ES2348238T3 - THERAPY PROCEDURES FOR SOLID TUMORS EXPRESSING THE CD40 CELL SURFACE ANTIGEN. - Google Patents

Abstract

A human anti-CD40 monoclonal antibody that is capable of specifically binding to the CD40 antigen, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can be obtained from the hybridoma cell line deposited in ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; and d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR-12.12 monoclonal antibody that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay. for use in the treatment of a human subject with a solid tumor comprising carcinoma cells expressing the CD40 antigen.

Description

Therapy procedures for solid tumors that express the CD40 cell surface antigen.

Field of the Invention

The invention relates to medicaments for treatment, and in vitro methods for the inhibition of the growth of solid tumors using human anti-CD40 antagonist antibodies, solid tumors comprising carcinoma cells expressing the CD40 cell surface antigen.

Background of the invention

CD40 is a cell surface antigen of 55 kDa present on the surface of normal B cells and B cells human neoplastic, dendritic cells, presenting cells of antigens (APC), endothelial cells, monocytic cells and epithelial, some epithelial carcinomas and many tumors solids, including lung, breast, ovarian and colon cancers. The transformed cells of patients with low B cell lymphomas  and high grade, acute B-cell lymphoblastic leukemia, myeloma multiple, chronic lymphocytic leukemia and Hodgkin's disease express the CD40 antigen. CD40 expression is also detected. in two thirds of cases of acute myeloblastic leukemia and in 50% of AIDS-related lymphomas. B cells Malignant of various B-cell lineage tumors express a high degree of CD40 and appear to depend on the CD40 signal for the Survival and proliferation Carcinomas expressing CD40 include urinary bladder carcinoma (Paulie et al. (1989) J. Immunol. 142: 590-595; Braesch-Andersen et al. (1989) J. Immunol. 142: 562-567), breast carcinoma (Hirano et al. (1999) Blood 93: 2999-3007; Wingett and cabbage. (1998) Breast Cancer Res. Treat. 50: 27-36); prostate cancer (Rokhlin et al. (1997) Cancer Res. 57: 1758-1768), renal cell carcinoma (Klutz and cabbage. (1997) Cancer Res. 57: 891-899), carcinoma nasopharyngeal undifferentiated (UNPC) (Agathanggelou et al. (1995) Am. J. Pathol. 147: 1152-1160), cell carcinoma Squamous (SCC) (Amo et al. (2000) Eur. J. Dermatol. 10: 438-442; Posner et al. (1999) Clin. Cancer Res. 5: 2261-2270), papillary thyroid carcinoma (Smith and cabbage. (1999) Thyroid 9: 749-755), malignant melanoma Cutaneous (van den Oord et al. (1996) Am. J. Parasol. 149: 1953-1961), multiple myeloma (Maloney et al. (1999) Semin. Hematol 36 (1 Suppl 3): 30-33), cells Hodgkin and Reed-Sternberg (primed cells) (Gruss and cabbage. (1994) Blood 84: 2305-2314), gastric carcinoma  (Yamaguchi et al. (2003) Int. J. Oncol. 23 (6): 1697-702), sarcomas (see, for example, Lollini and cabbage. (1998) Clin. Cancer Res. 4 (8): 1843-849, discussing human osteosarcoma and Ewing's sarcoma), and carcinoma of liver (see, for example, Sugimoto et al. (1999) Hepatology 30 (4): 920-26, discussing human hepatocellular carcinoma).

The CD40 antigen is related to the human nerve growth factor (NGF) receptor, the tumor necrosis factor? receptor (TNF-?), And Fas, suggesting that CD40 is a receptor for a ligand with important functions in activation of B cells. It has been shown to play a critical role in the development and function of normal B cells. CD40 expression in the APC plays an important costimulatory role in the activation of helper T lymphocytes and cytotoxic T lymphocytes. He CD40 receptor is expressed in activated T cells, platelets activated and in inflamed vascular smooth muscle cells. The CD40 receptors can also be found in eosinophils, synovial membranes in rheumatoid arthritis, in fibroblasts dermal and in other non-lymphoid cells. The binding of CDL40 to CD40 receptor stimulates proliferation and differentiation of B cells, antibody production, isotype change and memory generation of B cells. Through some cells of the carcinoma shows high levels of CD40 expression, it is not understand the role of CD40 signaling in relation to the CD40 expression in these cancer cells.

The document WO-A-96/18413 discloses the treatment of carcinomas in epithelial cells, and the prevention of epithelial cell proliferation, using a connector of the CD40 receiver. The CD40 receiver connector can be used together with at least one anti-neoplastic agent and / or inducer of CD40. Immunoregulatory antibodies are described in the WO-A-02/060485, which describes a combination antibody therapy for the treatment of B cell cancers using an antibody immunoregulatory, especially an antibody anti-B7, anti-CD23 or anti-CD40L and a cell removal antibody B, especially anti-CD19, anti-CD20, anti-CD22 or anti-CD37.

A majority of cancer cases are represented with the so-called solid tumors. Given its high incidence, it They need procedures to treat these cancers.

Brief Summary of the Invention

The invention provides an antibody monoclonal anti-human CD40 that is able to bind specifically to the CD40 antigen, said antibody being monoclonal free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group Constituted by:

a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543;

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b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12;

c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y

d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial,

for use in the treatment of a human subject with a solid tumor comprising carcinoma cells that expresses the CD40 antigen.

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The invention also provides the use of a effective amount of a human anti CD40 monoclonal antibody that is capable of specifically binding to the CD40 antigen in manufacturing of a medicament for the treatment of a human subject with a solid tumor comprising carcinoma cells that express the CD40 antigen, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543;

b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12;

c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y

d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial.

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The invention also provides an in vitro method for inhibiting the growth of a solid tumor comprising carcinoma cells expressing the CD40 antigen, said method comprising contacting said cells with an effective amount of a human anti CD40 monoclonal antibody that is capable of specifically binding to said CD40 antigen, said monoclonal antibody being free of significant agonist activity when binding to the CD40 antigen, said antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543;

b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12;

c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y

d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial.

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The invention also provides an antibody. monoclonal anti human CD40 that is capable of specifically binding to the CD40 antigen, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543;

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b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12;

c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y

d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial,

for use in the inhibition of the growth of a solid tumor comprising carcinoma cells that express CD40 antigen.

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The invention also provides the use of a effective amount of a monoclonal antibody human anti-CD40 that is capable of joining specifically to the CD40 antigen in the manufacture of a medicine for the inhibition of the growth of a solid tumor comprising carcinoma cells expressing the CD40 antigen, said being monoclonal antibody free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of:

a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543;

b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12;

c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y

d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial.

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

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Brief summary of the disclosure

Treatment procedures of a solid tumor in a subject, in which the carcinoma cells of the solid tumor express the CD40 cell surface antigen. The procedures include treating the subject with a anti-CD40 antagonist monoclonal antibody or a antigen binding fragment thereof that is free of significant agonist activity when bound to the CD40 antigen in a cell that expresses human CD40. Antibody binding monoclonal anti-CD40 antagonist (or fragment of binding to the appropriate antigen thereof comprising the Fc portion of anti-CD40 antibody antagonist) to antigen CD40 expressed in carcinoma cells results in cytotoxicity mediated by antibody-dependent cells (ADCC) -cytolysis dependent on these carcinoma cells. In some embodiments, anti-CD40 antibodies antagonists are administered in combination with one or more protocols of different cancer therapy, including, but not limited to these, surgery, radiation therapy; chemotherapy, therapy with cytosine, or other monoclonal antibody that is intended for use in the solid tumor treatment. Solid tumors that can be treated or prevent with the procedures of the present description include, but not limited to, ovarian cancer, lung (for example, non-small cell carcinoma lung cancer squamous cells, adenocarcinoma, and cell carcinoma types large, and small cell lung cancer), breast, colon, kidney (including, for example, renal cell carcinomas), bladder, liver (including, for example, carcinomas hepatocellular), gastric, cervical, prostate, nasopharyngeal, thyroid (for example, papillary carcinoma of thyroid), and skin cancers such as melanoma, and sarcomas (including, for example, Ewing osteosarcomas and sarcomas). Be also reveal procedures for growth inhibition of solid tumors comprising carcinoma cells that express CD40

Antibodies suitable for use in Methods of the invention include monoclonal antibodies.  described herein, which have a strong affinity by CD40 and are characterized by having an equilibrium constant of dissociation (K D) of at least 10-6 M, preferably at minus 10-7 M to about 10-8 M, of more preference at least about 10-8 M up to approximately 10-12 M. Monoclonal antibodies and their antigen binding fragments that are suitable for use in methods of the invention are capable of specifically binding to a human CD40 antigen expressed on the surface of a cell human, particularly a human carcinoma cell of a tumor solid. They are exempt from significant agonist activity but exhibit antagonistic activity when they bind to the CD40 antigen in human cells As demonstrated for normal B cells and human neoplastic expressing CD40. Antibodies Suitable monoclonal have constant human regions; from preference also have regions of the framework totally or partially humanized; and more preferably they are completely antibodies human or its antigen binding fragments.

Examples of such monoclonal antibodies are the antibodies referred to herein as CHIR-5.9 and CHIR-12.12 that can be produce recombinantly; monoclonal antibodies produced by the hybridoma cell lines called 131.2F8.5.9 (referred to in this document as cell line 5.9) and 153.8E2.D10.D6.12.12 (which is done reference herein as cell line 12.12); a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEC ID No. 6, the sequence shown in SEQ ID No. 7; sequence shown in SEQ ID No. 8, the sequences shown in SEQ ID No. 6 and SEQ ID NO. 7, and the sequences shown in SEQ ID NO. 6 and SEQ ID NO. 8; a monoclonal antibody comprising a sequence of amino acids 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 SEQ ID No. 2 and SEQ ID No. 4, and the sequences shown SEQ ID No. 2 and SEQ ID No. 5; a monoclonal antibody comprising a sequence of amino acids encoded by a nucleic acid molecule that comprises 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 sequences shown in SEQ ID No. 1 and SEQ ID No. 3; and fragments of these monoclonal antibodies that bind to the antigen that retain the ability to bind specifically to human CD40, and that they are exempt from activity significant agonist but exhibiting antagonistic activity when they bind to the CD40 antigen in human cells, as demonstrates for normal and human neoplastic B cells that express CD40. Examples of such monoclonal antibodies they also include a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the line 12.12 hybridoma cell or the monoclonal antibody produced by the hybridoma cell line 5.9; 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 antibody monoclonal CHIR-12.12 or CHIR-5.9 in a competitive binding assay; and a monoclonal antibody that is a monoclonal antibody antigen binding fragment CHIR-12.12 or any of the antibodies monoclonal anterior, where the fragment retains the ability to specific binding to human CD40 antigen and shows activity antagonist when it binds to this antigen, as demonstrated to Normal and human neoplastic B cells expressing CD40.

In one embodiment of the disclosure, the treatment procedures include administering to a patient a therapeutically effective dose of a pharmaceutical composition comprising anti-CD40 antagonist antibodies suitable or their antigen binding fragments. A dose Therapeutically effective anti-CD40 antibody or its fragment is in the range from about 0.01 mg / kg to about 40 mg / kg, from about 0.01 mg / kg to approximately 30 mg / kg, from approximately 0.1 mg / kg to approximately 30 mg / kg, from approximately 1 mg / kg to approximately 30 mg / kg, from approximately 3 mg / kg to approximately 30 mg / kg, from approximately 3 mg / kg to approximately 25 mg / kg, from approximately 3 mg / kg to approximately 20 mg / kg, from approximately 5 mg / kg to approximately 15 mg / kg, or from approximately 7 mg / kg to approximately 12 mg / kg It is recognized that the procedure of treatment may comprise a single dose administration therapeutically effective or multiple administrations of a dose therapeutically effective anti antagonist antagonist antibody CD40 or its antigen binding fragments.

Anti-CD40 antagonist antibodies identified herein as suitable for use in The methods of the invention can be modified. The modifications of these anti-CD40 antagonist antibodies include, but are not limited to, chimeric anti CD40 antibodies immunologically active, humanized anti-CD40 antibodies and immunologically active murine anti CD40 antibodies.

Brief description of the drawings

Figure 1 presents the sequences of amino acids for light and heavy chains of mAb CHIR-12.12. The leading regions (waste 1-20 of SEQ ID No. 2), variable (residuals 21-132 of SEQ ID No. 2), and constant (residues 133-239 of SEQ ID NO. 2) of the light chain is shown in Figure 1A. The leading regions (waste 1-19 of SEQ ID No. 4), variable (residuals 20-139 of SEQ ID No. 4), and constant (residues 140-469 of SEQ ID NO. 4) of the heavy chain is shown in Figure 1B. The alternative constant region for the CHIR-12.12 mAb heavy chain shown in the Figure 1B reflects a substitution of an alanine residue with a serine residue at position 153 of SEQ ID NO. 4. The sequence complete for this variant of the heavy chain of the mAb CHIR-12.12 is presented in SEQ ID No. 5.

Figure 2 shows the coding sequences for the light chain (Figure 2A; SEQ ID No. 1) and the heavy chain (Figure 2B; SEQ ID NO. 3) for the CHIR-12.12 mAb.

Figure 3 presents the sequences of amino acids for light and heavy mAb chains CHIR-5.9. The leading regions (waste 1-20 of SEQ ID No. 6), variable (residuals 21-132 of SEQ ID No. 6), and constant (residues 133-239 of SEQ ID NO. 6) of the light chain is shown in Figure 3A. The leading regions (waste 1-19 of SEQ ID No. 7), variable (residuals 20-144 of SEQ ID No. 7), and constant (residues 145-474 of SEQ ID NO. 7) of the heavy chain is shown in Figure 3B. The alternative constant region for the CHIR-5.9 mAb heavy chain shown in Figure 3B reflects a substitution of alanine residue for a residue serine at position 158 of SEQ ID NO. 7. The complete sequence for this variant of the heavy chain of mAB CHIR-5.9 It is presented in SEQ ID NO. 8.

Figure 4 shows the coding sequence (Figure 4A; SEQ ID No. 9) for the short isoform of human CD40 (amino acid sequence shown in Figure 4B; SEQ ID NO. 10), and the coding sequence (Figure 4C; SEQ ID No. 11) for the isoform length of human CD40 (amino acid sequence shown in the Figure 4D).

Figure 5 shows cytotoxicity activity antibody-dependent cell mediated (ADCC) antibody monoclonal CHIR-5.9 and CHIR-12.12 in the SKO3 ovarian cancer cell lines (Figure 5A) and Hey (Figure 5B), the squamous cell cancer cell line of the A431 skin (Figure 5C) and the HCT116 colon cancer cell line (figure 5D).

Figure 6 shows cytotoxicity activity antibody-dependent cell mediated (ADCC) antibody monoclonal CHIR-5.9 and CHIR-12.12 in breast cancer cell lines MDA-MB231 (figure 6A) and MDA-MB435 (figure 6B) and the lines NCI-H460 lung cancer cells (Figure 6C) and SK-MES-1 (figure 6D).

Figure 7 demonstrates the in vivo antitumor activity of the monoclonal antibodies CHIR-5.9 (donated 5.9 in the figure) and CHIR-12.12 (denoted 12.12 in the figure) using a xenograft colon cancer model based on the HCT116 cell line of the human colon carcinoma

Figure 8 shows the effects of antibody monoclonal anti-CD40 administered via intraperitoneal CHIR-12.12 (denoted 12.12 in the figure) or Herceptin® anti-HER2 monoclonal antibody in percent survival in an orthotopic murtin model not staged ovarian cancer using the cancer cell line of human ovaries SKOV3i.p.1.

Figure 9 compares the effects of antibody monoclonal administered intraperitoneally versus IV CHIR-12.12 (denoted 12.12 in the figure) or Herceptin® in percent survival in a model unstaged orthopic murine ovarian cancer using the line Human ovarian cancer cell SKOV3i.p.1.

Figure 10 demonstrates the in vivo antitumor activity of the monoclonal antibodies CHIR-12.12 (denoted 12.12 in the figure) and CHIR-5.9 (donated 5.9 in the figure) versus that observed for Herceptin® using a staged murine model of ovarian cancer based on the human ovarian cancer cell line SKOV3i.p.1.

Figure 11 shows the melting temperature of CHIR-12.12 in different pH formulations measured by differential scanning calorimetry (DSC).

Detailed description of the invention

"Tumor", as used herein document, refers to all cell growth and proliferation neoplastic, be it malignant or benign, and to all cells and tissues precancerous and cancerous. The term "solid tumor" is refers to a cancer or carcinoma of body tissues other than blood, bone marrow and lymphoid system.

The terms "cancer" and "cancerous" are refer to, or describe, the physiological condition in mammals that is typically characterized by non-cell growth regulated. Examples of cancer include, but are not limited to, lung cancer, breast cancer, ovarian cancer, colon cancer, liver cancer, gastric cancer, prostate and skin cancer.

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

The term "antibody" is used in the broader sense and encompasses antibodies completely structured, antibody fragments that can bind to antigen (e.g., Fab ', F' (ab) 2, Fv, antibodies of single chain, divalent fragments or diabodies), and peptides recombinants comprising the above.

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

The "native antibodies" and "native immunoglobulins" are usually glycoproteins heterotetramerics of approximately 150,000 dalton, composed of two identical light chains (L) and two heavy chains (H) identical. Each light chain is attached to a heavy chain by a covalent disulfide bond, while the number of bonds disulfide varies between heavy chains of different isotypes of immunoglobulins Each light and heavy chain also has links intra-chain disulfide spaced regularly. Each heavy chain it has at one end a variable domain (V_ {H}) followed by a number of constant domains Each light chain has a domain variable at one end (V_ {L}) and a constant domain at its other extreme; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the domain of light string variable is aligned with the variable domain of heavy chain It is believed that particular amino acid residues they form an interface between the variable domains of the strings Light and heavy

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

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

The term "hypervariable region" when used herein refers to waste from amino acids of an antibody that are responsible for the binding of antigen. The hypervariable region comprises amino acid residues of a "complementarity determining region" or "CDR" (i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the 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 residues of a "hypervariable loop" (ie residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the chain lightweight and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Clothia and Lesk (1987) J. Mol. Biol. 196: 901-917). Waste from the "frame" or "FR" are the residuals of the variable domain different from the residues of the hypervariable region.

"Antibody fragments" comprise a part of an intact antibody, preferably the region of binding to the antibody or the variable region of the intact antibody. The Examples of antibody fragments include Fab, Fab ', F (ab ') 2 and Fv fragments; divalent fragments; antibodies linear (Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062); chain antibody molecules simple; and multispecific antibodies formed from antibody fragments Digestion with papain of antibodies produces two identical fragments of antigen binding, called "Fab" fragments, each with a single binding site to the antigen, and a residual fragment "Fc", whose name reflects its ability to crystallize easily. Treatment with Pepsin gives an F (ab ') 2 fragment that has two sites of combination with the antigen and is still able to form a cross-linking with the antigen.

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

The Fab fragment also contains the domain light chain constant and the first constant domain (C_ {1}) of the heavy chain. Fab fragments differ from those Fab 'fragments by adding a few residues at the end carboxy terminal of domain C_ 1 of the heavy chain which includes one or more cysteines of the hinge region of the antibody. Fab'-SH is the denomination in this document for Fab 'in which the cysteine residue (s) of Constant domains have a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab 'fragments that have hinge cysteines between they. Other chemical fragment couplings are also known. of antibodies.

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

According to the amino acid sequence of the domain constant of their heavy chains, immunoglobulins can Assign to different classes. There are five main classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM and several of these can also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The constant domains of heavy chains that correspond to the different kinds of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The structures and configurations three-dimensional subunits of the different kinds of Immunoglobulins are well known. Different isotypes have different effector functions. For example, the IgG1 e isotypes Human IgG3 mediates cytotoxicity activity mediated by antibody dependent cells (ADCC).

The word "marker" when used herein refers to a detectable compound or composition that conjugates directly or indirectly to antibodies to generate a "labeled" antibody. The label can be detectable by itself (for example, radioisotope markers or fluorescent markers) or, in the case of an enzymatic label, it can catalyze the chemical alteration of a compound or substrate composition that is detectable. Radionuclides that can serve as detectable markers include, for example, I-131,
I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212 and Pd-109. The marker can also be an undetectable entity such as a toxin.

The term "antagonist" is used in the broader sense and includes any partial blocking molecule or totally, inhibits or neutralizes a biological activity of a native target disclosed in this document or its transcription or translation

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

A "host cell," as used in the This document refers to a microorganism or a cell or cultured eukaryotic cell line as a unicellular entity that it can be used, or has been used, as a vector receptor recombinant or other transfer polynucleotides, and includes the progeny of the original cell that has been transfected. Be understand that the progeny of a single cell may not be necessarily completely identical in morphology or in complement of genomic or total DNA to the original, by natural, accidental or deliberate mutations.

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

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

There are several ways to produce antibodies humans. For example, secretory cells can be immortalized by means of infection with Epstein-Barr virus (EBV). However, EBV infected cells are difficult to clone and usually produce only relatively low yields of immunoglobulin (James and Bell (1987) J. Immunol Methods 100: 5-40). In the future, it could possibly be achieved immortalization of human B cells by introducing a defined combination of transforming genes. That possibility stands out through 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 tumorigenic conversion of normal human fibroblast and epithelial cells (Hahn and cabbage. (1999) Nature 400: 464-468). Now it is possible produce transgenic animals (eg, mice) that are capable, after immunization, of producing a repertoire of human antibodies in the absence of endogenous production of immunoglobulins (Jakobovits et al. (1993) Nature 362: 255-258; Lonberg and Huszar (1995) Int. Rev. Immunol 13: 65-93; Fishwild et al. (1996) Nat. Biotechnol 14: 845-851; Mendez et al. (1997) Nat. Genet 15: 146-156; Green (1999) J. Immunol. Methods  231: 11-23; Tomizuka et al. (2000) Proc. Natl Acad. Sci. USA 97: 722-727; collected in Little and cabbage. (2000) Immunol. Today 21: 364-370). By example, it has been described that the homozygous deletion of the region of heavy chain binding (JH) of the antibody in chimeric mice and germline mutants result in inhibition Complete production of endogenous antibodies (Jakobovits et al. (1993) Proc. Natl Acad. Sci. USA 90: 2551-2555). The transfer of the human immunoglobulin gene set germline in such germline mutant mice gives as a result the production of human antibodies after the challenge with antigens (Jakobovits et al. (1993) Nature 362: 255-258). Mendez et al. (1997) (Nature Genetics 15: 146-156) have generated a line of mice GMOs that, when challenged with an antigen, generate fully human affinity antibodies. This was achieved by the integration in the germinal line of megabase chain loci heavy and light human chains in mice with deletion in the endogenous segment JH as described above. These mice (XenoMouse® II technology (Abgenix; Fremont, California)) house 1,020 kb of the human heavy chain locus containing approximately 66 VH genes, complete DH and JH regions, and three different constant regions, and also house 800 kb of the locus human? containing 32 V? genes, segments J? And C? Genes. The antibodies produced in these mice closely resemble those seen in humans in all aspects, including genetic reorganization, structure and repertoire. Human antibodies are expressed from preference over endogenous antibodies by deletion in the segment endogenous that prevents genetic reorganization in the murine locus. Such mice can be immunized with an antigen of interest. particular.

The sera of such immunized animals may selected for antibody reactivity against antigen initial. Lymphocytes can be isolated from lymph nodes or from Spleen cells and can also be selected for B cells selecting CD138 negative and CD19 positive cells. In a aspect, such B cell cultures (BCC) can be fused to myeloma cells to generate hybridomas as detailed previously.

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

This disclosure refers to procedures for the treatment of human subjects with tumors solids comprising carcinoma cells that express CD40 including, but not limited to, ovarian cancer, lung (for example, non-small cell carcinoma lung cancer of squamous cell, adenocarcinoma, and types of carcinoma of large cells, and small cell lung cancer), breast, colon, kidney (including, for example, cell carcinomas renal), bladder, liver (including, for example, carcinomas hepatocellular) gastric, cervix, prostate, nasopharyngeal, thyroid (for example, papillary carcinoma of thyroid), and skin cancers such as melanoma, and sarcomas (including, for example, Ewing osteosarcomas and sarcomas). The procedures involve treatment with an anti CD40 antibody described in this document, or one of its binding fragments to antigen, in which the administration of the antibody or its fragment antigen binding promotes a positive therapeutic response in the subject who undergoes this therapy procedure. The anti CD40 antibodies suitable for use in the procedures of the invention specifically bind to the expressed human CD40 antigen on the surface of a human carcinoma cell and are exempt of significant agonist activity, but exhibit activity antagonist when they bind to the CD40 antigen in a human cell expressing CD40, as demonstrated for normal human B cells and neoplastic expressing CD40. These anti CD40 antibodies and their antigen binding fragments are referred to herein. document anti CD40 antagonists. Such antibodies include, but are not limited to, monoclonal antibodies CHIR-5.9 and CHIR-12.12 totally human described below and the antibodies monoclonal that have the characteristics of union of the CHIR-5.9 monoclonal antibodies and CHIR-12.12. These monoclonal antibodies that they can produce recombinantly described to continuation.

The antibodies that have the characteristics of binding of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 include antibodies that interfere with competitive way with the union of CHIR-5.9 and CHIR-12.12 to CD40 and / or bind to the same epitopes than CHIR-5.9 and CHIR-12.12. A expert could determine if an antibody interferes so competitive with CHIR-5.9 or CHIR-12.12 using conventional procedures, known in the art.

When these antibodies bind to exposed CD40 on the surface of cells expressing CD40 of a tumor solid (also referred to in this invention as cells of the carcinoma expressing CD40), antibodies are exempt from important agonist activity; in some embodiments, their union to CD40 exposed on the surface of carcinoma cells that express CD40 results in ADCC-dependent death of these cells of carcinoma, and hence a reduction in tumor volume. By consequently, anti-CD40 antagonist antibodies suitable for use in the methods of the invention include antibodies monoclonal that may exhibit antagonistic activity towards human cells expressing the CD40 cell surface antigen, as demonstrated for normal and neoplastic human B cells that express CD40.

Anti CD40 Antagonist Antibodies

Monoclonal antibodies CHIR-5.9 and CHIR-12.12 represent anti CD40 antagonist antibodies suitable for use in Procedures of the present invention. Antibodies CHIR-5.9 and CHIR-12.12 are Isotype fully human anti-CD40 monoclonal antibodies IgG1 produced from hybridoma cell lines 131.2F8.5.9 (referred to in this document as cell line 5.9) and 153.8E2.D10.D6.12.12 (which is done reference herein as cell line 12.12). These cell lines were created using splenocytes from mice immunized xenotypics containing the heavy chain locus of Human IgG1 and the human \ kappa chain locus (technology XenoMouse®; Abgenix; Fremont, California). The spleen cells are fused with mouse SP2 / 0 myeloma cells (Sierra BioSource). The resulting hybridomas were subcloned several times to create stable monoclonal cell lines 5.9 and 12.12. Other antibodies of the invention can be prepared in a manner similar using transgenic mice for immunoglobulin loci human or by other methods known in the art and / or described in this document.

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

In addition to the antagonistic activity, it results from preference that anti-CD40 antibodies suitable for use in procedures of this invention have another mechanism of action against a tumor cell. For example, the antibodies CHIR-5.9 and CHIR-12.12 natives have ADCC activity. As an alternative, the variable regions of the CHIR-5.9 and CHIR-12.12 antibodies can be expressed in another antibody isotype that has activity  ADCC. It is also possible to conjugate native forms, forms recombinants or fragments of CHR-5.9 or CHIR-12.12 that bind the antigen to a cytotoxin

The monoclonal antibodies CHIR-5.9 and CHIR-12.12 bind to soluble CD40 in ELISA type assays, prevent binding of the CD40 ligand to the CD40 of the cell surface, and displace the previously bound CD40 ligand, as determined by assays of flow cytometry. The CHIR-5.9 and CHIR-12.12 antibodies compete with each other for binding to CD40 but not with 15B8, the anti-CD40 monoclonal antibody described in PCT International Application No. PCT / US01 / 30857, published as WO 02/28904, also entitled " Human Anti-CD40 Antibodies ", filed on October 2, 2001 (agent file no. PP 16092.003).

When the effects on B cell proliferation of normal human subjects are tested in vitro , CHIR-5.9 and CHIR-12.12 act as anti-CD40 antagonist antibodies. In addition, CHIR-5.9 and CHIR-12.12 do not induce strong proliferation of human lymphocytes from normal subjects. These antibodies are capable of killing CD40-expressing target cells (lymphoma lines and solid tumor cell lines) by antibody-dependent cell cytotoxicity (ADCC). The binding affinity of CHIR-5.9 for human CD40 is 1.2 x 10-8 M and the binding affinity of CHIR-12.12 is 5 x 10 -10 M, as determined by Biacore ™ assay medium.

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

It is understood as "CD40 antigen", "cell surface CD40 antigen", "CD40 receptor" or "CD40" a transmembrane glycoprotein belonging to the family of tumor necrosis factor (TNF) receptors (see, for example, US 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 (2nd ed .; Academic Press, San Diego)). Two have been identified human CD40 isoforms, encoded by transcription variants with alternative cuts and joints of this gene. The first isoform (also known as the "long isoform" or "isoform 1 ") is expressed as a 277 amino acid precursor polypeptide (SEQ ID No. 12 (first reported in GenBank with Access No. CAA43045, and identified as isoform 1 in GenBank with access No. NP 001241), coded by SEQ ID No. 11 (see Accession No. GenBank X60592 and NM 001250)), which has a signal sequence represented by the first 19 waste. 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), coded by SEQ ID No. 9 (GenBank Accession No. NM 152854)), which also has a signal sequence represented by the first 19 residues. The precursor polypeptides of these two isoforms of human CD40 share in common their first 165 waste (i.e. waste 1-165 of SEQ ID No. 10 and SEQ ID No. 12). He short isoform precursor polypeptide (shown in SEQ ID NO. 10) is encoded by a transcription variant (SEQ ID NO: 9) which lacks an encoder segment, which results in a change of translation framework; The resulting CD40 isoform contains a end C terminal shorter and different (waste 166-203 of SEQ ID No. 10) of which it is contained in the long isoform of CD40 (end C terminal shown in the residues 166-277 of SEQ ID NO. 12). For the purposes of the present invention, the term "CD40 antigen", "antigen CD40 cell surface "," CD40 receiver "or "CD40" encompasses both the short and the long isoform of CD40. The anti CD40 antibodies of the present invention bind to a human CD40 epitope that is in the same situation within the short isoform or the long isoform of this antigen of cell surface as noted below herein document.

CD40 antigen is exposed on the surface of a variety of cell types, as described in other Parties to this document. By "exposed in the surface "and" expressed on the surface "means that all or part of the CD40 antigen is exposed to the outside of the cell. The exposed or expressed CD40 antigen may be total or partially glycosylated.

The term "agonist activity" means that the Substance works like an agonist. An agonist is combined with a receptor in a cell and initiates a reaction or activity that is similar to the one that starts with the natural ligand of receiver. For example, a CD40 agonist induces, but is not limited a, any or all of the following responses: proliferation and B cell differentiation, antibody production, adhesion intercellular, B cell memory generation, isotype change, ascending regulation of cell surface expression of MHC class II and CD80 / 86, and proinflammatory cytokine secretion such as IL-8, IL-12 and TNF. By "antagonistic activity" means that the substance works as an antagonist A CD40 antagonist prevents or reduces the induction of any of the responses induced by the union of CD40 receptor to an agonist ligand, in particular CD40L. He antagonist may reduce the induction of one or more of the agonist binding responses by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more preference 70%, 80%, 85% and more preferably 90%, 95%, 99% or 100% The procedures for measuring binding specificity and antagonist activity of anti CD40 antibodies and ligands of CD40 are known to those skilled in the art and include, but They are not limited to, conventional competitive binding trials, assays to control the secretion of immunoglobulins by B cells, B cell proliferation assays, B-cell proliferation analogous to Banchereau, cell assays T helpers for the production of antibodies, assays of B-cell proliferation with costimulation and assays for ascending regulation of B cell activation markers. See, for example, the tests disclosed in WO 00/75348 and in US Patent No. 6,087,329.

By "significant" antagonistic activity an agonist activity of at least 30%, 35%, 40%, 45% is understood, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than agonist activity induced by a neutral substance or a control negative as measured in the assay of a B cell response. Preferably, the "significant" agonist activity is a agonist activity that is at least 2 times greater or at least 3 times greater than the agonist activity induced by a neutral substance or a negative control as measured in the test of a response of B cells. Therefore, for example, when the response of B cells of interest is B cell proliferation, 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 induced B cell proliferation by a neutral substance or a negative control. In one embodiment, a non-specific immunoglobulin, for example IgG1, that does not bind to CD40 serves as the negative control. A substance "free of significant agonist activity "will exhibit an agonist activity of no more than approximately 25% greater than the agonist activity induced by a neutral substance or a negative control, of preference no more than about 20% higher, 15% higher, 10% greater, 5% greater, 1% greater, 0.5% greater, or even no more than approximately 0.1% higher than the agonist activity induced by a neutral substance or a negative control as measured in a Assay of a B-cell response. Anti-CD40 antibodies antagonists useful in the methods of the present invention they are exempt from significant agonist activity as noted previously when they bind to a CD40 antigen in a cell human In an embodiment of the invention, the antibody anti CD40 antagonist is exempt from agonist activity significant in a B cell response. In another form of embodiment of the invention, the anti-CD40 antagonist antibody is exempt from significant agonist activity in trials of more of a B cell response (for example, proliferation and differentiation, or proliferation, differentiation and production of antibodies).

As used herein "anti CD40 antibody" encompasses any antibody that recognizes  specifically the CD40 cell surface antigen, including polyclonal antibodies, monoclonal antibodies, antibodies single chain, and its fragments such as Fab, F (ab ') 2, F_ {v}, and other fragments that retain the binding function to original anti CD40 antibody antigen. They are particular interest for the methods of the present invention the anti-CD40 antagonist antibodies disclosed herein document that share the union characteristics of the CHIR-5.9 monoclonal antibodies and CHIR-12.12 described above. Such Antibodies include, but are not limited to, the following: (1) monoclonal antibodies produced by the cell lines of hybridoma designated 131.2F8.5.9 (referred to in this document as cell line 5.9) and 153.8E2.D10.D6.12.12 (referred to in this document as a line cell 12.12), deposited with the ATCC as Patent Deposit No. PTA-5542 and Patent Deposit No. PTA-5543, respectively; (2) an antibody monoclonal comprising a selected amino acid sequence of 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 SEQ ID No. 2 and SEQ ID No. 4, and by the sequences shown in SEQ ID No. 2 and SEQ ID No. 5; (3) a monoclonal antibody comprising the amino acid sequence selected from the group consisting of the sequence shown in SEC ID No. 6, the sequence shown in SEQ ID No. 7, the sequence shown in SEQ ID NO. 8, the sequences shown in SEQ ID NO: 6 and SEQ ID No. 7, and by the sequences shown in SEQ ID No. 6 and SEQ ID No. 8; (4) a monoclonal antibody that has the sequence of amino acids encoded by a nucleic acid molecule that comprises a nucleotide sequence selected from the group constituted by the nucleotide sequence shown in SEQ ID No. 1,  the nucleotide sequence shown in SEQ ID No. 3, and the nucleotide sequences shown in SEQ ID NO. and SEQ ID NO. 3; (5) a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 5.9 or the hybridoma cell line 12.12; (6) an antibody monoclonal that binds to an epitope that comprises residues 82-87 of the amino acid sequence shown in SEC ID No. 10 or SEQ ID No. 12; (7) a competing monoclonal antibody with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding trial; and (8) a monoclonal antibody that is a fragment of the antibody monoclonal CHIR-12.12 or CHIR-5.9 that binds to the previous antigen or monoclonal antibodies in the previous points (1) - (7), in which the fragment retains the ability to specifically bind to human CD40 antigen. The experts will recognize that antagonistic antibodies and antigen binding fragments of these antibodies suitable for Use in the procedures described in this document include anti-CD40 antagonist antibodies and their fragments antigen-binding, which are produced recombinantly using procedures well known in the art and described in this document below, and include, for example, antibodies monoclonal CHIR-5.9 and CHIR-12.12 that have been produced recombinantly.

Production of anti-CD40 antagonist antibodies

Anti-CD40 antagonist antibodies for use in the processes of the present invention can occur by any antibody production procedure known to those skilled in the art. Therefore, they can prepare polyclonal sera by procedures conventional. In general, a solution is first used that contains the CD40 antigen to immunize a suitable animal, from Preferably a mouse, rat, rabbit or goat. Rabbits or goats they are preferably for the preparation of polyclonal sera by the volume of serum that can be obtained and the availability of labeled anti rabbit and anti goat antibodies.

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

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

Preferably the antibody is monoclonal in the nature. By "monoclonal antibody" is meant a antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible mutations that are naturally present that may be present in amounts minors The term is not limited with respect to the species or the source of the antibody. The term encompasses immunoglobulins. complete, as well as fragments such as Fab, F (ab ') 2, Fv and others that retain the antigen binding function of the antibody. Monoclonal antibodies are very specific, being directed against a single antigenic site, that is, the CD40 antigen from cell surface in the present invention. In addition, unlike of conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody It is directed against a single determinant in the antigen. He "monoclonal" modifier indicates the character of the antibody already which is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the antibody production by any particular procedure. By example, monoclonal antibodies that are used according to the present invention can be obtained by the process of hybridomas first described by Kohler et al. (1975) Nature 256: 495, or they can be produced by DNA procedures recombinant (see, for example, US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al. (1991) Nature 352: 624-628; Marks et al. (1991) J Mol. Biol. 222: 581-597; and in U.S. Patent No. 5,514,548.

"Epitope" means the part of the antigenic molecule for which an antibody is produced and to which the antibody will bind. Epitopes may comprise residues linear amino acids (i.e. residues in the epitope are arranged consecutively one after the other in a linear fashion), non-linear waste (referred to herein document as "nonlinear epitopes"; these epitopes are not consecutively organized), or amino acid residues both linear as nonlinear.

Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256: 495-496, or one of its modifications. Typically, a mouse is immunized with a solution containing the antigen. Immunization can be carried out by mixing or emulsifying the solution containing the antigen in saline solution, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally. Any immunization procedure known in the art can be used to obtain monoclonal antibodies of the invention. After immunization of the animal, the spleen is removed (and optionally, several large lymph nodes) and the individual cells are separated. Spleen cells can be selected by applying a cell suspension to a plate or well coated with the antigen of interest. B cells expressing the membrane-bound immunoglobulin specific for the antigen bind to the plate and are not cleared. The fusion of the resulting B cells, or all cells separated from the spleen, is then induced with myeloma cells to form hybridomas, and cultured in a selective medium. The resulting cells are plated by serial dilution and the production assay of antibodies that specifically bind the antigen of interest (and that do not bind unrelated antigens) is performed. Hybridomas secreting the selected monoclonal antibody (mAb) are then cultured in vitro (for example, in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

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

In some embodiments, the anti antibody CD40 antagonist, for example the antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment is produced in CHO cells using the GS gene expression system (Lonza Biologics, Portsmouth, New Hampshire), which uses glutamine synthetase as a marker. See also US Patents No. 5,122,464; 5,591,639; 5,658,759; 5,770,359; 5,827,739; 5,879,936; 5,891,693; and 5,981,216.

Monoclonal antibodies against CD40 are known in the art. See, for example, the dedicated sections  to B cell antigens in McMichael, ed. (1987; 1989). Leukocyte Typing III and IV (Oxford University Press, New York); Patents No. 5,674,492; 5,874,082; 5,677,165; 6,056,959; WO document 00/63395; International Publication documents No. WO 02/28905 and WO 02/28904; Gordon et al. (1988) J. Immunol. 140: 1425; Valley and cabbage. (1989) Eur. J. Immunol. 19: 1463; Clark et al. (1986) PNAS 83: 4494; Paulie et al. (1989) J. Immunol. 142: 590; Gordon et al. (1987) Eur. J. Immunol. 17: 1535; Jabara et al. (1990) J. Exp. Med. 172: 1861; Zhang et al. (1991) J. Immunol. 146: 1836; Gascan et al. (1991) J. Immunol. 147: 8; Banchereau et al. (1991) Clin. Immunol Spectrum 3: 8; and Banchereau et al. (1991) Science 251: 70. They are from Particular interest for the present invention is the anti-antibody CD40 antagonists disclosed herein that share the binding characteristics of antibodies monoclonal CHIR-5.9 and CHIR-12.12 described above.

The expression "epitope of the CD40 antigen" as used herein refers to a molecule that is able to have immunoreactivity with antibodies monoclonal anti CD40 of the present invention, excluding CD40 antigen itself. CD40 antigen epitopes can comprise proteins, protein fragments, peptides, hydrates of carbon, lipids and other molecules, but for the purpose of present invention are more commonly proteins, oligopeptides short, oligopeptide mimetics (i.e. organic compounds that mimic the antigen antibody binding properties CD40), or their combinations. The oligopeptide mimetics suitable are described, among others, in the PCT US application 04/19282.

In addition, the expression "anti CD40 antibody" as used herein encompasses antibodies anti CD40 chimeric; such chimeric anti CD40 antibodies to use in the methods of the invention have the binding characteristics of monoclonal antibodies CHIR-5.9 and CHIR-12.12 described in This document. By "chimeric" antibodies understand that more preferably they are obtained using acid techniques recombinant deoxyribonucleic and comprising both components humans (including immunologically "related" species, for example, chimpanzee) as nonhumans. Therefore, the region chimeric antibody constant is more preferably substantially identical to the constant region of an antibody natural human; the chimeric antibody variable region of more preference is obtained from a non-human source and has the desired antigen specificity for the CD40 antigen of cell surface The non-human source can be any source of vertebrate that can be used to generate antibodies against a human CD40 cell surface antigen or material comprising a human CD40 cell surface antigen. Such sources do not humans include, but are not limited to, rodents (for example, rabbit, rat, mouse, etc .; see, for example, US Pat. No. 4,816,567, incorporated herein by reference) and nonhuman primates (for example, old world monkey, ape, etc.; see, for example, U.S. Patent Nos. 5,750,105 and 5,756,096; incorporated in this disclosure by reference). As used herein, the phrase "immunologically  active "when used in reference to anti CD40 antibodies chimeric means a chimeric antibody that binds to CD40 human.

Chimeric anti CD40 antibodies and humanized are also encompassed by the term anti antibody CD40 as used herein. Antibodies chimerics comprise segments of antibodies obtained from different species Rituxan® (IDEC Pharmaceuticals Corp., San Diego, California) is an example of a chimeric antibody with a murine variable region and a human constant region.

By "humanized" are understood ways of anti CD40 antibodies containing a minimal derived sequence of non-human immunoglobulin sequences. Mostly, the Humanized antibodies are human immunoglobulins (antibody receiver) in which waste from a region is replaced hypervariable (also known as the determining region of Complementarity or CDR) of the receiver for waste from a region hypervariable of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate that has the specificity, affinity and capacity desired. The expression "region complementarity determinant "refers to the sequences of amino acids that together define the affinity and specificity of binding of the natural PV region of a binding site of native immunoglobulin. 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 expression "constant region" is refers to the part of the antibody molecule that confers effector functions. In previous works directed to the production of non-immunogenic antibodies for use in therapy human diseases, mouse constant regions were replaced by constant human regions. The constant regions of the Humanized antibodies of the subject were obtained from immunoglobulins human. However, these humanized antibodies still caused an unwanted and potentially dangerous immune response in beings humans and there was loss of affinity. Anti-CD40 antibodies humanized for use in the methods of the present invention they have similar binding characteristics to those exhibited by CHIR-5.9 monoclonal antibodies and CHIR-12.12 described herein.

Humanization can be carried out essentially following the procedure of Winter et al. (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science  239: 1534-1536), replacing sequences of a human antibody for the corresponding CDR or CDR sequences of rodents or rodent mutants. See also US Patent No. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. In some cases, waste within the framework regions of one or more Variable regions of human immunoglobulin are replaced by the corresponding non-human residues (see, for example, U.S. Patent No. 5,585,089; 5,693,761; 5,693,762; and 6,180,370). In addition, humanized antibodies may comprise residues that they are not found in the receptor antibody or in the antibody donor These modifications are made to further refine the antibody yield (for example, to obtain affinity desired). In general, humanized antibody will comprise substantially all of at least one, and typically two, domains variables, in which all or substantially all regions Hypervariables correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. Humanized antibody will also optionally comprise at least a part of a region immunoglobulin constant (Fc), typically that of a human immunoglobulin For more details, see Jones et al. (1986) Nature 331: 522-525; Riechmann et al. (1988) Nature 332: 323-329; and Presta (1992) Curr. Op. Struct. Biol. 2: 593-596. Therefore, such "humanized" antibodies may include antibodies in the that substantially less than a human intact variable domain is has replaced by the corresponding sequence of a species not human In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some frame residues are replaced by residues of similar sites in rodent antibodies. See for example, US Patent No. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also US Patent No. 6,180,370, and International Publication WO 01/27160, in which they are given to know humanized antibodies and techniques to produce antibodies humanized that have better affinity for an antigen predetermined.

They are also included in the term anti CD40 antibodies xenogenic anti CD40 antibodies or modified produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by loci of endogenous inactivated immunoglobulin (Ig). In such animals transgenic, the endogenous genes competent for the expression of light and heavy subunits of host immunoglobulins they become nonfunctional and are replaced with loci human immunoglobulin analogues. These transgenic animals produce human antibodies in substantial absence of subunits Light or heavy host immunoglobulin. See, for example, U.S. Patent Nos. 5,877,397 and 5,939,598.

Preferably, the antibodies fully Human against CD40 are obtained by immunizing transgenic mice. One such mouse is obtained using XenoMouse® technology (Abgenix; Fremont, California), and is disclosed in the Patents No. 6,075,181, 6,091,001 and 6,114,598. To produce the antibodies disclosed herein, are immunized transgenic mice for the heavy chain locus of Human IgG_1 and the human \ light chain locus with Sf9 cells expressing human CD40. Mice can also Be transgenic for other isotypes. Fully antibodies humans useful in the methods of the present invention are characterized by having binding properties similar to those displayed by the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein.

The anti-CD40 antibody fragments are suitable for use in the methods of the invention provided that retain the desired affinity of the full length antibody. Therefore, a fragment of an anti CD40 antibody will retain the ability to bind to the surface CD40 antigen of B cells. Such fragments are characterized by having properties similar to full length antagonist anti CD40 antibody corresponding, that is, the fragments will be specifically joined to a human CD40 antigen expressed on the surface of a cell human, and are exempt from significant agonist activity but exhibit antagonistic activity when they bind to a CD40 antigen in a human cell that expresses CD40. This document is done  reference to these fragments as fragments "that bind to antigen. "Fragments of an antibody that bind to the Suitable antigens comprise a part of an antibody of length total, usually the antigen binding region or one of its variable regions Examples of antibody fragments include, but are not limited to, Fab fragments, F (ab ') 2 and Fv and single chain antibody molecules. By "Fab" is understands a monovalent fragment of an immunoglobulin that binds to the antigen that is composed of the light chain and part of the heavy chain F (ab ') 2 means a fragment bivalent of an immunoglobulin that binds to the antigen that It contains both light chains and part of both heavy chains. By "Single chain Fv" or "sFv" antibody fragments fragments are understood that comprise the domains V_ {H} and V_ {L}  of an antibody, in which these domains are present in a simple polypeptide chain. See, for example, the Patents of U.S. No. 4,946,778, 5,260,203, 5,455,030 and 5,856,456. As usual, the Fv polypeptide further comprises a polypeptide linker between domains V_ {H} and V_ {L} that allows sFv to form the Desired structure for antigen binding. For a review of sFv see Pluckthun (1994) in The Pharmacology of Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pages 269-315. Fragments of anti antibodies CD40 antagonists that bind to the antigen disclosed in the This document can also be conjugated to a cytotoxin for produce lysis of the target cancer cells, as described Next in this document.

Antibodies or antibody fragments can be isolated from phage antibody libraries generated using the techniques described in, for example, McCafferty et al. (1990) Nature 348: 552-554 (1990) and in US Patent No. 5,514,548. Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Later publications describe the production of high affinity human antibodies (of the order of nM) by random chain mixing (Marks et al. (1992) Biol / Technology 10: 779-783), as well as by combinatorial infection and recombination in vivo as a strategy to build very large phage libraries (Waterhouse et al. (1993) Nucleic, Acids Res. 21: 2265-2266). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

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

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

The antibodies of the present invention can have sequence variations produced using the procedures described in, for example, Patent Publication documents No. EP 0 983 303 A1, WO 00/34317 and WO 98/52976. For example, it has shown that sequences within the CDR can cause a antibody binds to MAC Class II and trigger a response from unwanted helper T cells. A conservative substitution can allow the antibody to retain binding activity and still lose your ability to trigger a non-T cell response desired. Any such conservative substitutions or not Conservatives can be performed using procedures recognized in the technique, such as those indicated elsewhere herein document, and the resulting antibodies will be found within the scope of the invention. Variant antibodies can be tested. routinely to verify their antagonistic activity, affinity and specificity using the procedures described in the present document

An antibody produced by any of the procedures described above, or any other procedure not disclosed in this document, it you will find within the scope of this description if you have at least one of the following biological activities: inhibition of immunoglobulin secretion by human peripheral B cells normal stimulated by T cells; survival inhibition and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; survival inhibition and / or proliferation of normal human peripheral B cells stimulated by cells expressing CD40L or CD40 ligand soluble (sCD40L); intracellular intracellular signal inhibition apoptotic "survival" in any stimulated cell by solid phase sCD40L or CD40L; transduction inhibition of CD40 signal in any cell after binding with sCD40L or CD40L solid phase; and inhibition of B cell proliferation Human neoplastic as noted below. These essays they can be performed as described in Schultze et al. (1998) Proc. Natl Acad Sci. USA 92: 8200-8204; Denton et al. (1998) Pediatr. transplant 2: 6-15; Evans et al. (2000) J. Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol 3: 77-86; Coligan and cabbage. (1991) Current Protocols in Immunology 13: 12; Kwekkeboom and cabbage. (1993) Immunology 79: 439-444; and patents of U.S. No. 5,674,492 and 5,847,082.

A representative test to detect anti-CD40 antibodies specific for epitopes of the  CD40 antigen identified herein is an "assay of competitive union. "Competitive union trials are serological tests in which the unknown is detected and quantifies by its ability to inhibit ligand binding known label to its specific antibody. This is also known as a competitive inhibition trial. In a binding test Representative competitive, CD40 polypeptide is precipitated labeled by candidate antibodies in a sample, by example, in combination with monoclonal antibodies raised against one or more epitopes of the monoclonal antibodies of the invention. Anti-CD40 antibodies that react specifically with an epitope of interest they can be identified by selecting a series of antibodies prepared against a CD40 protein or fragment of the protein comprising the particular epitope of the protein CD40 of interest. For example, for human CD40, the epitopes of interest include epitopes comprising amino acid residues linear or non-linear short isoform of human CD40 (see GenBank Accession No. NP 690593) presented in Figure 4B (SEQ ID No. 10), encoded by the sequence presented in Figure 4A (SEC ID No. 9; see also GenBank Accession No. M 152854), or of the long isoform of human CD40 (see GenBank Accession No. CAA43045 and NP 001241) presented in Figure 4D (SEQ ID No. 12), encoded for the sequence presented in Figure 4C (SEQ ID NO: 11; see GenBank Accession No. X60592 and MN 001250). Alternatively, they could use competitive binding assays with anti CD40 antibodies suitable antagonists identified above to select monoclonal antibodies comparable to antibodies identified above.

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

Any of the anti CD40 antibodies antagonists or their antibody fragments described previously may be conjugated before use in the procedures herein invention. The procedures for producing conjugated antibodies They are known in the art. Therefore, the anti antibody CD40 can be marked using indirect dialing or an approach to indirect dialing. By "indirect dialing" or "approach of indirect marking "means that a chelating agent binds covalently to an antibody and at least one radionuclide in the chelating agent. See, for example, agents chelants and radionuclides described in Srivagtava and Mease (1991) Nucl. Med. Bio. 18: 589-603, incorporated into this invention as a reference. Suitable markers include fluorophores, chromophores, radioactive atoms (especially 32 P and125I), dense reagents to electrons, enzymes and ligands that They have specific binding partners. Enzymes are detected Normally for his activity. For example, horseradish peroxidase spicy is usually detected by its ability to convert 3,3 ', 5,5'-tetramethylbenzidine (TMB) in a pigment blue, quantifiable with a spectrophotometer. "Union Couple specific "refers to a protein capable of binding to a ligand molecule with high specificity, such as in the case of an antigen and a specific monoclonal antibody for East. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous pairs of receptor-ligand known in the art. Should It is understood that the above description is not intended to categorize different markers in different classes, since the same marker It can serve in several different modes. For example, 125 I it can serve as a radioactive marker or as a dense reagent in electrons HRP can serve as an enzyme or as an antigen for a mAb In addition, various markers can be combined for this purpose. wanted. For example, mAbs and avidin also require markers in the practice of this invention: therefore, it can mark a mAb with biotin, and detect its presence with avidin labeled with125I, or with an anti-biotin mAb marked with HRP. Other permutations and possibilities will be easily apparent to those skilled in the art, and it considered as equivalent within the scope of this invention.

Alternatively, the anti CD40 antibody can be labeled using "direct labeling" or a "direct labeling approach", in which a radionuclide is covalently fixed to an antibody (typically through an amino acid residue). Preferred radionuclides are provided in Srivagtava and Mease (1991) supra . The indirect dialing approach is particularly preferred. See also, for example, International Publication No. WO 00/52031 and WO 00/52473, in which a connector is used to attach the radioactive label to the antibodies; and the labeled forms of anti-CD40 antibodies described in US Patent No. 6,015,542.

In addition, an antibody (or its fragment) can conjugate to a therapeutic moiety such as a cytotoxin, an agent therapeutic, radioactive metal ions or radioisotopes. A Cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Some examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthracin dihydroxy diona, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and their analogues and counterparts Therapeutic agents include, but are not limited to, antimetabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), agents alkylating agents (eg, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (for example, dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)) and antimitotic agents (for example, vincristine and vinblastine) Radioisotopes include, but are not limited to, I-131, I-123, I-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 determined biological response; it should not be understood that the rest of the drug is limited to classical chemical therapeutic agents. For example, the rest of the drug can be a protein or a polypeptide having a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin; a protein such as tumor necrosis factor, interferon alfa, interferon beta, nerve growth factor, factor platelet-derived growth, tissue activator of plasminogen, or, biological response modifiers such as, for example, lymphokines, interleukin 1 ("IL-1"), interleukin 2 ("IL-2"), interleukin-6 ("IL-6"), colony stimulating factor of granulocytes and macrophages ("GM-CSF"), factor granulocyte colony stimulator ("G-CSF") or other growth factors.

The techniques to conjugate such remains Therapeutic antibodies are well known. See, for example, Amon et al. (1985) "Monoclonal Antibodies for Immunotargeting 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 and cabbage. (2nd ed; Marcel Dekker, Inc.), pages 623-653; Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review ", in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. pages 475-506 (Editrice Kurtis, Milan, 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. (Academic Press, New York, 1985), pages 303-316; and Thorpe et al. (1982) Immunol. Rev. 62: 119-158.

Alternatively, an antibody can be conjugated to a second antibody to form a heteroconjugate of antibodies as described in US Patent No. 4,676,980. In addition, connectors between the markers and the antibodies of the invention (see US Patent No. 4,831,175). Antibodies or their antigen binding fragments can mark directly with iodine, indium, radioactive yttrium or with others  radioactive particles known in the art (US Pat. No. 5,595,721). The treatment may consist of a combination of treatment with conjugated antibodies and not conjugates administered simultaneously or consecutively (WO 00/52031 and WO 00/52473).

Anti-CD40 antagonist antibody variants

In the methods of the present invention variants of anti CD40 antibodies can be used suitable biologically active antagonists. Such variants will retain the desired binding properties of the anti antibody CD40 original antagonist. Usually in the art they are procedures available to produce variants of antibodies

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, by medium of mutations in the cloned DNA sequence encoding the antibody of interest The procedures for mutagenesis and nucleotide sequence alterations are well known in the technique. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl Acad Sci. USA 82: 488-492; Kunkel et al. (1987) Methods Enzymol. 154: 367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); Patent US No. 4,873,192; and the references cited therein. At Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.) can guidance for adequate amino acid substitutions found that do not affect the biological activity of the polypeptide of interest. Conservative substitutions, such as exchanges of a amino acid with another that has similar properties, can be of preference. Examples of conservative substitutions include, but they are not limited to, Gly \ LeftrightarrowAla, Val \ LeftrightarrowIle \ LeftrightarrowLeu, Asp \ LeftrightarrowGlu, Lys \ LeftrightarrowArg, Asn \ LeftrightarrowGln and Phe \ LeftrightarrowTrpo \ LeftrightarrowTyr.

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

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

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

Regarding the optimal alignment of two amino acid sequences, the contiguous segment of the sequence of variant amino acids may have additional amino acid residues or amino acid residues deleted from the sequence of reference amino acids. The contiguous segment used for the comparison with the reference amino acid sequence will include at least 20 contiguous amino acid residues and may have 30, 40, 50 or more amino acid residues. Corrections can be made to sequence homology associated with gaps or substitutions waste conservators (see search algorithm for Smith-Waterman).

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

The technique provides substantial guidance with respect to the preparation and use of the variants of polypeptides In the preparation of antibody variants anti CD40, one skilled in the art can easily determine what modifications to the nucleotide or amino acid sequence of the native protein will result in a variant that is suitable for use as a therapeutically active component of a pharmaceutical composition used in the given procedures of the present invention

Therapy procedures using anti CD40 antibodies antagonists of the invention

The disclosure procedures are aimed at the use of anti-CD40 antagonist antibodies to treat subjects (i.e., patients) who have solid tumors that comprise cells that express the cell surface antigen CD40 By "carcinoma cells expressing CD40" is meant malignant cells (for example, neoplastic) or pre-malignant (for example, neoplastic) of a tumor solid that expresses the CD40 cell surface antigen. The procedures to detect CD40 expression in cells are very known in the art and include, but are not limited to, techniques PCR, immunohistochemistry, flow cytometry, transfer Western, ELISA and the like. Solid tumors that can be treated using the procedures of the present description include, but not limited to these, ovarian cancer, lung (for example, non-small cell lung cancer cell carcinoma squamous, adenocarcinoma, and types of large cell carcinoma, and small cell lung cancer), breast, colon, kidney (including, for example, renal cell carcinomas), bladder, liver (including, for example, hepatocellular carcinomas) gastric, cervical, prostate, nasopharyngeal, thyroid (by example, papillary thyroid carcinoma), and skin cancers such such as melanoma, and sarcomas (including, for example, osteosarcomas and Ewing sarcomas).

This document defines "treatment" as the application or administration of a anti-CD40 antagonist antibody or its binding fragment to antigen to a patient, or the application or administration of a anti-CD40 antagonist antibody or its fragment to an isolated tissue or cell line of a subject, in which the subject has a tumor solid, a symptom associated with a solid tumor, or a predisposition towards the development of a solid tumor, being the aim to cure, heal, calm, relieve, alter, remedy, improve or affect the solid tumor, any symptoms associated with the solid tumor or predisposition to solid tumor development. By "treatment" is also understood as the application or administration of a pharmaceutical composition comprising the anti-CD40 antagonist antibody or its fragments to a subject, or the application or administration of a pharmaceutical composition that comprises anti CD40 antibodies or their fragments to a tissue isolated or a subject's cell line, which has a tumor solid, a symptom associated with a solid tumor or a predisposition  to the development of the solid tumor, the objective being to cure, heal, soothe, relieve, alter, remedy, improve or affect the tumor solid, solid tumor symptoms or predisposition to solid tumor development.

"Antitumor activity" means a reduction in the rate of proliferation or accumulation of cells malignant expressing CD40, and therefore a decrease in growth rate of an existing tumor or a tumor that arises during therapy, and / or the destruction of neoplastic cells (tumor) existing or new formed neoplastic cells, and by consequently a decrease in the overall size of a tumor during therapy. Therapy with at least one anti CD40 antibody (or its antigen binding fragment) causes a physiological response which is advantageous with respect to the treatment of solid tumors in a human, where solid tumors comprise cells of carcinoma expressing CD40. It is recognized that the procedures of the description may be useful in preventing others tumor consequences that arise during therapy.

In accordance with the procedures of the present disclosure, at least one anti-CD40 antagonist antibody (or its antigen binding fragment) as defined in other parties in this document are used to promote a positive therapeutic response with respect to a solid tumor. By "positive therapeutic response" regarding treatment of cancer is understood as an improvement in the disease associated with the antitumor activity of these antibodies or their fragments, and / or an improvement in the symptoms associated with the disease. That is to say, an antiproliferative effect can be observed, prevention of others consequences of the tumor, a reduction in tumor size, a reduction in the amount of cancer cells and / or a decrease in one or more symptoms mediated by cell stimulation that express CD40. Therefore, for example, an improvement in Disease can be characterized as a complete response. By "complete response" means the absence of disease clinically detectable with normalization of any study radiographic, bone marrow and cerebrospinal fluid (CSF), previously abnormal Such response must persist for at least one month after treatment according to the procedures of the description. Alternatively, an improvement in the disease can be classified as a partial response. By "answer partial "means a decrease of at least 50% in all tumor loads that can be measured (i.e. the amount of tumor cells present in the subject) in the absence of new injuries and that persists for at least a month. Such an answer is Applicable only to tumors that can be measured.

The tumor response can be evaluated for changes in tumor morphology (i.e. general burden of tumor, tumor size and the like) using techniques detection such as resonance imaging scan magnetic (MR), x-ray images, tomography scanning computerized (CT), bioluminescent images, for example, Luciferase images, bone scintigraphy images and collection of tumor biopsy samples including bone marrow aspiration bone (I LOVE). In addition to these positive therapeutic responses, the subject undergoing therapy with anti CD40 antibody antagonist or its antigen binding fragment may experience the advantageous effect of an improvement in the symptoms associated with the disease.

By "therapeutically dose or quantity effective "or" effective amount "means an amount of anti-CD40 antagonist antibody or its binding fragment to antibody that, when administered, results in a response positive therapy regarding the treatment of a patient with a disease that involves the stimulation of the cells that express CD40. In some embodiments of the invention, a dose therapeutically effective of an anti CD40 antibody or its fragment is in the range from about 0.01 mg / kg to about 40 mg / kg, from about 0.01 mg / kg to approximately 30 mg / kg, from approximately 0.1 mg / kg to approximately 30 mg / kg, from approximately 1 mg / kg to approximately 30 mg / kg, from approximately 3 mg / kg to approximately 30 mg / kg, from approximately 3 mg / kg to approximately 25 mg / kg, from approximately 3 mg / kg to approximately 20 mg / kg, from approximately 5 mg / kg to approximately 15 mg / kg, or from approximately 7 mg / kg to approximately 12 mg / kg It is recognized that the procedure of treatment may comprise a single dose administration therapeutically effective or multiple administrations of a dose Therapeutically effective anti-CD40 antagonist antibody or its antigen binding fragment.

Another embodiment of the disclosure is the use of anti CD40 antagonist antibodies for diagnostic control of protein levels in tissue as part of a procedure of clinical trials, for example, to determine the effectiveness of a given treatment regimen. Detection can be facilitated by antibody coupling to a detectable substance. The Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, materials luminescent, bioluminescent materials and materials radioactive Examples of suitable enzymes include the horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; the Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; material examples Suitable fluorescents include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a fluorescent material includes luminol; material examples  Bioluminescent include luciferase, luciferin and aequorin; Y Examples of suitable radioactive materials include125I, 131 I 35 S or 3 H.

In some preferred embodiments, the anti-CD40 antagonist antibodies of the invention, or fragments thereof  antigen binding, are administered in combination with at least another cancer therapy, including but not limited to these, surgery, radiation therapy, chemotherapy, cytokine therapy, or other monoclonal antibody that is intended for use in the solid tumor treatment of interest, in which therapy Additional anticancer is given before, during, or after therapy with the anti CD40 antibody. By consequently, when combined therapies comprise the administration of an anti-CD40 antagonist antibody or its antigen binding fragment in combination with administration of another therapeutic agent, such as with chemotherapy, therapy with cytokine, or other monoclonal anticupero, the procedures of the disclosure encompass co-administration, using formulations separated from a single pharmaceutical formulation, and administration  consecutive in any order, in which there is preferably a period of time in which both (or all) active agents They simultaneously exercise their therapeutic activities. When the procedures of the present description comprise regimes combined therapies, these therapies can be given so simultaneous, that is, the anti-CD40 antibody or its fragment of antigen binding is administered simultaneously or within it period of time than the other cancer therapy (i.e. therapies take place simultaneously, but the anti antibody CD40 or its antigen binding fragment is not administered precisely at the same time as the other therapy against Cancer). Alternatively, the anti CD40 antibody of the present invention or its antigen binding fragment can also administered before or after other therapy against cancer. The sequential administration of the different therapies against cancer can be performed regardless of whether the treated subject responds to the first course of therapy to decrease the possibility of remission or relapse.

In some embodiments of the invention, the anti CD40 antibodies described herein, or their fragments antigen binding, are administered in combination with chemotherapy or cytokine therapy, being able to administer the antibody and agent (s) chemotherapeutic (s) or cytokine (s) sequentially, in any order, or simultaneously (that is, simultaneously or within the same period of time). Examples of agents Suitable chemotherapeutics include, but are not limited to, CPT-11 (irinotecano), which can be used, by example, in the treatment of colorectal cancer and cell cancer not small lung; gemcitabine, which can be used, for example, in the treatment of lung cancer, breast cancer, and epithelial ovarian cancer; and other chemotherapeutic agents suitable for the treatment of solid tumors. Cytokines of interest include, but not limited to these, alpha-interferon, gamma-interferone, interleukin-2 (IL-2), IL-12, IL-15 e IL-21, colony stimulation factor of granulocyte macrophage (GM-CSF) factor stimulation of granulocyte colony (G-CSF) or Biologically active variants of these cytokines.

In other embodiments of the invention, the anti CD40 antibodies described herein, or their fragments antigen binding, administered in combination with others monoclonal antibodies directed to the treatment of solid tumor. Therefore, for example, when the subject is subject to treatment of a breast cancer comprising cells of the carcinoma expressing CD40, therapy could include administration of effective amounts of an antibody anti-CD40 antagonist described herein, or its antigen binding fragment, in combination with administration of effective amounts of Herceptin® (Genentech, Inc., San Francisco, California), which targets protein Her2 receptor in Her2 + breast cancer cells. So similar, when the subject is undergoing cancer treatment colorectal comprising carcinoma cells expressing CD40, therapy could include administration of effective amounts of an anti-CD40 antagonist antibody described in this document, or its antigen binding fragment, in combination with administration of effective amounts of monoclonal antibody humanized Avastin ™ (also known as bevacizumab; Genentech, Inc., San Francisco, California), which binds and inhibits the vascular endothelial growth factor (VEGF), a protein that It plays a critical role in the tumor angiogenesis. Other examples of monoclonal antibodies directed to the treatment of tumors solids that can be used in combination with antibodies anti-CD40 of the present invention include, but without being limited to these, anti-EGFR antibody that direct the epidermal growth factor receptor (by example, IMC-C225 (ImClone Systems, New York, New York) (see, for example, Mendelsohn and Baselga (2000) Oncogene 19: 6550-6565 and Solbach et al. (2002) Int. J. Cancer 101: 390-394); antibody anti-IGF-1 receptor, which targets the IGF-1 receptor protein (see, for example, Maloney et al. (2003) Cancer Res. 63: 5073-5083 and Hailey et al. (2002) Mol. Cancer. Plier one: 1349-1353; anti-MUCl antibody, which the tumor-associated MUC1 antigen is targeted; anti-α5β1, anti-? v? 5, and anti-? v? 3, which targets those respective integrins, which regulate cell adhesion and processes signaling involved in proliferation and survival Cellular (see, for example, Laidler et al. (2000) Acta Biochimica Polonica 47 (4): 1159-1170 and Cruet-Hennequart et al. (2003) Oncogene 22 (11): 1688-1702); antibody anti-P-cadherin, which targets this member of the cadherin family (see, for example, the US patent application pending along with this 20030194406); and antibody anti-VE-cadherin, which targets the angiogenic related function of this adhesion molecule specific to the endothelial cell (see, for example, Liao and cabbage. (2002) Cancer Res. 62: 2567-2575).

The anti-CD40 antibodies of the invention and the other monoclonal antibody can be administered sequentially, in any order, or simultaneously (i.e. simultaneously or within the same period of time). When more than one type of monoclonal antibody is administered, the methods of the present invention may comprise additionally exposure to radiation and / or chemotherapy as specify for cancer treatment and how it Recommend by the responsible medical practitioner.

Pharmaceutical formulations and modes of administration

Anti-CD40 antibodies for use in procedures of the present invention are administered in a concentration that is therapeutically effective to prevent or treat solid tumors comprising carcinoma cells that express CD40, including ovarian cancer, lung (for example, cancer of non-small cell lung squamous cell carcinoma, adenocarcinoma, and types of large cell carcinoma, and cancer of small cells of the lung), breast, colon, kidney (including, by example, renal cell carcinomas), bladder, liver (including, for example, hepatocellular carcinomas) gastric, of cervix, prostate, nasopharynx, thyroid (for example, papillary thyroid carcinoma), and skin cancers such as melanoma, and sarcomas (including, for example, osteosarcomas and Ewing sarcomas). To achieve this goal, the antibodies can be formulated using a variety of acceptable excipients known in the art. Typically, antibodies are administered by injection, either intravenously or by intraperitoneal route. The procedures to carry out this Administration are known to those skilled in the art. Too it is possible to obtain compositions that can be administered via topically or orally, or that may be able to be transmitted to through mucous membranes.

Intravenous administration is performed in preference by infusion over a period of about 1 to about 10 hours, more preference for about 1 to about 8 hours,  more preferably still for about 2 until approximately 7 hours, even more preferably for approximately 4 to approximately 6 hours, depending on the anti CD40 antibody that is administered. The initial infusion with the Pharmaceutical composition can be administered during a period of approximately 4 to approximately 6 hours with infusions later administered more quickly. Infusions later can be administered during a period of about 1 to about 6 hours, including, for example, about 1 to about 4 hours, about 1 to about 3 hours, or about 1 Up to about 2 hours.

A pharmaceutical composition of the invention It is formulated to be compatible with its route of administration planned. Examples of possible routes of administration include parenteral administration (for example, intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), intraperitoneal, intratumoral or infusion), oral and pulmonary (for example, inhalation), nasal, transdermal (topical), transmucosal and rectal. The solutions or suspensions used for parenteral applications, Intradermal or subcutaneous may include the following Components: a sterile diluent such as water for injection, saline solution, nonvolatile oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; agents antibacterials such as benzyl alcohol or methylparabenos; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjust the tonicity such as sodium chloride or dextrose. PH it can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide Parenteral preparation can be included in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Anti CD40 antibodies are provided. typically by conventional techniques in a buffer pharmaceutically acceptable, for example, sterile saline solution, sterile buffered water, propylene glycol, combinations of previous, etc. In Remington's Pharmaceutical Sciences (18th ed .; Mack Publishing Company, Eaton, Pennsylvania, 1990) are described procedures for preparing agents that can be administered by parenteral route

See also, for example, WO 98/56418, which describes pharmaceutical antibody formulations stabilized suitable for use in the procedures described in This document.

One skilled in the art can easily determine without undue experimentation the amount of at least one anti-CD40 antibody or its fragment to be administered given the description herein. Factors that influence the mode of administration and the respective amount of at least one anti-CD40 antagonist antibody (or its fragment) include, but are limited to, the severity of the disease, the history of the disease, and the age, height , weight, health and physical condition of the individual undergoing treatment. Similarly, the amount of anti-CD40 antagonist antibody or its fragment to be administered will depend on the mode of administration and whether the subject will undergo a single dose or multiple doses of this antitumor agent. Generally, as the weight of the subject under treatment increases, a higher dose of the anti-CD40 antibody or its fragment is preferably preferred. The dose of the anti-CD40 antibody or its fragment to be administered is in the range from about 0.003 mg / kg to about 50 mg / kg, preferably in the range of 0.01 mg / kg to about 40 mg / kg. Therefore, for example, the dose may be 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg , 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg,
35 mg / kg, 40 mg / kg, 45 mg / kg or 50 mg / kg.

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

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

In other embodiments, the dose Therapeutically effective initial anti-CD40 antibody antagonist or its antigen binding fragment as defined in another part of this document may be in the range of lower dosage (i.e. from about 0.003 mg / kg up to approximately 20 mg / kg) with subsequent doses within higher dosage range (i.e. from approximately 20 mg / kg to about 50 mg / kg).

In alternative embodiments, the dose Therapeutically effective initial anti-CD40 antibody antagonist or its antigen binding fragment as defined in another part of this document may be in the range of higher dosage (i.e. from about 20 mg / kg up to approximately 50 mg / kg) with subsequent doses within lower dosage range (i.e. from 0.003 mg / kg up to about 20 mg / kg). Therefore, in a form of embodiment, the initial therapeutically effective dose of the antibody  anti CD40 antagonist or its antigen binding fragment is of approximately 20 mg / kg to approximately 35 mg / kg, including approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg and approximately 35 mg / kg, and subsequent doses Therapeutically effective anti-CD40 antagonist antibody or its antigen binding fragment are approximately 5 mg / kg until approximately 15 mg / kg, including approximately 5 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg and approximately 15 mg / kg.

In some embodiments of the disclosure, the Anti-CD40 antagonist antibody therapy is started by administering  a "loading dose" of the antibody or its binding fragment to the antigen to the subject that needs therapy with anti CD40 antibodies antagonists By "loading dose" is meant a dose initial of the anti-CD40 antagonist antibody or its fragment of binding to the antigen that is administered to the subject, in which the dose of the antibody or its antigen-binding fragment administered is within the upper dosage range (i.e., from approximately 20 mg / kg to approximately 50 mg / kg). The "loading dose" can be administered as a single administration, for example, a single infusion in which the antibody or its antigen binding fragment is administered by via IV, or as multiple administrations, for example, infusions multiple in which the antibody or its binding fragment to antigen is administered IV, provided that the "dose of full load "be administered in a period of approximately 24 hours. After administration of the "loading dose", then administer one or more therapeutically effective doses of the anti-CD40 antagonist antibody or its binding fragment to subject antigen. Dosages can be administered therapeutically subsequent effective, for example, according to a program of weekly dosage, or once every two weeks, once every three weeks, or once every four weeks. In such forms of realization, subsequent therapeutically effective doses they are generally in the lower dosage range (it is say, from 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 its antigen binding fragment are administered according to a "maintenance schedule", in which the dose is therapeutically Effective antibody or its antigen binding fragment is administered once a month, once every 6 weeks, once every two months, once every 10 weeks, once every three months, once every 14 weeks, once every four months, once every 18 weeks, once every five months, once every 22 weeks, once every six months, once every 7 months, once every 8 months, once every 9 months, once every 10 months , once every 11 months, or once every 12 months. In such embodiments, the therapeutically effective dose of the anti-CD40 antagonist antibody or its antigen binding fragment is in the lower dosage range (i.e., from 0.003 mg / kg to about 20 mg / kg), in particular when the Subsequent doses are administered at more frequent intervals, for example, once every two weeks up to once a month, or within the upper dosage range (i.e., from about 20 mg / kg to about
50 mg / kg), in particular when subsequent doses are administered at less frequent intervals, for example, when subsequent doses are administered separately for approximately one month to approximately 12 months.

Anti-CD40 antagonist antibodies present in the pharmaceutical compositions described herein document to use in the procedures disclosed in the This document can be native or can be obtained through of recombinant techniques and can be from any source, including sources of mammals such as, for example, mouse, Rat, rabbit, primate, pig and human. Preferably such polypeptides are obtained from a human source, and more preferably they are human proteins, recombinant cell lines of hybridoma

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

Any pharmaceutical composition that you understand an anti-CD40 antagonist antibody that has the properties of union described herein as the component Therapeutically active can be used in the procedures given to know in this document. Therefore, the liquid, lyophilized or spray dried compositions comprising one or more of the anti-CD40 antagonist antibodies of the invention can be prepared as a solution or suspension aqueous or non-aqueous for subsequent administration to a subject of in accordance with the procedures disclosed herein document. Each of these compositions will comprise at least one of the anti-CD40 antagonist antibodies of the present invention as a therapeutically or prophylactically active component. By "therapeutically or prophylactically active component" is understand that the anti CD40 antibody is specifically incorporated into the composition to elicit a response therapeutic or prophylactic with respect to treatment, the prevention or diagnosis of a solid tumor in a subject when The pharmaceutical composition is administered to that subject. From preferably the pharmaceutical composition comprises agents stabilizers, suitable loading agents, or both to reduce minimum problems associated with loss of stability and biological activity of proteins during preparation and storage.

Formulation compounds can be added to Pharmaceutical compositions comprising an anti CD40 antibody antagonist of the invention. These formulation compounds can include, but are not limited to, oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants or fillers. Carbohydrates of Preference include sugars or sugar alcohols such as mono-, di- or polysaccharides, or water soluble glucans. The saccharides or glucans may include fructose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulane, dextrin, cyclodextrin? and?, soluble starch, hydroxyethyl starch and carboxymethyl cellulose, or mixtures thereof. "Alcohol is defined of sugar "as a C4 to C8 hydrocarbon having a hydroxyl group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol and arabitol. These sugars or sugar alcohols They can be used individually or in combination. The Sugar or sugar alcohol concentration is between 1.0% and 7% p / v., more preferably between 2.0% and 6.0% p / v. Amino acids of Preference include levógira (L) forms of carnitine, arginine, and betaine; however, other amino acids can be added. From Preferably the polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. The surfactants that can be added to the formulation are shown in EP documents 270,799 and 268,110.

In addition, antibodies can be modified. chemically by covalent conjugation to a polymer for, by For example, increase your circulation half-life. Polymers of preference and the procedures for linking them to peptides, are shown in US Patent No. 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Preferred polymers are polyols. polyoxyethylated and polyethylene glycol (PEG). PEG is water soluble at room temperature and has the general formula: R (O-CH 2 -CH 2) n O-R where R can be hydrogen, or a group protector such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has preferably an average molecular weight between 1,000 and 40,000, of more preference between 2,000 and 20,000, more preferably between 3,000 and 12,000 Preferably, the PEG has at least one hydroxyl group, more preferably it is a terminal hydroxy group. This is the group hydroxy that is preferably activated to react with a group free amino in the inhibitor. However, it will be understood that the type and the amount of reactive groups can be varied to obtain a PEG / antibody of the present invention conjugated in a manner covalent

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

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

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

The procedures to control 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 disclosed herein document below. In general, the stability of Proteins are measured at a chosen temperature during a period of specified time. In preferred embodiments, a stable antibody pharmaceutical formulation provides stability of the anti-CD40 antagonist antibody or its fragment of antigen binding when stored at room temperature (approximately 25 ° C) for at least 1 month, at least 3 months, or at least 6 months, and / or is stable at approximately 2-8ºC for at least 6 months, at least 9 months, at At least 12 months, at least 18 months, at least 24 months.

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A protein such as an antibody, when formulated in a pharmaceutical composition, it is considered to maintain your physical stability at any given time if it shows no signs visual (i.e. discoloration or loss of transparency) or signs that can be measured (for example, using chromatography of size exclusion (SEC) or UV light scattering) of precipitation, aggregation, and / or denaturation in that composition Pharmaceutical With respect to chemical stability, a protein such as an antibody, when formulated in a composition pharmaceutical, it is considered to maintain its chemical stability in a given moment if chemical stability measures are indicative that the protein (i.e. the antibody) maintains its activity biological interest in that pharmaceutical composition. The procedures to control changes in chemical stability they are well known in the art and include, but are not limited to, procedures to detect chemically altered forms of the protein such as degradation result, using, by example, SDS-PAGE, SEC, and / or mass spectrometry per flight time of laser desorption / ionization assisted by matrix; and degradation associated with changes in molecular load (for example, associated with deamidation), using, for example, ion exchange chromatography. See, for example, the procedures disclosed in this document to continuation.

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

In some embodiments of the invention, the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment is formula in a liquid pharmaceutical formulation. Anti antibody CD40 antagonist or its antigen binding fragment can be prepared using any procedure known in the art, including the procedures disclosed earlier in the present document In one embodiment, the anti antibody CD40 antagonist, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment is produced recombinantly in a CHO cell line.

After preparation and purification, the anti-CD40 antagonist antibody or its antigen binding fragment  it can be formulated as a liquid pharmaceutical formulation of the manner set forth in this document. When the anti antibody CD40 antagonist or its antigen binding fragment must stored before formulation, can be frozen, by example, at ≤ -20 ° C, and then defrost at room temperature for another formulation. Formulation Liquid pharmaceutical comprises a therapeutically effective amount of the anti-CD40 antagonist antibody or its binding fragment to antigen. The amount of antibody or its binding fragment to antigen present in the formulation takes into account the route of administration and the desired dose volume.

In this way, the pharmaceutical composition liquid comprises the anti-CD40 antagonist antibody, for example, CHIR-12.12 or CHIR-5.9 antibody, or its antigen binding fragment in a concentration of about 0.1 mg / ml to about 50.0 mg / ml, approximately 0.5 mg / ml to approximately 40.0 mg / ml, approximately 1.0 mg / ml to approximately 30.0 mg / ml, approximately 5.0 mg / ml to approximately 25.0 mg / ml, about 5.0 mg / ml to about 20.0 mg / ml, or approximately 15.0 mg / ml to approximately 25.0 mg / ml. In some embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or its fragment of antigen binding at a concentration of approximately 0.1 mg / ml up to about 5.0 mg / ml, about 5.0 mg / ml up to approximately 10.0 mg / ml, approximately 10.0 mg / ml to approximately 15.0 mg / ml, approximately 15.0 mg / ml up to approximately 20.0 mg / ml, approximately 20.0 mg / ml to approximately 25.0 mg / ml, approximately 25.0 mg / ml up to approximately 30.0 mg / ml, approximately 30.0 mg / ml up to approximately 35.0 mg / ml, approximately 35.0 mg / ml to approximately 40.0 mg / ml, approximately 40.0 mg / ml up to approximately 45.0 mg / ml, or approximately 45.0 mg / ml to approximately 50.0 mg / ml. In other embodiments, the Liquid pharmaceutical composition comprises the anti CD40 antibody antagonist or its antigen binding fragment in a concentration of approximately 15.0 mg / ml, approximately 16.0 mg / ml, approximately 17.0 mg / ml, approximately 18.0 mg / ml, approximately 19.0 mg / ml, approximately 20.0 mg / ml, approximately 21.0 mg / ml, approximately 22.0 mg / ml, approximately 23.0 mg / ml, approximately 24.0 mg / ml, or 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 its antigen binding fragment and a buffer that maintains the pH of the formulation in the range of about pH 5.0 until approximately pH 7.0, including approximately 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 similar values within the range from about pH 5.0 to about pH 7.0. In some embodiments, the buffer maintains the pH of the formulation in the range of about pH 5.0 until approximately pH 6.5, approximately pH 5.0 to approximately  pH 6.0, approximately pH 5.0 to approximately pH 5.5, approximately pH 5.5 to approximately 7.0, approximately pH 5.5 to approximately pH 6.5, or approximately pH 5.5 to approximately pH 6.0.

Any buffer can be used in the formulation adequate to maintain the pH of the liquid formulation of the anti CD40 antibody in the range of approximately pH 5.0 to approximately pH 7.0, provided that the physicochemical stability and The desired biological activity of the antibody is maintained as noted earlier in this document. Tampons include but are not limited to conventional acids and their salts, where the counterion can be, for example, sodium, potassium, ammonium, calcium or magnesium Examples of conventional acids and their salts which can be used to buffer the liquid pharmaceutical formulation include, but are not limited to, succinic acid buffers or succinate, citric acid or citrate, acetic acid or acetate, acid tartaric 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 Buffer concentration in the formulation can be from approximately 1 mM to approximately 50 mM, including approximately 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other similar values within  from the range of about 1 mM to about 50 mM. In some embodiments, the concentration of the buffer in the formulation is from about 5 mM to about 15 mM, including approximately 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or other similar values within from the range of about 5 mM to about 15 mM.

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

When it is desirable that the formulation Liquid pharmaceutical is practically isotonic, the formulation liquid pharmaceutical comprising a therapeutically amount effective anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, and a buffer to keep the pH of the formulation within the range from about pH 5.0 to about pH 7.0 can further comprise an amount of an isotonicity agent enough to make the formulation practically isotonic By "practically isotonic" it is understood that the aqueous formulation has an osmolarity of approximately 240 mmol / kg to about 360 mmol / kg, preferably about 240 to about 340 mmol / kg, more preferably about 250 to about 330 mmol / kg, even more preferably about 260 to about 320 mmol / kg, more preferably still about 270 to approximately 310 mmol / kg. The procedures to determine the Isotonicity of a solution are known to those skilled in the technique. See, for example, Setnikar et al. (1959) J. Am. Pharm. Assoc. 48: 628.

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

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

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

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

Therefore, in some embodiments, the liquid pharmaceutical formulation comprises an amount Therapeutically effective anti-CD40 antagonist antibody, by example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, the buffer is sodium succinate buffer or sodium citrate in a concentration of about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 5 mM to about 15 mM; The formulation has a pH of approximately pH 5.0 to approximately pH 7.0, approximately pH 5.0 to approximately pH 6.0, or about pH 5.5 to about pH 6.5; and the formulation further comprises a surfactant, for example, polysorbate 80, in an amount from about 0.001% to approximately 1.0% or approximately 0.001% to approximately 0.5% Such formulations may optionally comprise an agent. of isotonicity, such as sodium chloride in a concentration of approximately 50 mM to approximately 300 mM, approximately 50 mM to approximately 200 mM, or approximately 50 mM to approximately 150 mM. In other embodiments, the Liquid pharmaceutical formulation comprises the anti CD40 antibody antagonist, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, in a concentration of about 0.1 mg / ml to about 50.0 mg / ml or approximately 5.0 mg / ml to approximately 25.0 mg / ml, including approximately 20.0 mg / ml; sodium chloride approximately 50 mM to approximately 200 mM, including approximately 150 mM sodium chloride; sodium succinate or sodium citrate at a concentration of approximately 5 mM until approximately 20 mM; including sodium succinate or citrate approximately 10 mM sodium; sodium chloride at a concentration from about 50 mM to about 200 mM, including approximately 150 mM; and optionally a surfactant, for example, polysorbate 80, in an amount from about 0.001% to approximately 1.0%, including approximately 0.001% up to about 0.5%; where the pharmaceutical formulation liquid has a pH of approximately pH 5.0 to approximately pH 7.0, approximately pH 5.0 to approximately pH 6.0, approximately pH 5.0 to approximately pH 5.5, approximately  pH 5.5 to approximately pH 6.5, or approximately pH 5.5 to approximately pH 6.0.

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

Once the liquid pharmaceutical formulation or other pharmaceutical composition described herein It is prepared, can be lyophilized to prevent degradation. The procedures for lyophilizing liquid compositions are known by those skilled in the art. Just before using, the composition can be reconstituted with a sterile diluent (solution of Ringer, distilled water or saline solution, for example) that can Include other components. After reconstitution, the composition is preferably administers subjects using procedures known to those skilled in the art.

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

The present invention also provides the use of an anti-CD40 antagonist antibody or its binding fragment to antigen in the manufacture of a medicament for the treatment of a subject with a solid tumor comprising carcinoma cells that express CD40 antigen, in which the drug is coordinated with treatment with at least one other cancer therapy. Examples of these tumors include, but are not limited to, ovarian cancer, lung (for example, cell lung cancer non-small squamous cell carcinoma, adenocarcinoma, and types of large cell carcinoma, and small cell cancer of the lung), breast, colon, kidney (including, for example, carcinomas of renal cells), bladder, liver (including, for example, hepatocellular carcinomas) gastric, cervix, prostate, nasopharyngeal, thyroid (for example, papillary carcinoma of thyroid), and skin cancers such as melanoma, and sarcomas (including, for example, Ewing osteosarcomas and sarcomas).

By "coordinated" it is understood that the medicament comprising the anti-CD40 antagonist antibody or its antigen binding fragment will be used before, during, or after of the treatment of the subject with at least one other therapy against Cancer. Examples of other cancer therapies include, but they are not limited to surgery; radiation therapy; chemotherapy; when appropriate chemotherapeutic agents include, but are not limited to, fludarabine or phosphate from fludarabine, chlorambucil, vincristine, pentostatin, 2-Chlorodeoxydenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations thereof, by example, anthracycline containing regimens such as CAP (cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincritsine, 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; cytosine therapy including, but not limited to these, interferon-alpha therapy, therapy with interferona-gamma therapy with interleukin-2 (IL-2), IL-12, IL-15 e IL-21, colony stimulation factor of granulocyte macrophage (GM-CSF) factor stimulation of granulocyte colony (G-CSF) or biologically active variants of these cytokines; or others monoclonal antibodies directed to the use in the treatment of solid tumors of interest, for example, Herceptin® (Genentech, Inc., San Francisco, California), which targets protein Her2 receptor in Her2 + breast cancer cells; the antibody humanized monoclonal Avastin? (also known as bevacizumab; Genentech, Inc., San Francisco, California), which binds and inhibits vascular endothelial growth factor (VEGF), and It is used in the treatment of colon cancer; antibody anti-EGFR that targets the factor receptor epidermal growth (for example, IMC-C225 (ImClone Systems, New York, New York); receptor antibody anti-IGF-1, which targets the IGF-1 receptor protein; antibody anti-MUCl, which targets the associated MUC1 antigen to the tumor; anti-α5β1, anti-? v? 5, and anti-? v? 3, which targets those respective integrins, which regulate cell adhesion and signaling processes involved in proliferation and cell survival; antibody anti-P-cadherin, which targets this member of the cadherin family (see, for example, the U.S. Patent Application No. 20030194406); and antibody anti-VE-cadherin, which targets the angiogenic related function of this adhesion molecule endothelial cell specific; where treatment with the additional cancer therapy, or cancer therapies additional, take place before, during or after the treatment of the subject with the medication comprising the anti-CD40 antagonist antibody or its fragment of antigen binding, as indicated herein previously.

Therefore, for example, in some embodiments, the invention provides the use of the antibody monoclonal CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, in the manufacture of a medication for the treatment of a subject with a solid tumor comprising carcinoma cells that express CD40 antigen, in the that the medication is coordinated with treatment by chemotherapy, the chemotherapeutic agent being selected from group consisting of CPT-11 (irinotecano), which you can use, for example, in the treatment of colorectal cancer and non-small cell lung cancer; gemcitabine, which can use, for example, in the treatment of lung cancer, cancer of breast, and epithelial ovarian cancer; and other agents Chemotherapeutic agents suitable for the treatment of tumors solids; where treatment with cancer therapy additional, or additional cancer therapies, take place before, during or after the treatment of the subject with the medicament comprising the anti-CD40 antibody antagonist or its antigen binding fragment, as indicated in This document above.

In other embodiments the invention provides the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, in the manufacture of a medicine for the treatment of a subject with a solid tumor comprising carcinoma cells that express CD40 antigen, in which the medication is coordinated with treatment with at least one other anti-cancer antibody selected from the group consisting of Herceptin® (Genentech, Inc., San Francisco, California), which targets the receptor protein Her2 in Her2 + breast cancer cells; the monoclonal antibody humanized Avastin ™ (also known as bevacizumab; Genentech, Inc., San Francisco, California), which binds and inhibits the vascular endothelial growth factor (VEGF), and is of use in the colon cancer treatment; anti-EGFR antibody which targets the epidermal growth factor receptor (for example, IMC-C225 (ImClone Systems, New York, NY); receptor antibody anti-IGF-1, which targets the IGF-1 receptor protein; antibody anti-MUCl, which targets the associated MUC1 antigen to the tumor; anti-α5β1, anti-? v? 5, and anti-? v? 3, which targets those respective integrins, which regulate cell adhesion and signaling processes involved in proliferation and cell survival; antibody anti-P-cadherin, which targets this member of the cadherin family (see, for example, the publication of US patent application 20030194406); Y anti-VE-cadherin antibody, which directs the angiogenic related function of this molecule of specific adhesion of the endothelial cell; where the treatment with additional cancer therapy, or therapies against Additional cancer occurs before, during or after treatment of the subject with the medication comprising the anti-CD40 antagonist antibody or its fragment of antigen binding, as indicated herein previously.

The invention also provides the use of a anti-CD40 antagonist antibody, for example, the antibody monoclonal CHIR-12.12 or CHIR-5.9 described herein, or its fragment binding to antigen in the manufacture of a medicament for the treatment of a subject with a solid tumor comprising cells expressing the CD40 antigen, in which the medication is used in a subject who has received prior treatment with at least one other therapy against Cancer. By "pretreated" or "pretreatment" is understands that the subject has received another or other therapies against cancer (that is, has been treated with at least one other therapy against cancer) before receiving the medicine that includes the anti-CD40 antagonist antibody or its binding fragment to antigen. "Pretreated" or "pretreatment" includes subjects who have been treated with at least one other therapy against cancer within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, in the 6-week period, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or even within 1 day before the start of treatment with the medicament comprising the anti-CD40 antagonist antibody, by example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 described in this document, or its antigen binding fragment. It is not necessary for the subject has been a patient who responds to previous treatment with the previous therapy against cancer, or previous therapies against Cancer. Therefore, the subject receiving the medication that comprises the anti-CD40 antagonist antibody or its fragment of antigen binding might have responded, or there might not be responded (ie cancer was refractory), to treatment prior to previous cancer therapy, or to one or more previous cancer therapies where pretreatment It comprised multiple cancer therapies. The examples of other cancer therapies for which a subject may have received prior treatment before receiving the medication that comprises the anti-CD40 antagonist antibody or its fragment of antigen binding include, but is not limited to, surgery; therapy with radiation; chemotherapy; when appropriate agents Chemotherapeutics include, but are not limited to, the listed above in this document; other therapies with anti-cancer monoclonal anti-cancer, including, but without being limited to these, the anticancer antibodies listed in this  document above, cytokine therapy, including cytokine therapies listed in this document above; or Any of your combinations.

"Treatment" in the context of use coordinated of a medicine described in this document with other or other cancer therapies is defined herein document such as the application or administration of the medication or of the other cancer therapy to a subject, or the application or administration of the medication or other cancer therapy to a isolated tissue or cell line of a subject, in which the subject have a solid tumor that comprises carcinoma cells that express CD40 antigen, a symptom associated with such cancer, or a predisposition to develop such cancer, the purpose being to cure, heal, calm, relieve, alter, remedy, improve or affect the cancer, any associated symptoms of cancer, or predisposition to develop cancer

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

Experimental part Introduction

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

In summary, splenocytes of mice immunized with murine myeloma cells SP 2/0 or P 3 x 63Ag8.653 in a 10: 1 ratio 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 supplemented with hypoxanthine (0.1 mM), aminopterin (0.01 mM), thymidine (0.016 mM), and hIL-6 0.5 ng / ml (Genzyme, Cambridge, Massachusetts). Then you they distributed the fused cells between the wells of plates of 96-well tissue culture, so that each contained 1 growing hybridoma on average.

After 10-14 days they were analyzed supernatants of hybridoma populations to detect the production of specific antibodies. For the detection of specific antibodies for the hybridoma clones, were combined supernatants from each well and tested for specificity of anti-CD 40 activity by ELISA first. TO then the positives for fluorescent staining of B cell cells transformed with EBV as described for the FACS test above. Hybridoma cells positive were cloned twice by limiting dilution in IMDM / FBS containing hIL-6 0.5 ng / ml.

A total of 31 spleens of mice were fused with SP2 / 0 mouse myeloma cells to generate 895 antibodies that recognize the recombinant CD40 in ELISA. On average approximately 10% of hybridomas produced using Abgenix XenoMouse® technology may contain the light chain mouse lambda instead of the human kappa chain. Be selected antibodies containing mouse lambda light chain and it discarded. A subset of 260 antibodies was selected that they also showed binding to the cell surface CD40 for another analysis. Stable hybridomas selected during a series of subcloning procedures were used for further characterization in binding and functional tests.

Clones of another 7 hybridomas were identified that had antagonistic activity. Based on its antagonistic potency relative and ADCC activities, two clones of hybridoma for further evaluation. They are called 131.2F8.5.9 (5.9) and 153.8E2.D10, D6.12.12 (12.12).

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TABLE 1 Summary of initial antibody data series IgG1 anti CD40 CHIR-5.9 and CHIR-12.12

one

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The hybridoma line 131.2F5.8.5.9 (CMCC # 12047) and the hybridoma line 153.8E2.D10.D6.12.12 they have been deposited in the American Type Culture Collection [ATCC; 10801 University Blvd., Manassas, Virginia 20110-2209 (USA)] with the Deposit Number of Patent No. PTA-5542 and No. PTA-5543, respectively.

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

As expected for antibodies derived from independent hybridomas, there is a substantial variation in the nucleotide sequences in the determining regions of complementarity (CDR). It is believed that diversity in the region of CDR3 of VH significantly determines the specificity of the antibody.

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

The monoclonal antibodies CHIR-12.12 and CHIR-5.9 are strong antagonists and inhibit in vitro CD40-mediated ligand proliferation of normal B cells, as well as inhibit CD40-mediated ligand proliferation in vitro of cancer cells in NHL and CLL patients. . In vitro , both antibodies kill primary cancer cells of NHL patients by ADCC. Dose-dependent anti-tumor activity was observed in a model of human xenograft lymphoma.

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Example 1 CD40 is expressed in a high proportion of solid tumors

A variety of lines have been found cultured cells derived from solid tumor and biopsies of patient to express CD40. It was found that a high% of samples of biopsy of patients with breast, lung, ovarian, and skin cancer express CD40.

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Example 2 Candidate monoclonal antibody capacity CHIR-12.12 to join CD40 expressed in various human carcinomas

Carcinoma tissue samples were obtained from individuals (n = 10) with ovarian, lung, breast, or colon cancer and frozen for subsequent analysis of antibody binding using immunohistochemistry The percentage of cells was determined neoplastic in the various human carcinomas capable of joining CHIR-12.12 mAb. As can be seen in the table 2, 60% of ovarian and lung carcinoma samples are they were in the top category of union in percentage (that is, 50 to 100% of the cells in these samples were able to join CHIR-12.12 mAb). Thirty per percent of breast carcinoma samples and 10% of samples of colon carcinoma were in the upper category of the percentage union.

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TABLE 2 CHIR-12.12 mAb ability to join to various human carcinomas

2

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Example 3 CHIR-5.9 and CHIR-12.12 are capable of causing cytolysis of target cells that carry CD40 by ADCC

Candidate antibodies can cause cytolysis of target cells (lymphoma lines and solid tumor cell lines) through the ADCC mechanism. Both CHIR-5.9 and CHIR-12.12 are fully human IgG1 isotype antibodies and are expected to have the ability to induce cytolysis of target cells through the ADCC mechanism. Its ability to cause cytolysis of cancer cell lines was tested in in vitro assays . Two human lymphoma cell lines (Ramos and Daudi) and a human colon cancer cell line (HCT116) were initially selected as target cells for these assays. PBMC or enriched NK cells from 8 normal voluntary donors were used as effector cells in these assays. Greater ADCC was observed against lymphoma cell lines than with the colon cancer cell line. A more potent ADCC response was observed with CHIR-12.12 compared to CHIR-5.9 against the target cells of both lymphoma cancer cell lines. The lymphoma cell lines also express CD20, the target antigen for rituximab (Rituxan®, IDEC Pharmaceuticals Corp., San Diego, California), which made it possible to compare the ADCC activity of these two candidate mAbs with the ADCC activity of rituximab . For the lymphoma cell line target, an average specific lysis of 35%, 59% and 47% was observed for CHIR-5.9, CHIR-12.12, and rituximab, respectively, when used at a concentration of 1 µg / ml . A mean specific lysis of 20% and 39% for CHIR-5.9 and CHIR12.12 were observed respectively for colon cancer cell line. See table 3 below. The two antibodies did not show much activity in the complement-dependent cytotoxicity (CDC) assays.

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Additional activity test was carried out ADCC of these two monoclonal anti-CD40 antibodies in the HCT116 colon cancer cell line and seven other lines carcinoma cells, including cancer cell lines of ovaries SKOV3 and HEY, the squamous skin cancer cell line A431, breast cancer cell lines MDA-MB231 and MDA-MB435, and the lines NCI-H460 lung cancer cells and SK-MES-1 using the procedures described above. As seen in the figures 5A-D and 6A-D, the antibody monoclonal CHIR-12.12 usually showed more ADCC activity than the monoclonal antibody CHIR-5.9 at any given concentration and for any given cell line rehearsed

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Example 4 CHIR-5.9 and CHIR-12.12 show antitumor activity in animal models Pharmacology / efficacy in vivo

Candidate mAbs are expected to produce Desired pharmacological effects to reduce tumor burden by either of two anti-tumor mechanisms, signal blockade of ADCC proliferation / survival and induction. The models of Human lymphoma xenograft currently available use long-term lymphoma cell lines that, unlike the primary cancer cells are not dependent on stimulation of CD40 for growth and survival. Therefore, it expect the component of this antitumor activity of these mAbs based on the proliferation / survival signal block Tumor does not contribute to antitumor efficacy in these models. The efficacy in these models is dependent on the ADCC, the second anti-tumor mechanism associated with CHIR-5.9 mAbs and CHIR-12.12.

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Models of human B-cell lymphoma xenograft

The antitumor activities of candidate mAbs with two models of human lymphoma xenograft on Namalwa and Daudi cell lines. To demonstrate more your activity therapeutic, these mAbs were evaluated in a non-xenograft model statized (ie prophylactic) and staged xenograft (ie. therapeutic) of human lymphoma based on the cell line Daudi

In summary, the entire body of nude mice deficient in T-3 Gy cells was irradiated to further suppress the immune system one day before tumor inoculation. Tumor cells were inoculated subcutaneously on the right side at the rate of 5 x 10 6 cells per mouse. Treatment began one day after tumor implantation (non-staged subcutaneous human B-cell lymphoma xenograft models, Namalwa and Daudi) or when the tumor volume reached 200-400 mm 3 (Daudi staging model , usually 15 days after tumor inoculation). Mice carrying the tumor were injected with the anti-CD40 mAbs intraperitoneally (ip) once a week at the indicated doses. The doses for the unstaged Namalwa model were as follows: 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 1 mg / kg and 10 mg / kg (mAb CHIR-12.12); 1 mg / kg (mAb CHIR-5.9); Y
10 mg / kg (rituximab). Doses for the unstaged Daudi model were as follows: 0.01 mg / kg, 0.1 mg / kg and 1 mg / kg (CHIR-12.12 mAb) and (CHIR-5.9 mAb); and 10 mg / kg; 1 mg / kg (rituximab). The doses for the staged Daudi model were as follows: 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg and 10 mg / kg (mAb CHIR-12.12); 1 mg / kg (mAb CHIR-5.9); and 1 mg / kg (rituximab). Tumor volumes were recorded twice a week. When the tumor volume in any group reached 2500 mm3, the study was terminated. Note that in Daudi's staged model, tumor volume data was analyzed until day 36 for the death of some mice after that day. Complete regression (CR) was counted until the end of the study. Data were analyzed using ANOVA or Kruskal-Wallis test and the corresponding subsequent test for comparison of multiple groups.

In the unstaged Namalwa model, the mAb anti CD40 CHIR-12.12, but not Rituxan® (rituximab), significantly increased (p = <0.01) the growth of Namalwa tumors (60% reduction in tumor volume compared to 25% for rituximab, n = 10 mice / group) (data not shown). By consequently, in this model, the anti CD40 mAb CHIR-12.12 was more potent than rituximab. Fits highlight that the second candidate mAb, CHIR-5.9, was at least as effective as rituximab in a dose of 1/10 of that of rituximab. Both anti CD40, CHIR-12.12 and rituxan mAb significantly prevented tumor development in the model of Daudi's unstaged tumor (14/15 challenge resistance tumor) (data not shown).

When these anti CD40 monoclonal antibodies they were also compared in a staged xenograft model of Daudi, in which the treatment commented when the subcutaneous tumor was palpable, the anti-CD40 CHIR12.12 mAb at a dose of 1 mg / kg caused significant reduction of the tumor (p = 0.003) with regression 60% complete (6/10), while rituximab in the same dose does not significantly inhibited tumor growth or caused regression complete (0/10) (data not shown).

In summary, the anti CD40 mAb CHIR-12.12 significantly inhibited tumor growth in experimental models of lymphoma. At the same dose and the same regimen, the CHIR-12.12 mAb showed better anticancer activity than Rituxan® (rituximab). In addition, no signs of toxicity were observed in this dose and in this regimen. These data suggest that CHIR-12.12 anti CD40 mAb has potent human anti-lymphoma activity in vitro and in xenograft models and could be clinically effective for the treatment of lymphoma.

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Xenograft model of human colon carcinoma

These candidate mAbs were further evaluated in relation to its therapeutic antitumor activity in a model of solid tumor Similar to many human solid tumors, the HCT116 human colon carcinoma cell line expresses CD40 and it selected for a model of colon cancer xenograft. Be inoculated tumor cells subcutaneously in the side right of nude mice deficient in T cells (this tumor can grow on naked tattoos without prior irradiation) at the rate of 5 x 10 6 cells per mouse. One day after tumor inoculation the mice received intraperitoneal (i.p.) injection of anti-CD40 mAbs once a week with a total of 5 dose.

Treatment with anti-CD40 mAbs showed a reproducible tendency towards tumor growth inhibition in two repeated studies. Data from one of these two studies are shown in Figure 7. Interestingly, an inversion of antitumor activity was observed with the highest dose (10 mg / kg) in this model, suggesting that a dose may be necessary. / optimal regimen to achieve the best inhibition of tumor growth. The monoclonal CHIR-12.12 antibody, which showed higher ADCC activity in vitro and increased anti-tumor efficacy in a lymphoma model, was tested only with a single dose of 1 mg / kg in the colon carcinoma model. A dose assessment of CHIR-12.12 was carried out to determine the total potential of its anti-tumor efficacy in this human colon cancer xenograft model.

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Orthotopic ovarian cancer model not staged (prophylactic)

The CHIR-12.12 mAb was evaluated also in relation to its therapeutic antitumor activity in a unstaged (prophylactic) orthotopic murine model of cancer ovaries using the ovarian cancer cell line SKOV3i / p.1. Be they inoculated tumor cells intraperitoneally (i.p.) in nude mice deficient in T cells at 2 X 10 6 cells per mouse. Starting the first day after tumor inoculation the mice received injections i.p. of various doses of CHIR-12.12 mAbs or Herceptint® (Genentech, Inc., San Francisco, California), which is under clinical investigation for the treatment of ovarian cancer. The antibody was dosed once a week with a total of 6 doses. He calculated the Survival percentage over time.

Treatment with CHIR-12.12 mAb prolonged survival time dependently on the dose (figure 8). Fifty-four days after inoculation of the tumor, the survival percentage was significantly higher for the group receiving 30 mg / kg of CHIR-12.12 mAb which for the control group does not treaty. Although a similar dose of Herceptin® showed a tendency towards prolongation of survival, the percentage survival at 54 days after inoculation was not significantly greater than that observed for the control group untreated

Figure 9 shows a comparison of effects of CHIR-12.12 mAb on the percentage of survival in this non-staged orthotopic murine model of ovarian cancer when the antibody is administered intraperitoneally (i. p.) versus intravenous (i.v.). He Treatment protocol was as described above. How It can be seen in this figure, the injection via i.p. from CHIR-12.12 mAb gave better survival rate with respect to that observed with administration via i.v. of this antibody.

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Staged (therapeutic) murine model of cancer ovaries

The CHIR-5.9 and CHIR-12.12 mAbs in relation to its therapeutic anti-tumor activity in a staged (therapeutic) murine model of ovarian cancer using the ovarian cancer cell line SKOV3i.p.1. For this one study tumor cells were inoculated subcutaneously in the right side of nude mice deficient in T cells at a rate of 5 x 10 6 cells per mouse with 10% matrigel. Starting 6 days after tumor inoculation (when tumor volume reached 100-200 mm3), the mice received injections of these mAbs intraperitoneally once a week with a total of 4 doses. Tumor volume two was measured times a week after the first dosing day of the antibody.

The two candidate mAbs inhibited significantly tumor growth compared to observed for the untreated control group (figure 10) to the older antibody concentrations tested (1 mg / kg for CHIR-5.9 mAb, and 10 mg / kg for CHIR-12.12 mAb). For CHIR-12.12 mAb (the only antibody for which the dose was varied), the tumor growth inhibition occurred dependently of the dose, with the greatest tumor reduction happening at the dose higher (i.e. 10 mg / kg). With the higher dose the CHIR-12.12 mAb was just as effective as a equivalent dose of Herceptin®.

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Example 5 CHIR-5.9 and CHIR-12.12 join a different epitope than 15B8 in CD40

Candidate monoclonal antibodies CHIR-5.9 and CHIR-12.12 compete one with the other by binding to CD40 but not with 15B8, an anti IgG2 mAb CD40 (see International Publication No. WO 02/28904). Be designed studies of competitive antibody binding using Biacore with CM5 biosensor chips with protein A immobilized by amine coupling medium, which was used to capture anti CD40, CHIR-12.12, or 15B8. The curves of normal association / dissociation binding with concentrations CD40-his variables (data not shown). For the competition studies, CHIR-12.12 or 15B8 on the surface of protein A. It was subsequently flowed a CD40-his / Fab complex of CHIR-5.9 (CD40 100 nM: Fab of CHIR-5.9 1 µM), in Variable concentrations, across the modified surface. In In the case of CHIR-12.12, no association of complex, which indicates that CHIR-5.9 blocks the junction from CHIR-12.12 to CD40-his. For 15B8, association of the Fab CHIR-5.9 complex was observed that indicates that CHIR-5.9 does not block the binding of 15B8 to the CD40 binding site. However, the rate of dissociation of complex increased dramatically (data not shown).

It was also determined that 15B8 and CHIR-12.12 do not compete for the union of CD40-his. This experiment was carried out. capturing CHIR-12.12 on the biosensor chip of protein A, blocking residual sites of protein A with hIgG1 control, joining CD40-his and then doing flow 15B8 over the modified surface. 15B8 joined under these conditions indicating that CHIR-12.12 does not block the union of 15B8 to CD40.

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Example 6 Binding properties of CHIR-12.12 and CHIR-5.9 mAb

Protein A was immobilized in biosensing chips CM5 through amines coupling. Antibodies were captured monoclonal anti-human CD40, in a concentration 1.5 µg / ml, on the modified surface of the biosensor for 1.5 minutes at 10 µl / min. It flowed Surface-soluble recombinant CD40-his of the biosensor in varying concentrations. The antibody and the antigen were diluted in 0.01 M HEPES pH 7.4, 0.15 M NaCl, EDTA 3 mM, Surfactant P 20.005% (HBS-EP). Be determined the kinetic and affinity constants using the Biaevaluation software with an interaction setting 1/1 model / global.

As shown in Table 4 below, there is a 121-fold difference in the rate of dissociation of CHIR-5.9 and CHIR-12.12 which gives as result 24 times higher affinity for CHIR-12.12.

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Example 7 Epitope characterization for monoclonal antibodies CHIR-12.12 and CHIR-5.9

To determine the location of the epitope in CD40 recognized by monoclonal antibodies CHIR-12.12 and CHIR-5.9, were performed SDS-PAGE analysis and Western transfer. Separated Purified CD40 (0.5 µg) on a NUPAGE gel 4-12% under reducing and non-reducing conditions, it was transferred to PVDF membranes, and the survey was performed with monoclonal antibodies in a concentration of 10 µg / ml. The transfers were probed with anti human IgG conjugated with alkaline phosphatase and was developed using the stabilized substrate Western Blue® for alkaline phosphatase (Promega).

The results indicate that the antibody monoclonal anti CD40 CHIR12.12 recognizes epitopes both in the form not reduced as in the reduced form of CD40, exhibiting the form non-reduced CD40 greater intensity than the reduced form of CD40 (Table 15; transfers not shown). The fact that the Positive recognition for both forms of CD40 indicates that this antibody interacts with a conformational epitope, part of the which is a linear sequence Monoclonal antibody CHIR-5.9 mainly recognizes the form no reduced CD40 suggesting that this antibody interacts mainly with a conformational epitope (Table 5; transfers not shown).

TABLE 5 Domain Identification

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

Monoclonal antibody CHIR-12.12 recognizes an epitope in Domain 2 both under reducing and non-reducing conditions (Table 6; transfers not shown). On the contrary, the antibody monoclonal CHIR-5.9 exhibits very weak recognition for Domain 2 (Table 6; transfers not shown). None these antibodies recognize Domains 1, 3 or 4 in this analysis.

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TABLE 6 Domain Analysis2

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To more precisely define the epitope recognized by the CHIR-12.12 mAb, peptides from CD40 extracellular Domain 2 were synthesized, corresponding to the sequence PCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGT
SETDTICT (residues 61-104 of the sequence shown in SEQ ID No. 10 or SEQ ID No. 12). SPOTs (Sigma) synthesis membranes containing thirty-five 10-mer peptides with a 1 amino acid shift were generated. Western blot analysis was performed with CHIR-12.12 mAb and anti-human IgG labeled with beta galactosidase as secondary antibody. The transfer was dismantled and re-probed with CHIR-5.9 mAb to determine the region recognized by this antibody.

SPOTs assays probing with anti-CD40 monoclonal antibody CHIR-12.12 at a concentration of
10 µg / ml gave positive reactions with transfers 18 to 22. The region of the sequence covered by these peptides is shown in Table 7.

TABLE 7 SPOT analysis results with sounding with anti CD40 monoclonal antibody CHIR-12.12

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These results correspond to an epitope linear 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 CD40-CD40 ligand.

SPOTs analysis with mAb CHIR-5.9 showed a weak recognition of peptides represented by transfers 20-22 shown in Table 8, suggesting involvement of the region YCDPNLGL (residues 82-89 of the sequence shown in SEQ ID No. 10 or SEQ ID No. 12) in its binding to CD40. It should be noted that the CHIR-12.12 and CHR-5.9 mAbs They compete with each other for binding to CD40 in the analysis BIACORE.

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TABLE 8 SPOT analysis results with sounding with anti CD40 monoclonal antibody CHIR-5.9

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The linear epitopes identified by the SPOTs analysis are inside the B1 module of CD40. The sequence of CD40 module B1 is:

HKYCDPNLGLRVQQKGTSETDTIC (waste 80-103 of SEQ ID No. 10 or 12).

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

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Example 8 CHIR-12.12 blocks the survival pathways and of CD40 signal mediated by CD40L in human B cells normal

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

The following studies show that CHIR-12.12 directly inhibited all these effects of stimulation in normal human B cells. For example, CHIR-12.12 treatment resulted in increased caspase-9 cleavage, caspasa-3 and PARP as well as the reduction of XIAP and Mcl-1 in a time and manner dependent manner dose, restoring apoptosis of B cells. Treatment with CHIR-12.12 also inhibited phosphorylation of I? Kinase (IKK)? And? (NF? B path), ERK, Akt and p38 in response to CD40 stimulation mediated by CD40L In addition, it was found that CHIR-12.12 did not cause these apoptotic effects without the initial stimulation of CD40 mediated by CD40L.

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

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

In summary, it was observed that the stimulation of CD40 mediated by CD40L provided survival signals as did not lead to increases in split 9 caspase, split 3 caspase or PARP cleaved over time, indicating that the cells were not suffering apoptosis However, treatment with CHIR-12.12 resulted in an increase in these cleavage products, indicating that treatment with CHIR-12.12 nullified the effects of CD40L binding on survival signals in normal stimulated B cells with cCD40L, restoring apoptosis of B cells (data not shown).

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CHIR-12.12 inhibited protein expression "survival" antiapoptotic drugs

In these experiments, 0.6 x were stimulated 10 6 normal human B cells from healthy donors (percentage purity between 85 and 95%) with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK). Then it was added CHIR-12.12 (10 µg / ml) and control IgG. The cells were collected at 0 and 20 minutes, at 2 hours, 6 hours, 18 hours and 26 hours. Controls were detected Mcl-1, XIAP, CD40 and β actin in lysates Western cell phones. In short, stimulation with sCD40L resulted in the sustained expression of Mcl-1 and XIAP over time. However the treatment of sCD40L stimulated cells with CHIR-12.12 resulted in a decrease in expression of these proteins over time (data not shown). As Mcl-1 and XIAP are signs of "survival" capable of blocking the apoptotic pathway, these results show that treatment with CHIR-12.12 eliminates blockade against apoptosis in normal B cells stimulated with sCD40L.

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Treatment with CHIR-12.12 inhibited the phosphorylation of IKK? (Ser180) and IKK? (Ser 181) in normal B cells

In these experiments, 1.0 x 10 6 normal human B cells from healthy donors (purity percentage between 85 and 95%) were stimulated with 1CD / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK) . CHIR-12.12 (10 µg / ml) and control IgG were then added. Cells were collected at 0 and 20 minutes. IKK? (Ser180) and IKK? (Ser 181) and total IKK? Controls were detected in cell lysates by transfer
Western.

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

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

In these experiments, 0.6 x were stimulated 10 6 normal human B cells from healthy donors (percentage purity between 85 and 95%) with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK). Then it was added CHIR-12.12 (0.01, 0.1, 0.2, 0.5, 1.0 µg / ml) e IgG control. Cells were collected at 24 hours. Be detected controls of cleaved PARP and β actin in the Western cell lysates.

Briefly, treatment with CHIR-12.12 resulted in increased excision of PARP in cells stimulated with sCD40L in a dependent manner of the dose and, therefore, canceled the signal path of Survival in normal B cells stimulated with sCD40L (data not revealed).

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CHIR-12.12 inhibited protein expression "survival" antiapoptotic drugs in a manner dependent on the dose

In these experiments, 0.6 x were stimulated 10 6 normal human B cells from healthy donors (percentage purity between 85 and 95%) with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK). Then it was added CHIR-12.12 (0.5, 2 and 10 µg / ml) and control IgG. Cells were collected at 22 hours. They were detected Mcl-1, XIAP, cleaved PARP and β controls Actin in cell lysates by Western blotting.

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

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CHIR-12.12 does not affect protein expression antiapoptotic, split PARP and XIAP, in the absence of signs of Soluble CD40L

In these experiments, 1.0 x 10 6 normal human B cells from healthy donors (purity percentage between 85 and 95%) were stimulated with CHIR-12.12 (10 µg / ml) and control IgG alone (ie ie, cells were not previously stimulated with sCD40L before adding the antibody). Cells were collected at 0, 4, 14 and 16 hours. XIAP, cleaved PARP and β actin controls were detected in cell lysates by transfer
Western.

Briefly, the results show that without sCD40L stimulation, cells expressed concentrations increased split PARP while XIAP expression remained constant, both in cells treated with IgG of control as in those treated with CHIR-12.12 (data not shown). These data indicate that CHIR-12.12 does not causes apoptosis in normal human B cells without stimulation of CD40L.

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

In these experiments, they were deprived of serum in medium containing 1% FBS 1.0 x 10 6 human B cells normal healthy donors (purity percentage between 85 and 95%) and they were stimulated with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK). The cultures were treated with CHIR-12.12 (1 and 10 µg / ml) and control IgG. The cells were collected at 0 and 20 minutes. Were detected phospho-IKK?, phospho-IKK?, Total IKK?, PhosfoERK, total ERK, phospho-Akt, Total Akt, phospho-p38 and total p38 in lysates Western cell phones.

In summary, stimulation with sCD40L gave as result increases in phosphorylation of IKK? /?, ERK phosphorylation, Akt phosphorylation and p38 phosphorylation, consequently giving rise to the survival and / or proliferation of the cells. The treatment of cells with CHIR-12.12 nullified the effects of stimulation of sCD40L in these signal pathways in normal B cells (data not shown).

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

In these experiments, they were deprived of serum in medium containing 1% FBS 1.0 x 10 6 human B cells normal healthy donors (purity percentage between 85 and 95%) and they were stimulated with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK). The cultures were also treated with CHIR-12.12 (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) and PD 98095 (a MEK inhibitor, 10 and 30 \ mug / ml). Cells were collected at 0 and 20 minutes. Be detected phosphoerk, phospho-Akt, total Akt, phospho-IKK? /? and total in lysates Western cell phones.

In summary, the results show that CHIR-12.12 canceled the phosphorylation of all these signal transduction molecules while inhibitors signal transduction showed only specific cancellation of the signals, indicating that CHIR-12.12 probably it inhibits these transduction molecules in an earlier stage signals mediated by CD40L stimulation (data not shown).

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

In these experiments, they were deprived of serum for four hours in medium containing 1% FBS 4.0 x 10 6 Normal human B cells from healthy donors (percent purity between 85 and 95%) and were stimulated with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK) for 20 minutes. The cells They were collected at 0 and 20 minutes. CD40 was immunoprecipitated using anti CD40 polyclonal (Santa Cruz Biotechnology, CA), and was probed at a Western transfer with anti TRAF2 mAb (Santa Cruz Biotechnology, CA), anti TRAF3 mAb (Santa Cruz Biotechnology, CA), and mAb anti CD40 (Santa Cruz Biotechnology, CA).

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In summary, the results show that TRAF2 and TRAF3 precipitated together with CD40 after stimulation of sCD40L. On the contrary, treatment with CHIR-12.12 canceled the formation of the signal complex CD40-TRAF2 / 3 in normal B cells stimulated with sCD40L. There were no changes in the expression of CD40 (data not shown).

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

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Example 9 Liquid pharmaceutical formulation for anti CD40 antibodies antagonists

The objective of this study was to investigate the pH effects of the solution on antibody stability anti CD40 antagonist CHIR-12.12 by means of biophysical and biochemical procedures to select the environment of optimal solution for this antibody. The results of differential scanning calorimetry (DSC) showed that the conformation stability of CHIR-12.12 is optimal in formulations that have pH 5.5-6.5. Based on a combination of SDS-PAGE, HPLC analysis size exclusion (SEC-HPLC) and HPLC of cation exchange (CEX-HPLC), stability Physicochemical of CHIR-12.12 is optimal at a pH of approximately 5.0-5.5. In view of these results, a recommended liquid pharmaceutical formulation that comprises this antibody is a formulation that comprises CHIR-12.12 at a concentration of approximately 20 mg / ml formulated in approximately 10 mM sodium succinate, approximately 150 mM sodium chloride and having a pH of approximately pH 5.5.

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Materials and procedures

CHIR-12.12 antibody used in formulation studies is a monoclonal antibody human produced by a CHO cell culture process. This mAb It has a molecular weight of 150 kDa and consists of two light chains and two heavy chains joined together by links disulfide It is directed against the surface CD40 receiver cell in cells expressing CD40, including B cells normal and malignant, for the treatment of various types of cancer and autoimmune / inflammatory diseases.

The substance of the anti-CD40 drug used for this study it was a bulk batch of anti CD40 (CHIR-12.12) purified obtained from CHO cells. The composition of the drug substance was antibody CHIR-12.12 9.7 mg / ml in 10 mM sodium citrate, 150 mM sodium chloride, at pH 6.5. The control sample in the study was the substance of the drug received, followed by freezing at ≤ -60 ° C, thawing at RT and tested together with stability samples at predetermined time points. Stability samples were prepared by dialysis of the drug substance against solutions of different pH and the CHIR-12.12 concentration in each of the Samples were determined by UV 280 as presented in the Table 9.

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TABLE 9 Formulations of CHIR-12.12

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

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Differential Scanning Calorimetry (DSC)

The conformational stability of different Formulations samples were monitored using a MicroCal VP-DSC after heating from 15ºC to 90ºC at the rate of 1ºC / min.

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SDS-PAGE

Fragmentation and aggregation was estimated using 4-20% Tris-glycine gel under non-reducing and reducing conditions. The proteins by means of staining with Coomassie blue.

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Size exclusion chromatography (SEC-HPLC)

Fragmentation and aggregation of proteins it was also measured by means of an HPLC Water Alliance system with a Tosohaas TSK-GEL 3000SWXL column, with phosphate of 100 mM sodium, pH 7.0 as the mobile phase at a flow rate of 0.7 ml / min

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Cation Exchange Chromatography (CEX-H PLC)

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

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Results and Discussion Conformational stability study

The thermal deployment of CHIR-12.12 revealed at least two transitions thermal, probably representing the deployment and fusion of the Fab and Fc domains, respectively. At higher temperatures, the protein presumably added, leading to loss of the DSC signal In order to select the formulation, it defined the lowest thermal transition temperature as the melting temperature, Tf, in this study. Figure 11 shows the thermal melting temperature as a function of the pH of the formulations Formulations at pH 5.5-6.5 provided anti CD40 with superior conformational stability as demonstrated by thermal melting temperatures more high.

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SDS-PAGE analysis

Samples of formulations of CHIR-12.12 at pH 4.5-9.0 were incubated at 40 ° C for 2 months and subjected to analysis SDS-PAGE (data not shown). Under conditions no reductive, 23 kDa molecular weight (PM) species were observed and 2.7 kDa in formulations with pH higher than 5.5, and were observed species with PM of 51 kDa in all formulations, but They appeared less at pH 5.0-5.5. Could you see one species with PM of 100 kDa at pH 7.5 and pH 9.0.

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

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SEC-HPLC analysis

SEC-HPLC analysis detected the CHIR-12.12 intact as the species of the beak main, a kind of aggregation as a kind of beak anterior separated from the main beak species, a kind of large fragment like a peak plateau at the back of the species of the main beak, and fragment species were detected small subsequent to the species of the main peak. Behind the incubation at 5 ° C and 25 ° C for 3 months, amounts were detected insignificant protein fragments and aggregates (<1.0%) in the above formulations and the species of the main peak of CHIR-12.12 continued to have more than 99% purity (data not revealed). However, they developed gradually fragments after storage at 40 ° C and more were formed fragments at pH 4.5 and pH 6.5-9.0, as shown in Table 21. After incubating the formulations of CHIR-12.12 at 40 ° C for 3 months, was detected approximately 2-3% aggregates at pH 7.5 and pH 9.0, while less than 1% of aggregates were detected in the formulations at other pH (data not shown). The results of SEC-HPLC indicate that CHIR-12.12 is more stable at a pH of about 5.0-6.0,

TABLE 10 Results of SEC-HPLC samples CHIR-12.12 stability under weather conditions real and accelerated storage

16

CEX-FIPLC analysis

CEX-HPLC analysis detected the CHIR-12.12 intact as the species of the beak main, acid variants eluted earlier than main peak species and lysine addition variants in the C-terminus eluted after the peak species principal. Table 11 shows the dependence of the percentages of the remaining species of CHIR-12.12 from the peak main and acidic variants in the pH of the solution. The control sample already contained a high degree of acidic species (approximately 33%), probably due to the processes of early stage fermentation and purification. The susceptibility of CHIR-12.12 at the highest pH solutions were evidence for two facts. First, the formulation sample initial at pH 9 (t = 0) already generated 12% more acidic species than the control. Second, the percentage of acidic species increased sharply with the increase in pH. Degradation related to Change of charges is probably due to deamidation. The data above indicate that this type of degradation of CHIR-12.12 was minimized at pH of approximately 5.0-5.5.

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TABLE 11 Percentage of peak area by CEX-HPLC for CHIR-12.12 in different pH formulations under real-time conditions and of accelerated storage

17

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conclusion

The pH has a significant effect on the conformational and physicochemical stability of CHIR-12.12. Degradation was determined related to changing loads is the main route of degradation for CHIR-12.12, which was minimized at pH 5.0-5.5. Based on stability data In general, a recommended liquid pharmaceutical formulation that comprises this antibody is a formulation that comprises CHIR-12.12 at a concentration of approximately 20 mg / ml formulated in approximately 10 mM sodium succinate, chloride of sodium approximately 150 mM, and it has a pH of approximately 5.5.

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Example 10 Clinical studies with CHIR-5.9 and CHIR-12.12 Clinical objectives

The general objective is to provide a therapy effective for solid tumors comprising carcinoma cells that express CD40 by turning them into targets with an anti CD40 IgG1. These tumors include lung, breast, colon, ovaries, and carcinomas. of skin. The signal for these diseases is determined in phase II although some measurement of activity in phase I can be obtained. Initially, the agent is studied as a single agent, but it will combine with other agents, chemotherapeutics, and other antibodies as development proceeds.

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Phase I

Evaluate the safety and Pharmacokinetics - dose increase in subjects with solid tumors previously cited.

Choose the dose based on the safety, tolerability and change in serum markers of CD40. In general, a maximum tolerable dose (DMT) is sought but may other indications of efficacy (cell depletion) B CD40 +, etc.) to look for the dose.

Consider more than one dose especially for different indications, for example, the dose For breast cancer it may be different than for ovarian cancer. Therefore, some dose search may be necessary in phase II

Patients are dosed weekly with sampling for pharmacokinetics (Pk) in real time. Initially the maximum allowed dose is a cycle of 4 weeks Pk can be very variable depending on the disease studied, density of CD40, etc.

This essay (s) is open to subjects with solid tumors that express CD40, including carcinomas of the lung, breast, colon, ovaries, and skin.

The decision to interrupt or continue studies is based on safety, dose and testing Preliminary antitumor activity.

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

Identify dosage (s) for the Phase II

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Phase II

Several trials will be initiated in the types of tumors mentioned above with concentration in cancer of lung, ovary and breast. You can test more than one dose and more than one program in a randomized phase II configuration.

In each disease, target a population which has failed with the current conventional treatment:

? lung: surgery, radiation therapy, chemotherapy

ovaries: surgery, therapy radiation, chemotherapy

breast: surgery, radiation therapy, chemotherapy, hormone therapy

\ ding {51} The decision to interrupt or continue with the study is based on proof of concept Therapeutic Phase II

\ ding {51} Determine if a substitute marker as an early indication for efficacy clinic

\ ding {51} Identify the doses for Phase III

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Phase III

Phase III will depend on when the signal in phase II and of which competing therapies are considered the reference pattern. If the signal is detected at a stage of the disease in which there is no reference pattern of therapy, then you can serve a study of a single arm, well controlled, As a fundamental essay. If there are competing agents that consider reference standards, then studies are conducted direct.

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32

Claims (16)

1. A monoclonal antibody human anti-CD40 that is capable of joining specifically to the CD40 antigen, said antibody being monoclonal free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group Constituted by: a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial. for use in the treatment of a human subject with a solid tumor comprising carcinoma cells that expresses the CD40 antigen.

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2. Use of an effective amount of an antibody monoclonal anti human CD40 that is capable of specifically binding to the CD40 antigen in the manufacture of a medicament for treatment of a human subject with a solid tumor comprising carcinoma cells expressing the CD40 antigen, said being monoclonal antibody free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of: a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial.

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3. An in vitro method for inhibiting the growth of a solid tumor comprising carcinoma cells expressing the CD40 antigen, said method comprising contacting said cells with an effective amount of a human anti CD40 monoclonal antibody that is capable of specifically binding to said CD40 antigen, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of: a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y

           \ newpage
        

d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial.

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4. A monoclonal antibody human anti-CD40 that is capable of joining specifically to the CD40 antigen, said antibody being monoclonal free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group Constituted by: a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial. for use in the inhibition of the growth of a solid tumor comprising carcinoma cells that express CD40 antigen.

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5. Use of an effective amount of an antibody monoclonal anti-human CD40 that is able to bind specifically to the CD40 antigen in the manufacture of a medicine for the inhibition of the growth of a solid tumor comprising carcinoma cells expressing the CD40 antigen, said being monoclonal antibody free of significant agonist activity when bound to the CD40 antigen, said antibody being selected from the group consisting of: a) a monoclonal antibody that binds to a epitope capable of binding to the monoclonal antibody CHIR-5.9 that can be obtained from the cell line of hybridoma deposited with the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to a epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to a epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; Y d) a monoclonal antibody that competes with the CHIR-5.9 monoclonal antibody that can be obtained of the hybridoma cell line deposited in the ATCC as Deposit Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive union trial.

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6. The antibody, use or procedure of any one of claims 1 to 5, wherein said antibody or its antigen binding fragment is selected from the group Constituted by: (i) an antibody or its binding fragment to antigen comprising a light chain variable domain that contains residues 44-54, 70-76 and 109-117 of SEQ ID No. 2 or SEQ ID No. 6; (ii) an antibody or its binding fragment to antigen comprising a heavy chain variable domain that Contains residues 50-54, 69-84 and 114-121 of SEQ ID No. 4 or SEQ ID No. 7; (iii) an antibody or its binding fragment to antigen comprising a light chain variable domain that contains residues 46-52, 70-72 and 111-116 of SEQ ID No. 2 or SEQ ID No. 6; Y (iv) an antibody or its binding fragment to antigen comprising a heavy chain variable domain that Contains residues 45-51, 72-74 and 115-120 of SEQ ID No. 4 or SEQ ID No. 7.

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7. The antibody, use or procedure of any one of claims 1 to 5, wherein said antibody comprises an amino acid sequence selected from the group Constituted by: (i) SEC residues 21-132 ID No. 2; (ii) SEC waste 21-239 ID No. 2; (iii) SEQ ID NO: 2; (iv) SEC residues 20-139 ID No. 4; (v) SEC residues 20-469 ID No. 4; (vi) SEQ ID NO: 4; (vii) SEC residues 20-469 ID No. 5; (viii) SEQ ID NO: 5; (ix) SEC waste 21-132 ID No. 2 and residues 20-139 of SEQ ID No. 4; (x) SEC residues 21-239 ID No. 2 and residues 20-469 of SEQ ID No. 4; (xi) SEC residues 21-239 ID No. 2 and residues 20-469 of SEQ ID No. 5; (xii) SEQ ID No. 2 and SEQ ID No. 4; Y (xiii) SEQ ID No. 2 and SEQ ID No. 5.

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8. The antibody, use or procedure of any one of claims 1 to 5, wherein said antibody or its antigen binding fragment comprises a sequence of amino acids selected from the group consisting of: (i) SEC residues 21-132 ID No. 6; (ii) SEC waste 21-239 ID No. 6; (iii) SEQ ID No. 6; (iv) SEC residues 20-144 ID No. 7; (v) SEC residues 20-474 ID No. 7; (vi) SEQ ID No. 7; (vii) SEC residues 20-474 ID No. 8; (viii) SEQ ID NO: 8; (ix) SEC waste 21-132 ID No. 6 and residues 20-144 of SEQ ID No. 7; (x) SEC residues 21-239 ID No. 6 and residues 20-474 of SEQ ID No. 7; (xi) SEC residues 21-239 ID No. 6 and residues 20-474 of SEQ ID No. 8 (xii) SEQ ID No. 6 and SEQ ID No. 7; Y (xiii) SEQ ID No. 6 and SEQ ID No. 8.

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9. The antibody, use or procedure of any preceding claim, wherein said antibody is the CHIR-5.9 monoclonal antibody that can Obtain from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or CHIR-12.12 that can be obtained from the cell line of hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543. 10. The antibody, use or procedure of any preceding claim, wherein said solid tumor is select from the group consisting of lung carcinoma, carcinoma breast, ovarian carcinoma, skin carcinoma, carcinoma of colon, urinary bladder carcinoma, liver carcinoma, gastric carcinoma, prostate cancer, cell carcinoma renal, nasopharyngeal carcinoma, squamous cell carcinoma, papillary thyroid carcinoma cervical carcinoma and sarcomas 11. The antibody, use or procedure of the claim 10, wherein said medicament is administered in a procedure further comprising administration to said subject of at least one other cancer therapy protocol selected from group consisting of surgery, radiation therapy; chemotherapy, therapy with cytokine, or other monoclonal antibody designed for use in the treatment of said solid tumor. 12. The antibody, use or procedure of any one of claims 1 to 5, wherein said antibody monoclonal is an antigen binding fragment of an antibody monoclonal, in which said fragment retains the ability to bind specifically to said human CD40 antigen. 13. The antibody, use or procedure of the claim 12, wherein said binding antibody fragment the antigen is selected from the group consisting of a fragment Fab, an F (ab ') 2 fragment, an Fv fragment and a single chain PV fragment. 14. The antibody, use or method of any preceding claim, wherein said monoclonal antibody or its antigen binding fragment binds to the human CD40 antigen with an affinity (K D) of at least
10-6 M. 15. The antibody, use or procedure of the claim 14, wherein said monoclonal antibody or its antigen binding fragment binds to human CD40 antigen with an affinity (K D) of at least 10-8 M. 16. The antibody, use or procedure of any preceding claim, wherein said antibody monoclonal or its antigen binding fragment is produced in a CHO cell line.