MM) • Cytotoxicity dependent on supplements (4 patients with NHL, 4 patients with CLL and 4 patients with MM) • Antibody-dependent cytotoxicity (6 patients with NHL, 6 patients with CLL and 6 patients with MM) Example 22: Identification of Relevant Animal Species for Toxicity Studies How these two candidate antibodies do not cross-react with the CD40 antigen of rodent, other species must be identified to test the toxicological effects. The ability of the two candidate anti-CD40 antibodies to cross-react with the animal CD40 antigen is tested by means of flow cytometry assays. Rats, rabbits, dogs, cynomolgus monkeys and marmosets are tested for this study. The candidate antibodies show antagonistic activity with binding to the CD40 antigen in human B cells. To identify an animal species that has a similar response to candidate antibodies, lymphocytes from species that show binding to candidate antibodies are tested in proliferation assays for antagonist activity. Lymphocytes of the species selected for antagonistic binding of the candidate antibodies are further tested for their ability to serve as effector cells for the elimination of lymphoma cell lines expressing the CD40 antigen through the ADCC mechanism. Finally, the selected animal species are tested in an IHC study for the tissue-binding pattern of the candidate antibodies. Animal species that respond to candidate antibodies in these assays in a manner similar to that observed for human cells are selected for toxicology studies. Initial studies indicate that the candidate anti-CD40 mAbs cross-react with the cynomolgus monkey CD40 antigen. Example 23: Fixation Profile as Target in Tumors of the Antibodies CHIR-5.9 and CHIR-12.12 To determine the target binding profile in relative tumors of mAbs CHIR-12.12 and CHIR-5.9, the candidate mAbs labeled with fluorescence and the Isotypic control antibodies are administered in mice bearing tumors. Specimens of tumors and normal organs are collected at different time points after dosing. The accumulation of the labeled antibody in tumors and normal organs is analyzed. Example 24: Mechanism of Action of the Antibodies CHIR-5.9 and CHIR-12.12 To clarify the mechanism (s) mediating the inhibition of tumor growth by CHIR-5.9 and mAbs CHIR-12.12, they underwent to the following studies: Selected segment separation model or Fc receptor block: ADCC is measured by the binding of effector cells such as NK, macrophages and monocytes to the Fc portion of the antibody through the Fc receptor. Mice deficient in the activation of Fc receptors as well as antibodies designed to alter the binding of Fc to these receptors will block the inhibition of tumor growth mediated by ADCC. The loss or inhibition of tumors significantly reduced in this model will suggest that the inhibition of tumor growth by these two candidate mAbs is mediated primarily by the mechanism of ADCC. Example 25. Pharmaceutical Formulation, Liquid for Anti-CD40 Antibodies Antagonists The objective of this study was to investigate the effects of pH of the solution on the stability of anti-CD40 antagonist CHIR-12.12 by means of both biophysical and biochemical methods in order to to select the optimal environment of the solution for this antibody. The results of Differential Exploration Calorimetry (DSC), for its acronym in English) showed that the stability of conformation of the CHIR-12.12 antibody 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 CLAR (CEX-HPLC), the physicochemical stability of the antibody CHIR-12.12 is optimum at approximately pH 5.0-5.5. In view of these results, a recommended liquid pharmaceutical formulation comprising this antibody is a formulation comprising the CHIR-12.12 antibody at approximately 20 mg / ml formulated in sodium succinate at approximately 10 mM, sodium chloride at approximately 150 mM. and having a pH of about pH 5.5. Materials and Methods The CHIR-12.12 antibody used in the formulation studies is a human monoclonal antibody that is produced by means of a CHO cell culture process. This MAb has a molecular weight of 150 kDa and consists of two light chains and two heavy chains linked together by disulfide bands. It targets the CD40 cell surface receptor on cells expressing the CD40 antigen, including normal and malignant B cells for the treatment of various cancers and autoimmune / inflammatory diseases. The substance of the anti-CD40 drug used for this study was a bulk lot of purified anti-CD40 antibody that is derived from CHO (CHIR-12.12). The composition of the drug substance was 9.7 mg / ml of the CHIR-12.12 antibody in 10 mM sodium citrate, 150 mM sodium chloride at pH 6.5. The control sample in the study was the drug substance received, followed by the freezing to <-60 ° C, thawing at room temperature and testing together with stability samples at predetermined time points. The stability samples were prepared by means of dialysis of the drug substance against solutions of different pH and the concentration of the CHIR-12.12 antibody in each sample was determined by means of UV 280 as presented in Table 20.

Table 20. Formulations of CHIR-12.12 antibody The physicochemical stability of the CHIR-12.12 antibody in various formulations was tested using the following protocols. Differential Scanning Calorimetry (DSC) The conformational stability of different formulation samples was monitored using a MicroCAl VP-DSC device with heating from 15 ° C to 90 ° C at 1 ° C / minute. SDS-PAGE Fragmentation and aggregation were estimated using Tris-Glycine gel 4-20% under non-reducing and reducing conditions. The protein was detected by staining with Coomassie blue.

Size Exclusion Chromatography (SEC-HPLC) Protein fragmentation and aggregation were also measured by means of a CLAR Water Alliance ™ * with a Tosohaas TSK-GEL 3000SWXL column, 100 mM sodium phosphate, pH 7.0 as a mobile phase to a Flow rate of 0.7 ml / minute. Cationic Exchange Chromatography (CEX-HPLC) The degradation related to the charge change was measured using a Waters 600s ™ * CLAR system with a Dionex Propac WCX-10 column, 50 mM HEPEs, pH 7.3 as the mobile phase A and HEPES 50 mM containing 500 mM NaCl, pH 7.3 as mobile phase B at a flow rate of 0.5 ° C / minute. Results and Discussion Conformational stability study The thermal splitting of the CHIR-12.12 antibody revealed at least two thermal transitions, which probably represent the split fusion of the Fab and Fc domains, respectively. At higher temperatures, the protein was presumably added, resulting in the loss of DSC signal. For the purpose of examining the formulation, the lowest thermal transition temperature was defined as the melting temperature, Tm, in this study. Figure 13 shows the thermal melting temperature as a function of the pHs of the formulation. Formulations at pH 5.5-6.5 provided an anti-CD40 antibody with higher conformational stability as demonstrated by the higher thermal melting temperatures. SDS-PAGE Analysis Samples of the CHIR-12.12 antibody formulation 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 27 kDa were observed in formulations above pH 5.5 and species with MW of 51 kDa were observed in all formulations, but appeared lower at pH 5.0- 5.5 A species with a MW of 100 kDa could be observed at pH 7.5 and pH 9.0. Under reducing conditions, the CHIR-12.12 antibody was reduced in the heavy chains and free light chains with a MW of 50 kDa and 24 kDa, respectively. The 100 kDa species did not appear completely reducible and increased with the increasing pH of the solution, suggesting that a different covalent association of disulfide in the molecules could occur. Since there were other species with unknown identities in the SDS-PAGE analysis, the stability comparison of each formulation is based on the remaining purity of the CHIR-12.12 antibody. Formulations at pH 5.0-6.0 provided a more stable environment for the CHIR-12.12 antibody.

Some aggregates were detected by SDS-PAGE analysis (data not shown). SEC-HPLC analysis SEC-HPLC analyzes detected the intact CHIR-12.12 antibody as the main peak species, a species of aggregation as a species of the front peak separated from the main beak species, a species of large fragment as a Beak peak at the rear of the main beak species and a small fragment species were detected after the main beak species. After incubation at 5 ° C and 25 ° C for 3 months, insignificant amounts of fragments and protein aggregates (< 1.0%) were detected in the above formulations and the main peak species of CHIR-12.12 remained with more than 99% purity (data not shown). Nevertheless, protein fragments were gradually developed with storage at 40 ° C and more fragments were formed at pH 4.5 and pH 6.5-9.0, as shown in Table 21. After incubating the CHIR-12.12 antibody formulations at 40 ° C for 3 months, approximately 2-3% aggregates were detected at pH 7.5 and pH 9.0, while less than 1% aggregates were detected in other pH formulations (data not shown). The results of the SEC-HPLC analysis indicate that the CHIR-12.12 antibody is more stable at about pH 5. 0-6. 0 Table 21. Results of the SEC-HPLC analysis of CHIR-antibody stability samples Analysis of CEX-EPLC. CEX-HPLC analysis detected the intact CHIR-12.12 antibody as the main peak species, the acid variants eluted faster than the main peak species and the C-terminal lysine addition variants were eluted after the main peak. Table 22 shows the dependence of the percentages of the CHIR-12.12 antibody species on the remaining main peak and the acid variants on the pH of the solution. The control sample already contained a high degree of acid species (~ 33%), probably due to early stage fermentation and purification processes. The susceptibility of CHIR-12.12 antibody to higher pH solutions is evidenced by two facts. First, the initial formulation sample at pH 9.0 (t = 0) already generated 12% more acidic species than the control. Second, the percentage of acid species increased clearly with the increase in pH. The degradation related to the change of the load is probably due to deamidation. The above data indicate that this type of degradation of the CHIR-12.12 antibody was minimized to approximately pH 5.0-5.5. Table 22. Percentage of peak area by means of CEX-HPLC analysis for CHIR-12.12 antibody in formulations of different pH under accelerated real-time storage conditions Conclusion The pH has a significant effect on the conformational and physicochemical stabilities of the CHIR-12.12 antibody. The degradation related to the charge change was determined to be the main degradation pathway for the CHIR-12.12 antibody, which was minimized to pH 5.0-5.5. Based on the total stability data, a recommended liquid pharmaceutical formulation comprising this antibody is a formulation comprising the CHIR-12.12 antibody at approximately 20 mg / ml formulated in sodium succinate at approximately 10 mM, sodium chloride a about 150 mM and having a pH of about pH 5.5. Example 26: Clinical Studies with Antibodies CHIR-5.9 and CHIR-12.12 Clinical Objectives The overall objective is to provide an effective therapy for B-cell tumors by targeting them with an anti-CD40 IgGl. These tumors include B-cell lymphoma, Chronic Lymphocytic Lymphoma (CLL), Acute Lymphoblastic Leukemia (ALL), Multiple Myeloma (MM), Waldenstrom's Macroglobulinemia, and Castleman's Systemic Disease. The signal for these diseases is determined in phase II although some measurement of the activity in phase I can be obtained. Initially, the agent is studied as an individual agent, but will be combined with other agents, chemotherapeutic products and other antibodies, to As development proceeds. Phase I • Evaluate safety and pharmacokinetics - dose escalation in patients with B cell malignancies.

• Select the dose based on the safety, tolerability and change in the markers in the serum of the CD40 antigen. In general, a BAT is sought but other indicators of efficacy (depletion of CD40 + B cells, etc.) may be adequate to find the dose. • Consideration of more than one dose especially for different indications, for example, the dose for CLL may be different from the dose for NHL. In this way, some dose discovery in phase II may be necessary. • Patients are dosed weekly with a pharmacokinetic sampling in real time (Pk) Initially, a cycle of 4 weeks is the maximum dosage allowed. The Pk can be highly variable depending on the disease studied, density of CD40, etcetera. • This test (s) is open to subjects with B-cell lymphoma, CLL and potentially other B-cell malignancies. "The decision to discontinue or continue the studies is based on safety, dose and preliminary evidence of antitumor activity. • The activity of the drug determined by the speed of response is determined in phase II. • Identify the dose (s) for phase II. Phase II Several tests will be initiated on the types of tumors mentioned above with concentration in B-cell lymphoma, CLL and multiple myeloma (MM). Separate tests may be required in low-grade and intermediate / high-grade NHL since the CD40 antigen may have a different function depending on the degree of lymphoma. In low-grade disease, the CD40 antigen acts more as a survival factor, preventing apoptosis. In a higher grade disease, disruption of CD40 antigen signaling can lead to cell death. More than one dose and more than one program can be tested in a randomized Phase II setting. In each disease, target a population in which a current standard of care has failed: • CLL: patients who were resistant to Campath and chemotherapy. • Low-grade NHL: Rituxan ™ * or CHOP-R failures • Intermediate NHL: CHOP-R failures • Multiple myeloma: Failures of Chemotherapy X The decision to discontinue or continue the study is based on a proof of concept therapeutic in phase II ^ The determination if a marker is canceled can be used as the first indication of clinical efficacy X Identify the doses for phase III. Phase III Phase III will depend on where the signal is detected in phase II and which competent therapies are considered to be the standard. If the signal is in a disease stage where there is no standard of therapy, then a well-controlled single-arm study could serve as an essential test. If there are competent agents that are considered standard, then direct comparative studies are conducted. Many modifications and other embodiments of the inventions set forth in this document will occur to a person skilled in the field to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and associated drawings. Therefore, it is to be understood that the inventions should not be limited to the specific embodiments that are described and that it is proposed that the modifications and other embodiments be included within the scope of the appended claims and a list of modalities described herein. Although specific terms are used in this document, they are used in a generic and descriptive sense only and not for purposes of limitation. All publications and patent applications mentioned in the specification are indicative of the level of those persons skilled in the field to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same degree as if each publication or individual patent application was specifically and individually indicated to be incorporated by reference. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (54)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A human monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing the antigen Human CD40, the monoclonal antibody is free of significant agonist activity, characterized in that the monoclonal antibody exhibits increased antitumor activity relative to an equivalent amount of the monoclonal anti-CD20 monoclonal antibody IDEC-C2B8, wherein the antitumor activity is tested in a nude mouse xenograft tumor model staged using the human B-cell lymphoid cell line Daudi.
2. 2. The human monoclonal antibody according to claim 1, characterized in that the antibody is selected from the group consisting of: a) the monoclonal antibody CHIR-12.12; b) the monoclonal antibody produced by the 12.12 hybridoma cell line, deposited with the ATCC as Patent Deposit No. PTA-5543; c) 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 , the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5: d) a monoclonal antibody having a sequence of amino acids encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID NO-1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 as in SEQ ID NO: 3, e) a monoclonal antibody that binds to an epitope capable of binding the monoclonal antibody produced by the hybridoma cell line 12.12, f) a monoclonal antibody that binds to a epitope comprising residues 82-87 of an antigen sequence Human CD40 shown in SEQ ID NO: 10 or SEQ ID NO: 12; g) monoclonal antibody that competes with the monoclonal antibody CHIR-12.12 in a competitive binding assay; h) the monoclonal antibody of the preceding article a) or a monoclonal antibody of any of the above items c) -g), wherein the antibody is produced recombinantly; and i) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -h), wherein the fragment retains the ability to specifically bind to the human CD40 antigen.
3. 3 . The monoclonal antibody according to claim 1, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (K__) of at least about 10 ~ 6 M to about 10"12 M.
4. 4. A hybridoma cell line, characterized in that it is capable of producing the monoclonal antibody according to claim 1.
5. 5. A method for treating a cancer, characterized in that the expression of the CD40 antigen, which comprises administering to an human patient an effective amount of an anti-CD40 monoclonal antibody. human according to claim 1.
6. 6. The method according to claim 5, characterized in that the cancer is selected from the group consisting of a non-Hodgkin lymphoma, chronic lymphocytic leukemia, multiple myeloma, B-cell lymphoma, cell lymphoma. High-grade B, intermediate-grade B-cell lymphoma, low-grade B-cell lymphoma, acute lymphoblastic leukemia of B cells, myeloblastic leukemia and Hodgkin's disease.
7. 7 A human monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing the human CD40 antigen, the monoclonal antibody is free of significant agonist activity, whereby, when the monoclonal antibody binds to the CD40 antigen expressed on the surface of the cell, cell growth or differentiation is inhibited, characterized in that the antibody is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown both in SEQ ID NO: as in SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO-1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen.
8. 8. The antigen-binding fragment according to claim 7, characterized in that the fragment is selected from the group consisting of a Fab fragment, an F (ab ') 2 fragment. an Fv fragment and an individual chain Fv fragment.
9. 9. The monoclonal antibody according to claim 7, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (KD) of at least about 10"6 M to about 10" 12 M.
10. 10. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide encoding an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO. : 6, SEQ ID NO: 7 and SEQ ID NO: 8. eleven . A hybridoma cell line capable of producing a human monoclonal antibody having specificity for a human CD40 antigen expressed on the surface of a cell expressing the human CD40 antigen, whereby the monoclonal antibody is free of significant agonist activity, thereby , when the monoclonal antibody binds to the CD40 antigen expressed on the surface of the cell, the growth or differentiation of the cell is inhibited and characterized in that the monoclonal antibody is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR- 12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; and j) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of a) -i), wherein the fragment retains the ability to specifically bind to the human CD40 antigen. 12. A method for inhibiting the growth or differentiation of a normal human B cell, characterized in that it comprises contacting the B cell with an effective amount of a human anti-CD40 monoclonal antibody that is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO. : 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) "or a monoclonal antibody of any of the above items c) -i), wherein the antibody is produced recombinantly; and k) a monoclonal antibody that is an antigen-binding fragment; of a monoclonal antibody of any of the above articles a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen, the antibody or a fragment thereof is free of significant agonist activity and whereby, when the antibody or a fragment thereof binds to the CD40 antigen in the B cell, the growth or differentiation of the B cell is inhibited. The method according to claim 12, characterized in that the monoclonal antibody or a fragment thereof is binds the human CD40 antigen with an affinity (Kb) of at least about 10"6 M to about 10 ~ 12 M. The method according to claim 12, acted because the fragment is selected from the group consisting of a Fab fragment, a F (ab ') 2 fragment, an Fv fragment and an individual Fv fragment. 15. A method for inhibiting the proliferation of a normal human B cell, wherein the proliferation is increased by the interaction of a CD40 ligand with a CD40 antigen expressed on the surface of the B cell, the method is characterized in that it comprises putting in contacting the B cell with an effective amount of a human anti-CD40 monoclonal antibody that is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 antigen sequence shown in SEQ ID NO: 10 or SEQ ID NO -.12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen; the antibody or a fragment thereof is free of significant agonist activity and whereby, when the antibody or a fragment thereof binds to the CD40 antigen in the B cell, the growth or differentiation of the B cell is inhibited. method according to claim 15, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (Kb) of at least about 10"6 M to about 10" 12 M. 17. The method according to claim 15 , characterized in that the 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. A method for inhibiting the production of antibodies by B cells in a human patient, characterized in that it comprises administering to a human patient an effective amount of a human anti-CD40 monoclonal antibody or a fragment thereof that is selected from the group consisting of from: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen; the antibody or a fragment thereof is free of significant agonist activity and whereby, when the antibody or a fragment thereof binds to the CD40 antigen in the B cell, the growth or differentiation of the B cell is inhibited. 19 The method according to claim 18, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (KD) of at least about 10"6 M to about 10" 12 M. twenty . The method according to claim 18, characterized in that the fragment is selected from the group consisting of a Fab fragment, an F (ab ') fragment 2. an Fv fragment and a single chain Fv fragment. 21. A method for inhibiting the growth of cancer cells of a B cell lineage, characterized in that it comprises contacting the cancer cells with an effective amount of a human anti-CD40 monoclonal antibody that is selected from the group consisting of: a) the antibody monoclonal CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO-3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen; the antibody or a fragment thereof is free of significant agonist activity and whereby, when the antibody or a fragment thereof binds to the CD40 antigen in the B cell, the growth or differentiation of the B cell is inhibited. 22 The method according to claim 21, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (KD) of at least about 10"6 M to about 10" 12 M. 23. The method according to claim 21, characterized in that the fragment is selected from the group consisting of a Fab fragment, an F (ab ') 2 fragment, an Fv fragment and an individual Fv fragment. 24 The method according to claim 21, characterized in that the cancer is selected from the group consisting of a non-Hodgkin lymphoma, chronic lymphocytic leukemia, multiple myeloma, B-cell lymphoma, high-grade B-cell lymphoma, B-cell lymphoma, intermediate grade, low-grade B-cell lymphoma, acute B-cell lymphoblastic leukemia, myeloblastic leukemia and Hodgkin's disease. 25. A method for treating an autoimmune disease, characterized in that it comprises administering to a human patient an effective amount of a human anti-GD40 monoclonal antibody that is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen; the antibody or a fragment thereof is free of significant agonist activity and whereby, when the antibody or a fragment thereof binds to the CD40 antigen in the B cell, the growth or differentiation of the B cell is inhibited. The method according to claim 25, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (KD) of at least about 10"6 M to about 10" 12 M. 27. The method according to claim 25, characterized in that the 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. 28. The method according to claim 25, characterized in that the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, autoimmune thrombocytopenic purpura, rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, myasthenia gravis and pemphigus vulgaris. 29. A method for treating a cancer distinguished by expression of the CD40 antigen, characterized in that it comprises administering to a human patient an effective amount of a human anti-CD40 monoclonal antibody that is selected from the group consisting of: a) the monoclonal antibody CHIR -5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) an onoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen; the antibody or a fragment thereof is free of significant agonist activity and whereby, when the antibody or a fragment thereof binds to the CD40 antigen in the B cell, the growth or differentiation of the B cell is inhibited. method in accordance with the claim 29, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (KD) of at least about 10"6 M to about 10 ~ 12 M. 31. The method according to claim 29, characterized in that the fragment is selects from the group consisting of a Fab fragment, an F (ab ') 2 fragment, an Fv fragment, and an individual chain Fv fragment 32. A method for identifying an antibody that has an antagonistic activity towards the cells expressing the antigen CD40, characterized in that it comprises performing a competitive binding assay with a monoclonal antibody that is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12, b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5. 9 or 12 12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; and i) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -h), wherein the antibody is recombinantly produced; and j) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -i), wherein the fragment retains the ability to specifically bind to the human CD40 antigen. 33. An antagonist anti-CD40 monoclonal antibody, characterized in that it binds specifically to domain 2 of the CD40 antigen. 34. The monoclonal antibody according to claim 33, characterized in that it is a human antibody. 35. The monoclonal antibody according to claim 34, characterized in that it is free of significant agonist activity. 36 The monoclonal antibody according to claim 33, characterized in that it has the binding specificity of an antibody selected from the group consisting of the antibody produced by the hybridoma cell line 5. 9 and the antibody produced by the hybridoma cell line 12. 12 37 The monoclonal antibody according to claim 33, characterized in that it is selected from the group consisting of the antibody produced by the hybridoma cell line deposited in the ATCC as the deposit of patent No. PTA-5542 and the hybridoma cell line deposited in ATCC as the patent deposit No. PTA-5543. 38. The monoclonal antibody according to claim 33, characterized in that it has the binding specificity of the monoclonal antibody CHIR-12.12 or CHIR-5.9. 39. The monoclonal antibody according to claim 33, characterized in that it binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12. 40 The monoclonal antibody according to claim 33, characterized in that it is selected from the group consisting of: a) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 as in SEQ ID NO: 5; b) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; c) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 12.12; d) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; e) a monoclonal antibody that competes with the monoclonal antibody CHIR-12.12 in a competitive binding assay; and f) a monoclonal antibody that is an antigen-binding fragment of the monoclonal antibody CHIR-12. 12 or the above monoclonal antibodies in the above articles (a) - (e), wherein the fragment retains the ability to specifically bind to the human CD40 antigen. 41 A method for inhibiting a CD40 antigen signaling pathway mediated by the CD40 ligand in a cell expressing the human CD40 antigen, the method is characterized in that it comprises contacting the cell with an effective amount of a human anti-CD40 monoclonal antibody. which is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5. 9 or 12 12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 in SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) an onoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen. 42 The method in accordance with the claim 41, characterized in that the monoclonal antibody binds to the human CD40 antigen with an affinity (KD) of at least about 10"M to about 10" 12 M. 43 The method according to claim 41, characterized in that the fragment is selected from the group consisting of a Fab fragment, an F (ab ') fragment 2. an Fv fragment and a single chain Fv fragment. Four . The method according to claim 41, characterized in that the cell expressing the human CD40 antigen is a human, normal B cell or a malignant human B cell and the signaling pathway of the CD40 antigen is the survival of B cells. Four. Five . A pharmaceutical composition, characterized in that it comprises a human monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing the human CD40 antigen, the monoclonal antibody is free of significant agonist activity, wherein the antibody it is selected from the group consisting of: a) the monoclonal antibody CHIR-5.9 or CHIR-12.12; b) the monoclonal antibody produced by the hybridoma cell line 5.9 or 12.12; c) 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, the sequence shown in both SEQ ID NO: 6 and SEQ ID NO: 7 and the sequence shown both in SEQ ID NO: 6 and SEQ ID NO: 8; d) 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, the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in both SEQ ID NO: 2 and SEQ ID NO: 5; e) 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 sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in both SEQ ID NO: 1 and SEQ ID NO: 3; f) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridomas 5.9 or 12.12 cell line; g) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; h) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the sequence of the human CD40 antigen shown in SEQ ID NO: 10 or SEQ ID NO: 12; i) a monoclonal antibody that competes with the monoclonal antibody CHIR-5. 9 or CHIR-12. 12 in a competitive binding assay; j) the monoclonal antibody of preceding article a) or a monoclonal antibody of any of the above items c) -i), wherein the antibody is recombinantly produced; and k) a monoclonal antibody that is an antigen-binding fragment of a monoclonal antibody of any of the above items a) -j), wherein the fragment retains the ability to specifically bind to the human CD40 antigen. 46. The pharmaceutical composition according to claim 45, characterized in that the composition is a liquid, pharmaceutical formulation comprising a buffer in an amount to maintain the pH of the formulation in a range from about pH 5.0 to about pH 7.0. 47. The pharmaceutical composition according to claim 46, characterized in that the formulation further comprises an isotonicity agent in an amount to render the same composition almost isotonic. 48. The pharmaceutical composition according to claim 47, characterized in that the isotonicity agent is sodium chloride, the sodium chloride is present in the formulation in a concentration of about 50 mM to about 300 mM. 49. The pharmaceutical composition according to claim 48, characterized in that the sodium chloride is present in the formulation in a concentration of approximately 150 mM. 50. The pharmaceutical composition according to any of claims 46 to 49, characterized in that the buffer is selected from the group consisting of succinate, citrate and phosphate buffer. 51. The pharmaceutical composition according to claim 50, characterized in that the formulation comprises the buffer in a concentration of about 1 mM to about 50 mM. 52. The pharmaceutical composition according to claim 51, characterized in that the buffer is sodium succinate or sodium citrate in a concentration of about 5 mM to about 15 mM. 53. The pharmaceutical composition according to any of claims 46 to 52, characterized in that the formulation further comprises a surfactant in an amount from about 0.001% to about 1.0%. 54. The pharmaceutical composition according to claim 53, characterized in that the surfactant is polysorbate 80, which is present in the formulation in an amount from about 0.001% to about 0.5%.