ES2348237T3 - USE OF ANTI-CD40 ANTI-BODIES FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA. - Google Patents

Abstract

A human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40, said monoclonal antibody being free of significant agonist activity, whereby when said monoclonal antibody is bound to the CD40 antigen expressed on the surface of said cell, growth or differentiation of said cell is inhibited, for use in a method of treating a human subject with chronic lymphocytic leukemia (CLL), the method comprising administering to said subject a quantity effective of the human anti-CD-40 monoclonal antibody, said human monoclonal anti-CD40 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 line hybridoma cell deposited in the ATCC as Patent Deposit No. PTA-5542 or to the mon antibody CHIR-12.12 oclonal that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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.

Description

FIELD OF THE INVENTION

The invention relates to methods for the treatment of chronic lymphocytic leukemia using anti-CD40 antagonist monoclonal antibodies. BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (CLL) is a B-cell cancer characterized by proliferation and accumulation of neoplastic cells in bone marrow, blood, lymph nodes and the spleen. CLL is the most common type of leukemia in adults in the western hemisphere. The incidence of CLL increases in the aging population, with the mean age at the time of diagnosis being 65 years. Current treatment protocols include chemotherapeutic agents such as fludarabine, 2-chlorodeoxyadenosine (cladribine), chloroambucil, vincristaine, pentostatin, cyclophosphamide, alemtuzumab (campath-1H), doxorubicin, and prednisone. Fludarabine is the most effective chemotherapeutic with response rates of 17 to 74%, but CLL often becomes refractory to repeated courses of the drug (Rozman and Montserrat (1995) NEJM 2133: 1052).

The average survival rate of patients with CLL is new years, although some patients with mutated immunoglobulin genes have a more favorable prognosis. See, for example, Rozman and Montserrat (1995) NEJM 2133: 1052) and Keating et al. (2003) Hematol. 2003: 153. In cases where CLL has been transformed into large cell lymphoma, the average survival drops to less than one year; Similarly, cases of prolymphocytic leukemia have a worse prognosis than classical CLL (Rozman and Montserrat (1995) NEJM 2133: 1052). To date no evidence of a cure has been obtained.

CD40 is a 55 kDa cell surface antigen present on the surface of normal B cells and human neoplastic B cells, dendritic cells, antigen presenting cells (APC), endothelial cells, monocytic and epithelial cells. The binding of the CD40 ligand to the CD40 antigen in the B cell membrane provides a positive costimulatory signal that stimulates the activation and proliferation of B cells, resulting in the maturation of B cells in a plasma cell that secretes high levels of soluble immunoglobulin . Transformed cells of patients with low and high grade B-cell lymphomas, acute lymphoblastic B-cell leukemia, CLL, myeloblastic leukemia and Hodgkin's disease express CD40. Malignant B cells of various B cell lineage tumors express a high level of CD40 and appear

depend on CD40 signaling for survival and proliferation. This makes the CD40 antigen a potential target for anti-cancer therapy. Given the poor prognosis for patients with chronic lymphocytic leukemia, alternative treatment protocols are needed.

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

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

BRIEF SUMMARY OF THE INVENTION

The invention provides a human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40, said monoclonal antibody being free of significant agonist activity, whereby when said monoclonal antibody to the CD40 antigen expressed on the surface of said cell, the growth or differentiation of said cell is inhibited, for use in a method of treating a human subject with chronic lymphocytic leukemia (CLL), the method comprising administering to said subject of an effective amount of the human anti-CD-40 monoclonal antibody, said human monoclonal anti-CD40 antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding the antibody

monoclonal CHIR-5.9 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543;

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

c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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 monoclonal antibody CHIR-12.12

which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay. The invention also provides the use of an effective amount of an antibody.

human anti-CD40 monoclonal that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40, said monoclonal antibody being free of significant agonist activity, in the manufacture of a medicament for the treatment of a subject human with chronic lymphocytic leukemia (CLL), whereby when said monoclonal antibody is bound to the CD40 antigen expressed on the surface of said cell, the growth or differentiation of said cell is inhibited, said human monoclonal anti-CD40 antibody being selected from the group constituted by: a) a monoclonal antibody that binds to an epitope capable of binding to the antibody

monoclonal CHIR-5.9 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543;

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

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

which is capable of specifically binding to a CD40 antigen, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen, for use in a method of inhibiting the growth of chronic lymphocytic leukemia (CLL) cells expressing CD40 antigen, the method comprising contacting said cells with an effective amount of the monoclonal anti-CD40 antibody, 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 Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543;

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

c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. The invention also provides the use of an effective amount of an antibody.

human anti-CD40 monoclonal that is capable of specifically binding to CD40 antigen

in the manufacture of a medicament for the inhibition of cell growth of

chronic lymphocytic leukemia (CLL) expressing CD40 antigen, said antibody being

monoclonal free of significant agonist activity when bound to 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 Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543;

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

c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. The invention also provides an in vitro method for the inhibition of

growth of chronic lymphocytic leukemia (CLL) cells expressing CD40 antigen, said method comprising contacting said cells with an effective amount of a human monoclonal anti-CD40 antibody that is capable of specifically binding to said CD40 antigen, said 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 an epitope capable of binding the antibody

monoclonal CHIR-5.9 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543;

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

c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. Other aspects of the invention are presented in the appended claims.

BRIEF SUMMARY OF THE DISCLOSURE

Methods are described for the treatment of a human subject with chronic lymphocytic leukemia (CLL), which comprise administration to the subject of an anti-CD40 antibody or an antigen-binding fragment thereof that is free of significant agonist activity when bound to a CD40 antigen or a cell that expresses human CD40. Methods for the inhibition of the growth of CLL cells expressing CD40 antigen are also described.

Anti-CD40 antagonist antibodies suitable for use herein

The invention has a strong affinity for CD40 and is characterized by a dissociation equilibrium constant (KD) of at least 10-6 M, preferably at least 10-7 M to about 10-8 M, more preferably at least about 10 -8 M to about 10-12 M. These monoclonal antibodies and their antigen binding fragments are capable of specifically binding to human CD40 antigen expressed on the surface of a human cell. They are exempt from significant agonist activity but exhibit antagonistic activity when they bind to the CD40 antigen in human cells. In one embodiment, the anti-CD40 antibody or its fragment shows antagonistic activity when it binds to CD40 antigen in normal human B cells. In another embodiment the anti-CD40 antibody or its framgneto shows antagonistic activity when it binds to CD40 antigen in malignant human B cells. Suitable monoclonal antibodies have human constant regions; preferably they also have fully or partially humanized framework regions; and most preferably they are fully human antibodies or their 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 produced recombinantly; monoclonal antibodies produced by hybridoma cell lines designated 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.D10.D6.12.12 (referred to in the present document as cell line 12.12); a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID No. 6, the sequence shown in SEQ ID No. 7; the sequence shown in SEQ ID No. 8, the sequences shown in 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 an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID No. 2, the sequence shown in SEQ ID No. 4, the sequence shown in SEQ ID No. 5, the sequences shown SEQ ID No. 2 and SEC ID No. 4, and the sequences shown SEQ ID No. 2 and SEQ ID No. 5; a monoclonal antibody comprising an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID No. 1, the sequence shown in SEQ ID No. 3, and the sequences shown in SEQ ID No. 1 and SEQ ID No. 3; and the fragments of these monoclonal antibodies that bind to the antigen that retain the ability to specifically bind to human CD40, and that are free of significant agonist activity but that exhibit antagonistic activity when they bind to the CD40 antigen in human cells, as demonstrated for normal B and human neoplastic B cells expressing CD40. Examples of such monoclonal antibodies also include a monoclonal antibody that binds to an epitope capable of binding to the monoclonal antibody produced by the hybridoma cell line 12.12 or 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 monoclonal antibody CHIR-12.12 or CHIR-5.9 in a competitive binding assay; and a monoclonal antibody that is an antigen binding fragment of the monoclonal antibody CHIR-5.9 or CHIR

12.12 or any of the above monoclonal antibodies, where the fragment retains the specific binding capacity to the human CD40 antigen.

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

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

Figure 1 shows the amino acid sequences for the light and heavy chains of the CHIR-12.12 mAb. The leading regions (residues 1-20 of SEQ ID No. 2), variable (residues 21-132 of SEQ ID No. 2), and constant (residues 133-239 of SEQ ID No. 2) of the light chain are shown in Figure 1A. The leading regions (residues 1-19 of SEQ ID No. 4), variable (residues 20-139 of SEQ ID No. 4), and constant (residues 140-469 of SEQ ID No. 4) of the heavy chain are shown in Figure 1 B. The alternative constant region for the CHIR-12.12 mAb heavy chain shown in Figure 1B reflects a substitution of an alanine residue with a serine residue at position 153 of SEQ ID NO: 4. The complete sequence for this heavy chain variant 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 shows the amino acid sequences for the light and heavy chains of the CHIR-5.9 mAb. The leading regions (residues 1-20 of SEQ ID No. 6), variable (residues 21-132 of SEQ ID No. 6), and constant (residues 133-239 of SEQ ID No. 6) of the light chain are shown in Figure 3A. The leading regions (residues 1-19 of SEQ ID No. 7), variable (residues 20-144 of SEQ ID No. 7), and constant (residues 145-474 of SEQ ID No. 7) of the heavy chain are shown in Figure 3B. The alternative constant region for the CHIR-5.9 mAb heavy chain shown in Figure 3B reflects a substitution of an alanine residue with a serine residue at position 158 of SEQ ID NO.

7. The complete sequence for this variant of the heavy chain of mAB CHIR-5.9 is presented in SEQ ID NO: 8.

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

Figure 5 shows that the CHIR-12.12 monoclonal antibody inhibits CD40L mediated proliferation of cancer cells in patients with CLL (n = 9) at 48 hours (Figure 5A) and 72 hours (Figure 5B).

Figure 6 shows that the CHIR-12.12 monoclonal antibody does not show a stimulatory effect in CLL patient cells (n = 9) at 48 hours (Figure 6A) and 72 hours (Figure 6B).

Figure 7 shows ADCC-mediated cell lysis of CLL EHEB cell line by CHIR-12.12 monoclonal antibody against Rituxan® monoclonal antibody.

Figure 8 shows the melting temperature of CHIR-12.12 in different pH formulations measured by differential scanning calorimetry (DSC). DETAILED DESCRIPTION

"Tumor," as used herein, refers to all growth and

proliferation of neoplastic cells, whether malignant or benign, and to all precancerous and cancerous cells and tissues.

The terms "cancer" and "cancerous" refer to, or describe, the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, lymphs and leukemias.

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

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

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

"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each light and heavy chain also has regularly spaced intra-chain disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the variable domains of the light and heavy chains.

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

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

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues of a "complementarity determining region" or "CDR" (ie residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and 31-35 (H1), 50-65 (H2) and 95102 (H3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th ed., Public Health Service, National Institute of Health, Bethesda, MD) and / or residues of a “hypervariable loop” (ie residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and 2632 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Clothia and Lesk (1987) J. Mol. Biol. 196: 901 -917) The residues of the "frame" or "FR" are the residues of the variable domain different from the residues of the hypervariable region.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antibody binding region or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ’, F (ab’) 2 and Fv fragments; divalent fragments; linear antibodies (Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062); single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of the antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects their ability to crystallize easily. Pepsin treatment gives an F (ab ’) 2 fragment that has two combination sites with the antigen and is still capable of forming a cross-link with the antigen.

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

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

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

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

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

or substrate composition that is detectable. 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 broadest sense and includes any molecule that partially or totally blocks, inhibits or neutralizes a biological activity of a native target disclosed herein or its transcription or translation.

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

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

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

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

The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al. (1976) J. Immunol. 117: 587 and Kim et al. (1994) J. Immunol. 24 : 249 (1994)).

There are several ways to produce human antibodies. For example, secretory cells can be immortalized by infection with the Epstein-Barr virus (EBV). However, EBV infected cells are difficult to clone and usually produce only relatively low immunoglobulin yields (James and Bell (1987) J. Immunol Methods 100: 5-40). In the future, the immortalization of human B cells could possibly be achieved by introducing a defined combination of transforming genes. This possibility is highlighted by a recent demonstration that the expression of the telomerase catalytic subunit together with the large SV40 oncoprotein and an oncogenic H-ras allele resulted in the tumorigenic conversion of normal human fibroblast and epithelial cells (Hahn et al. (1999) Nature 400: 464-468). It is now possible to produce transgenic animals (eg, mice) that are capable, after immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production (Jakobovits et al. (1993) Nature 362: 255-258; Lonberg and Huszar (1995) Int. Rev. Immunol. 13: 65-93; Fishwild et al. (1996) Nat. Biotechnol. 14: 845-851; Mendez et al. (1997) Nat. Genet. 15: 146-156 ; Green (1999) J. Immunol. Methods 231: 11-23; Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-727; compiled in Little et al. (2000) Immunol. Today 21: 364-370). For example, it has been described that homozygous deletion of the heavy chain (JH) binding region of the antibody in chimeric mice and germline mutants results in complete inhibition of endogenous antibody production (Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90: 25512555). Transfer of the set of human germline immunoglobulin genes in such germline mutant mice results in the production of human antibodies after challenge with antigens (Jakobovits et al. (1993) Nature 362: 255-258). Mendez et al. (1997) (Nature Genetics 15: 146-156) have generated a line of transgenic mice that, when challenged with an antigen, generate fully human antibodies of high affinity. This was achieved by the integration in the germline of the megabase heavy chain and human light chain loci in mice with deletion in the endogenous JH segment as described above. These mice (XenoMouse® II technology (Abgenix; Fremont, California)) house 1,020 kb of the human heavy chain locus that contains approximately 66 VH genes, complete DH and JH regions, and three different constant regions, and also houses 800 kb of the Human κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. The antibodies produced in these mice closely resemble those seen in humans in all aspects, including genetic reorganization, structure and repertoire. Human antibodies are preferably expressed on endogenous antibodies by deletion in the endogenous segment that prevents genetic reorganization in the murine locus. Such mice can be immunized with an antigen of particular interest.

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

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

The present invention relates to compositions and methods for the treatment of human subjects presenting with chronic lymphocytic leukemia (CLL). The methods involve treatment with an anti-CD40 antibody described herein, or one of its antigen-binding fragments, in which the administration of the antibody or its antigen-binding fragment promotes a positive therapeutic response in the subject undergoing to this therapy procedure. Anti-CD40 antibodies suitable for use in the methods of the invention specifically bind to the human CD40 antigen expressed on the surface of a human cell and are free of significant agonist activity, but exhibit antagonistic activity when bound to the CD40 antigen in a human cell. which expresses CD40, as demonstrated for normal and neoplastic human B cells expressing CD40, including CLL cells. These anti CD40 antibodies and their antigen binding fragments are referred to herein as anti CD40 antagonist antibodies. Such antibodies include, but are not limited to, the fully human CHIR-5.9 and CHIR-12.12 monoclonal antibodies described below and the monoclonal antibodies having the binding characteristics of the CHIR-5.9 and CHIR-12.12 monoclonal antibodies. also described below.

These monoclonal antibodies that can be produced recombinantly are described below.

Antibodies that have the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12 include antibodies that competitively interfere with the binding of CD40 and / or bind to the same epitopes as CHIR-5.9 and CHIR-12.12. An expert could determine whether an antibody interferes competitively with CHIR-5.9 or CHIR-12.12 using conventional procedures, known in the art.

When these antibodies bind to exposed CD40 on the surface of human cells, such as human B cells, the antibodies are free of significant agonist activity; In some embodiments, their binding to CD40 exposed on the surface of human cell cells results in the inhibition of proliferation and differentiation of these human cells. Accordingly, anti-CD40 antagonist antibodies suitable for use in the invention include those monoclonal antibodies that can exhibit antagonistic activity towards human cells that express the CD40 cell surface antigen. Anti CD40 Antagonist Antibodies

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

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

In addition to the antagonistic activity, it is preferred that the anti-CD40 antibodies of this invention have another mechanism of action against a tumor cell. For example, the native CHIR-5.9 and CHIR-12.12 antibodies have ADCC activity. Alternatively, the variable regions of the CHIR-5.9 and CHIR-12.12 antibodies can be expressed in another antibody isotype that has ADCC activity. It is also possible to conjugate native forms, recombinant forms or fragments of CHR-5.9 or CHIR-12.12 that bind the antigen to a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope, as indicated herein below.

The monoclonal antibodies CHIR-5.9 and CHIR-12.12 bind to soluble CD40 in ELISA type assays, prevent binding of the CD40 ligand to the CD40 of the cell surface, and displace the previously bound CD40 ligand, as determined by assays of flow cytometry. The CHIR-5.9 and CHIR-12.12 antibodies compete with each other for binding to CD40 but not with 15B8, the anti-CD40 monoclonal antibody described in WO 82/28904. When the effects on B cell proliferation of normal human subjects are tested in vitro, CHIR-5.9 and CHIR-12.12 act as anti-CD40 antagonist antibodies. In addition, CHIR-5.9 and CHIR-12.12 do not induce strong proliferation of human lymphocytes from normal subjects. These antibodies are capable of killing CD40-expressing target cells by antibody-dependent cellular 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 the Biacore ™ assay.

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

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

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

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

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

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

Therefore, in addition to the monoclonal antibodies CHIR-5.9 and CHIR12.12, other antibodies that would be useful in the practice of the present invention described herein include, but are not limited to, the following: (1) the monoclonal antibodies produced by hybridoma cell lines designated 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.D10.D6.12.12 (referred to herein as cell line 12.12), deposited with the ATCC as Patent Deposit No. PTA-5542 and Patent Deposit No. PTA-5543, respectively; (2) a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID No. 2, the sequence shown in SEQ ID No. 4, the sequence shown in SEQ ID No. 5, the sequences shown in 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 SEQ 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 having the amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in SEQ ID No. 1, the nucleotide sequence shown in SEQ ID NO. 3, and 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) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the amino acid sequence shown in SEQ ID No. 10 or SEQ ID No. 12; (7) a monoclonal antibody that competes with the monoclonal antibody CHIR-5.9 or CHIR-12.12 in a competitive binding assay; and (8) a monoclonal antibody that is a fragment of the CHIR monoclonal antibody

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

Anti-CD40 antagonist antibodies for use in the present invention can be produced by any antibody production method known to those skilled in the art. Polyclonal sera can be prepared by conventional procedures. In general, a solution containing the CD40 antigen is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits or goats are preferably for the preparation of polyclonal sera because of 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 g x for 10 minutes). Approximately 20-50 ml can be obtained by drawing blood from rabbits.

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

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

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

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

Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256: 495-496, or 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 is then induced,

or of all cells separated from the spleen, with myeloma cells to form hybridomas, and grown 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).

As an alternative to the use of hybridomas, the antibody can be produced in a cell line such as a CHO cell line, as described 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.

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

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

Humanized anti-CD40 antibodies represent additional anti-CD40 antibodies suitable for use in the invention. By "humanized" are meant forms of anti-CD40 antibodies that contain a minimal sequence derived from non-human immunoglobulin sequences. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues of a hypervariable region (also known as complementarity determining region or CDR) of the receptor are replaced by residues of a hypervariable region of a non-human species (antibody donor) such as mouse, rat, rabbit or non-human primate that has the desired specificity, affinity and capacity. The term "complementarity determining region" refers to the amino acid sequences that together define the affinity and binding specificity of the natural Fv region of a native immunoglobulin binding site. See, for example, Chothia et al. (1987) J. Mol. Biol

196: 901-917; Kabat et al. (1991) U.S. Dept of Health and Human Services, NIH Publication No. 91-3242). The term "constant region" refers to the part of the antibody molecule that confers effector functions. In previous studies aimed at the production of non-immunogenic antibodies for use in therapy of human diseases, mouse constant regions were replaced by human constant regions. The constant regions of the subject's humanized antibodies were obtained from human immunoglobulins. However, these humanized antibodies still caused an unwanted and potentially dangerous immune response in humans and there was loss of affinity. Humanized anti-CD40 antibodies for use in the present invention have binding characteristics similar to those exhibited by the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described herein.

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

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

5,939,598.

Preferably, fully human antibodies against CD40 are obtained by immunizing transgenic mice. One such mouse is obtained using XenoMouse® technology (Abgenix; Fremont, California), and is disclosed in US Patent Nos. 6,075,181, 6,091,001 and 6,114,598. To produce the antibodies disclosed herein, transgenic mice were immunized for the human IgG1 heavy chain locus and the human κ light chain locus with Sf9 cells expressing human CD40. Mice can also be transgenic for other isotypes. Fully human antibodies useful in the methods of the present invention are characterized by having binding properties similar to those exhibited by the monoclonal antibodies CHIR-5.9 and CHIR-12.12 described herein.

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

Suitable fragments of an antibody that bind to the antigen comprise a portion of a full length antibody, usually the antigen binding region

or one of its variable regions. Examples of antibody fragments include, but are not limited to, Fab, F (ab ’) 2 and Fv fragments and single chain antibody molecules. By "Fab" is meant a monovalent fragment of an immunoglobulin that binds to the antigen that is composed of the light chain and part of the heavy chain. F (ab ’) 2 means a bivalent fragment of an immunoglobulin that binds to the antigen that contains both light chains and part of both heavy chains. By "single chain Fv" or "sFv" fragments of the antibody is meant fragments comprising the VH and VL domains of an antibody, in which these domains are present in a single polypeptide chain. See, for example, U.S. Patent Nos. 4,946,778, 5,260,203, 5,455,030 and 5,856,456. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun (1994) in The Pharmacology of Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pages 269-315. Fragments of the anti-CD40 antagonist antibodies that bind to the antigen disclosed herein may also be conjugated to a cytotoxin to produce lysis of the target cancer cells, as described hereinbelow.

Antibodies or antibody fragments can be isolated from phage antibody libraries generated using the techniques described in, for example, McCafferty et al. (1990) Nature 348: 552-554 (1990) and in US Patent No. 5,514,548. Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol

222: 581-597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Later publications describe the production of high affinity human antibodies (of the order of nM) by random chain mixing (Marks et al. (1992) Biol / Technology 10: 779-783), as well as by combinatorial infection and recombination in vivo as a strategy to build very large phage libraries (Waterhouse et al. (1993) Nucleic, Acids Res. 21: 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 chemically coupled to form F (ab ’) 2 fragments (Carter et al. (1992) Bio / Technology 10: 163-167). According to another approach, F (ab ’) 2 fragments can be isolated directly from recombinant host cell culture. Another technique for the production of antibody fragments will be apparent to the person skilled in the art.

Anti-CD40 antagonist antibodies useful in the present invention include the monoclonal antibodies CHIR-5.9 and CHIR 12.12 disclosed herein as well as antibodies that differ from these antibodies but retain CDRs; and antibodies with one or more 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 antibodies. anti-CD40 de-immunized antagonists, 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 convert to antibodies in non-immunogenic or less immunogenic to humans while maintaining their 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 may 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 been shown that sequences within the CDR can cause an antibody to bind to MAC Class II and trigger an unwanted helper T cell response. A conservative substitution may allow the antibody to retain binding activity and still lose its ability to trigger an unwanted T-cell response. Any such conservative or non-conservative substitutions may be made using methods recognized in the art, such as those set forth elsewhere herein, and the resulting antibodies will be within the scope of the invention. Variant antibodies can be routinely tested to verify their antagonistic activity, affinity and specificity using the procedures described herein.

An antibody produced by any of the procedures described above, or any other procedure not disclosed herein, will be within the scope of this description if it has at least one of the following biological activities: inhibition of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by cells expressing CD40L or soluble CD40 ligand; and inhibition of the proliferation of human malignant B cells as noted below. See the tests described in Schultze et al. (1998) Proc. Natl Acad Sci. USA 92: 8200-8204; Denton et al. (1998) Pediatr. transplant 2: 6-15; Evans et al. (2000) J. Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13: 12; Kwekkeboom et al. (1993) Immunology 79: 439-444; and U.S. Patent Nos. 5,674,492 and 5,847,082.

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

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

Any of the anti-CD40 antagonist antibodies or their antibody fragments described previously can be conjugated before use in the present invention. Methods for producing conjugated antibodies are known in the art. Accordingly, the anti CD40 antibody can be labeled using indirect labeling or an indirect labeling approach. By "indirect labeling" or "indirect labeling approach" is meant that a chelating agent covalently binds to an antibody and at least one radionuclide is inserted into the chelating agent. See, for example, the chelating agents and radionuclides described in Srivagtava and Mease (1991) Nucl. Med. Bio. 18: 589-603. Suitable markers include fluorophores, chromophores, radioactive atoms (especially 32P and 125I), electron-dense reagents, enzymes and ligands that have specific binding partners. Enzymes are normally detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3 ’, 5,5 ′ tetramethylbenzidine (TMB) into a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding to a ligand molecule with high specificity, such as in the case of an antigen and a specific monoclonal antibody for it. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not intended to categorize the various markers into different classes, since the same marker can serve in several different ways. For example, 125I can serve as a radioactive label or as a dense electron reagent. HRP can serve as an enzyme or as an antigen for a mAb. In addition, various markers can be combined for the desired effect. For example, mAbs and avidin also require markers in the practice of this invention: therefore, a mAb can be labeled with biotin, and its presence detected with avidin labeled with 125I, or with an antibiotic mAb labeled with HRP. Other permutations and possibilities will be readily apparent to those skilled in the art, and are considered as equivalent within the scope of the present invention.

Alternatively, the anti CD40 antibody can be labeled using "direct labeling" or a "direct labeling approach", in which a radionuclide is covalently attached 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 Pat. No.

6,015,542.

In addition, an antibody (or its fragment) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, 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, dihydroxy anthracin dione, mitoxantrone, mitramycin, D-streptococcal, 1-dihydrocortin, glutathicotrophinin, 1-dihydrocortin, glutathin-acetonic acid procaine, tetracaine, lidocaine, propranolol and puromycin, and their analogues and homologues. Therapeutic agents include, but are not limited to, antimetabolites (for example, fludarabine, 2-chlorodeoxyadenosine, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5fluorouracildecarbacin), alkylating agents (for example, mechlorethamine, thioepam carmelom, chloramoleamine, chloramoleamine, chloramoleamine, chloramoleamine, chlorambalimine, chloramoleamine, chloramoleamine, chloramoleamine, chloramoleamine, chlorambalimine, chloramoleamine, chlorambalimine, chlorambalimine, chloramoleamine, chloramoleamine, chloramoleamine, chlorambalimine, chlorambalimine, chloramoleamine (chloramoleamine) 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 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, I125, Y-90 , Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111 and the like. The conjugates of the invention can be used to modify a given biological response; 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 that has 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, alpha interferon, beta interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, or, biological response modifiers such as, for example, lymphokines, interleukin 1 (“IL-1”), interleukin 2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte and macrophage colony stimulating factor (“GM-CSF”), stimulator factor granulocyte colonies ("G-CSF") or other growth factors.

Techniques for conjugating such therapeutic moieties to antibodies are well known. See, for example, Amon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pages 243-256; ed. Hellstrom et al. (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, ed. Robinson et al. (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 an antibody heteroconjugate as described in US Pat. No.

4,676,980. In addition, connectors between the markers and the antibodies of the invention can be used (see US Patent No. 4,831,175). Antibodies or their antigen binding fragments can be directly labeled with iodine, indium, yttrium radioactive or with other radioactive particles known in the art (US Patent No. 5,595,721). The treatment may consist of a combination of treatment with conjugated and unconjugated antibodies administered simultaneously or consecutively (WO 00/52031 and WO 00/52473). Anti-CD40 antagonist antibody variants

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

For example, variants of amino acid sequences of a

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

In the construction of variants of the polypeptide of interest of the anti-CD40 antagonist antibody, the modifications are made such that the variants continue to have the desired activity, that is, similar binding affinity and are capable of specifically binding to 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 Patent Application Publication No. 75,444.

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

Preferably, variants of an anti-CD40 antagonist reference antibody have amino acid sequences that have at least 70% or 75% sequence homology, preferably at least 80% or 85% sequence homology, more preferably at least 90%, 91%, 92%, 93%, 94% or 95% sequence homology with the amino acid sequence for the reference antagonist anti CD40 antibody molecule, for example, the monoclonal antibody CHIR-5.9 or CHIR-12.12 described herein, or with a shorter part of a reference antibody molecule. More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence homology. For the purposes of the present invention, the percentage of sequence homology is determined using the Smith-Waterman homology search algorithm using a related hollow search with open hole penalty parameters of 12 and gap extension penalty of 2 , BLOSUM matrix of

62. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math 2: 482-489. A variant may, for example, differ from the reference antagonist anti CD40 antibody in as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

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

The exact chemical structure of a polypeptide capable of specifically binding to CD40 and retaining antagonistic activity, particularly when it binds to the CD40 antigen in malignant B cells, depends on a number of factors. Since ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide can be obtained as an acidic or basic salt, or in a neutral form. All preparations that retain their biological activity when placed under 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 regarding the preparation and use of polypeptide variants. In the preparation of anti-CD40 antibody variants, one skilled in the art can readily determine which modifications to the nucleotide or amino acid sequence of the native protein will result in a variant that is suitable for use as a therapeutically active component of a composition. pharmaceutical used in the procedures described in this document. Therapy methods using the anti-CD40 antagonist antibodies of the invention

The procedures disclosed herein are directed to the use of anti-CD40 antagonist antibodies to treat subjects (i.e., patients) who have chronic lymphocytic leukemia (CLL), wherein the cells of this cancer express the CD40 antigen. "Chronic lymphocytic leukemia cells expressing CD40" means CLL cells expressing the CD40 antigen. The successful treatment of CLL depends on how advanced the cancer is at the time of diagnosis, and whether the subject has undergone or will undergo other therapy procedures in combination with administration of anti-CD40 antibody. Methods for detecting CD40 expression in cells are well known in the art and include, but are not limited to, PCR techniques, immunohistochemistry, flow cytometry, Western blotting, ELISA and the like.

A plurality of criteria can be used to classify the degree of CLL. The procedures described in this invention can be used to treat cased CLL according to the Rai-Binet classification system. In Rai's system, there are five grades: grade 0 in which only lymphocytosis is present; grade I in which lifadenopathy is present; grade II in which splenomegaly, lymphadenopathy are present

or both; grade III in which anemia, organomegaly or both are present (progression is defined by weight loss, fatigue, fever, massive organomegaly, and a rapid increase in the number of lifes); and grade IV in which anemia, thrombocytopenia, organomegaly or a combination thereof are present. With the Binet classification system there are only three categories: grade A in which lymphocytosis is present and less than three lymph nodes are enlarged (this grade is included in all grade 0 patients according to Rai, half of patients in grade 1 according to Rai, and a third of patients in grade II according to Rai); grade B, in which three or more lymph nodes are involved; and grade C in which anemia or thrombocytopenia are present, or both. The Rai-Binet classification system can be combined with measures of mutation of immunoglobulin genes to provide a more accurate characterization of the extent of the disease. The presence of mutated immunoglobulin genes correlates with a better prognosis.

The procedures of the present disclosure are applicable to the treatment of CLL classified according to any of the above criteria. Precisely because these criteria can be used to characterize the progressive degrees of the disease, these same criteria, that is, anemia, lymphadenopathy, organomegaly, thrombocytopenia and mutation of immunoglobulin genes, can be controlled to assess the efficacy of the treatment.

"Treatment" is defined herein as the application or administration of an anti-CD40 antagonist antibody or its antigen binding fragment to a patient, or the application or administration of an anti-CD40 antagonist antibody or its antigen binding fragment a an isolated tissue or cell line of a subject, in which the subject has CLL, a symptom associated with CLL, or a predisposition towards the development of CLL, the goal being to heal, heal, calm, relieve, alter, remedy, improve or affect CLL, any symptoms associated with CLL or the predisposition to the development of CLL. By "treatment" is also meant the application or administration of a pharmaceutical composition comprising the anti-CD40 antagonist antibody or its antigen binding fragment to a subject, or the application or administration of a pharmaceutical composition comprising the anti-CD40 antagonist antibody or its fragment of binding to the antigen to an isolated tissue or a cell line of a subject, which has CLL, a symptom associated with CLL or a predisposition to the development of CLL, being the objective to cure, heal, calm, relieve, alter, remedy, improve or affect CLL, the symptoms of CLL or the predisposition to the development of CLL.

By "antitumor activity" is meant a reduction in the rate of proliferation or accumulation of malignant cells expressing CD40, and therefore a decrease in the growth rate of an existing tumor or a tumor that arises during therapy, and / or the destruction of existing neoplastic cells (tumor) or new formed neoplastic cells, and 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) elicits a physiological response that is advantageous with respect to the treatment of CLL in a human, where the disease comprises cells expressing CD40 antigen. It is recognized that the procedures described herein may be useful in preventing other tumor consequences of CLL cells that arise during therapy.

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

Tumor response can be evaluated for changes in tumor morphology (i.e., general tumor burden, tumor size and the like) using screening techniques such as magnetic resonance imaging (MRI) scanning, x-ray images, computed tomography (CT) scan, flow cytometry

or fluorescence activated cell sorter (FACS) analysis, bioluminescent images, for example, luciferase images, bone scintigraphy images and collection of tumor biopsy samples including bone marrow aspiration (AMO). In addition to these positive therapeutic responses, the subject undergoing therapy with the anti-CD40 antagonist antibody or its antigen binding fragment may experience the advantageous effect of an improvement in the symptoms associated with the disease.

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

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

Anti-CD40 antagonist antibodies can be used in combination with chemotherapeutics, alone or in combination with surgery or surgical procedures (for example, splenectomy, hepatectomy, lymphadenectomy, leukophoresis, bone marrow transplant, and the like); radiation therapy; Chemotherapy, another anti-cancer therapy with monoclonal antibodies, steroids, IL-2 therapy and interferonaalpha for the treatment of CLL. Thus, the anti-CD40 antagonist antibodies described herein, or their antigen-binding fragments, are administered in combination with at least one other cancer therapy, including, but not limited to, surgery, radiation therapy, chemotherapy. , another anti-cancer therapy with monoclonal antibodies (for example, alemtuzumab (Campath®), which targets the CD52 cell surface antigen in malignant B cells; rituximab (Rituxan®), which targets the CD20 cell surface antigen in cells Malignant B, or other therapeutic anti-CD20 antibody, for example, the fully human HuMax-CD20 antibody, R1594, IMMU-106, TRU-015, AME-133, tositumomab / I-131 tositumomab (Bexxar®), ibritumomab tiuxetan ( Zevalin®), or anti CD20 antibody directed to the CD23 antigen in malignant B cells); interferon-alpha therapy, interleukin-2 (IL-2) therapy, IL2, IL-15 or IL-21 therapy, or steroid therapy, in which additional anticancer therapy is given before, during, or after to therapy with the anti CD40 antibody. Therefore, when the combination therapies comprise the administration of an anti-CD40 antibody or its antigen-binding fragment in combination with the administration of another therapeutic agent, such as with chemotherapy, radiation therapy, or interferon-alpha therapy, IL-2 and / or steroids, the procedures described in this document encompass co-administration, using separate formulations of a single pharmaceutical formulation, and consecutive administration in any order. When the methods described herein comprise combined therapeutic regimens, these therapies can be given simultaneously, that is, the anti-CD40 antagonist antibody or its antigen binding fragment is administered simultaneously or within the same period of time as the other anti-therapy. cancer (ie, therapies take place simultaneously, but the anti-CD40 antagonist antibody or its antigen binding fragment is not administered precisely at the same time as the other cancer therapy). Alternatively, the anti-CD40 antibody of the present invention or its antigen-binding fragment can also be administered before or after other cancer therapy. The sequential administration of the different cancer therapies can be carried out regardless of whether the treated subject responds to the first course of therapy to reduce the possibility of remission or relapse. When the combined therapies comprise administration of the anti-CD40 antibody or its antigen-binding fragment with administration of a cytotoxic agent, preferably the anti-CD40 antibody or its antigen-binding fragment, it is administered before administering the cytotoxic agent.

In some embodiments of the disclosure, the anti-CD40 antagonist antibodies described herein, or their antigen binding fragments, are administered in combination with chemotherapy, and optionally in combination with autologous bone marrow transplantation, in which the antibody and the (the) chemotherapeutic agent (s) may be administered sequentially, in any order, or simultaneously (ie, simultaneously or within the same period of time). Examples of suitable chemotherapeutic agents include, but are not limited to, fludarabine, chlorambucil, vincristine, pentostatin, 2-chlorodeoxyadenosine (cladribine), cyclophosphamide, doxorubicin and prednisone.

Therefore, for example, in some embodiments, the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CH1R-5.9, or its antigen-binding fragment is administered in combination with fludarabine. In one such embodiment, the anti-CD40 antagonist antibody is administered before administration of fludarabine. In alternative embodiments, the anti-CD40 antagonist antibody is administered after treatment with fludarabine. In still other embodiments, fludarabine is administered simultaneously with the anti-CD40 antagonist antibody.

In other embodiments of the disclosure, chlorambucil, an alkylating agent, is administered in combination with an anti-CD40 antagonist antibody described herein, for example, the monoclonal antibody CHIR 12.12 or CHIR-5.9, or its antigen binding fragment. In one such embodiment, the anti-CD40 antagonist antibody is administered before administration of chlorambucil. In alternative embodiments, the anti-CD40 antagonist antibody is administered after treatment with chlorambucil. In still other embodiments, chlorambucil is administered simultaneously with the anti-CD40 antagonist antibody.

In still other embodiments, anthracycline-containing regimens such as CAP (cyclophosphamide, doxorubicin plus prednisone) and CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin) can be combined with the administration of an antagonistic anti-CD40 antibody described herein, for example the antibody. monoclonal CHIR 12.12 or CHIR-5.9, or its antigen binding fragment. In one such embodiment, the anti-CD40 antagonist antibody is administered before administration of anthracycline containing regimens. In other embodiments, the anti-CD40 antagonist antibody is administered after treatment with anthracycline containing regimens. In still other embodiments, the anthracycline containing regimen is administered simultaneously with the anti-CD40 antagonist antibody.

In alternative embodiments, an anti-CD40 antagonist antibody described herein, for example, the CHIR 12.12 or CHIR-5.9 monoclonal antibody, or its antigen binding fragment, is administered in combination with alemtuzumab (Campath®; distributed by Berlex Laboratories , Richmond, California). Alemtuzumab is a humanized recombinant monoclonal antibody (Campath-1H) that targets the CD52 antigen expressed in malignant B cells. In one such embodiment, the anti-CD40 antagonist antibody is administered before administration of alemtuzumab. In other embodiments, the anti-CD40 antagonist antibody is administered after treatment with alemtuzumab. In still other embodiments, alemtuzumab is administered simultaneously with the anti-CD40 antagonist antibody.

In other embodiments, the anti-CD40 antagonist antibodies described herein, for example the CHIR 12.12 or CHIR-5.9 monoclonal antibody, or its antigen binding fragment, can be used in combination with another agent having anti-angiogenic properties, such as thalidomide, or interferon-alpha. The latter agents can be effective when a subject is resistant to first-line therapy. Alternatively, anti-CD40 antagonist antibodies can be administered to a subject in combination with high-dose chemotherapy, alone or with autologous bone marrow transplantation.

In alternative embodiments, an anti-CD40 antagonist antibody described herein, for example the monoclonal antibody CHIR 12.12 or CHIR-5.9, or its antigen binding fragment, can be used in combination with other immunotherapeutic agents, notably IL-2. , IL-2, an agent known to increase the amount of natural cytolytic effector cells (NK) in treated patients, can be administered before, or in conjunction with, the anti-CD40 antagonist antibody of the invention. This increased amount of effector NK cells may result in increased ADCC activity of the administered anti-CD40 antagonist antibody.

In addition, combination therapy with two or more therapeutic agents and an anti-CD40 antibody described herein can also be used for the treatment of CLL. Without being limited, examples include triple combination therapy, in which two chemotherapeutic agents are administered in combination with an anti-CD40 antagonist antibody described herein, and in which a therapeutic agent and another anti-cancer monoclonal antibody (e.g., alemtuzumab, rituximab, or other anti-CD20 antibody, for example, the fully human HuMax-CD20 antibody, R-1594, IMMU-106, TRU-015, AME-133, tositumomab / I-131 tositumomab (Bexxar®), ibritumomab tiuxetan (Zevalin®), or anti-CD23 antibody) are administered in combination with an anti-CD40 antagonist antibody described herein. Examples of such combinations include, but are not limited to, combinations of fludarabine, cyclophosphamide, and the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR 12.12 or CHIR-5.9, or its antigen binding fragment; and combinations of fludarabine, an anti-CD20 antibody, for example, rituximab (Rituxan®; IDEC Pharmaceuticals Corp., San Diego, California), and the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR 12.12 or CHIR-5.9, or its antigen binding fragment. Pharmaceutical formulations and modes of administration

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

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

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion), oral and pulmonary (e.g., inhalation), nasal, transdermal administration (topical), transmucosal and rectal. The solutions

or suspensions used for parenteral, intradermal or subcutaneous applications 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; antibacterial agents such as benzyl alcohol or methylparabenos; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparation can be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

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

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

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

course of a treatment period.

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

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

In some embodiments of the disclosure, anti-CD40 antagonist antibody therapy is initiated by administering a "loading dose" of the antibody or its antigen-binding fragment to the subject in need of therapy with anti-CD40 antagonist antibodies. By "loading dose" is meant an initial dose of the anti-CD40 antagonist antibody or its antigen-binding fragment that is administered to the subject, in which the dose of the antibody or its antigen-binding fragment administered is within the dosage range. higher (that is, from about 20 mg / kg to about 50 mg / kg). The "loading dose" can be administered as a single administration, for example, a single infusion in which the antibody or its antigen binding fragment is administered via IV, or as multiple administrations, for example, multiple infusions in which the antibody or its antigen binding fragment is administered IV, provided that the full "loading dose" is administered over a period of approximately 24 hours. After administration of the "loading dose", one or more therapeutically effective doses of the anti-CD40 antagonist antibody or its antigen-binding fragment is then administered to the subject. Subsequently therapeutically effective doses may be administered, for example, according to a weekly dosing schedule, or once every two weeks, once every three weeks, or once every four weeks. In such embodiments, subsequent therapeutically effective doses are generally in the lower dosage range (ie, from 0.003 mg / kg to about 20 mg / kg).

Alternatively, in some embodiments, after the "loading dose", subsequent therapeutically effective doses of the 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 higher 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 for use in the methods disclosed herein may be native or may be obtained by recombinant techniques and may be from any source, including mammalian sources. such as, for example, mouse, rat, rabbit, primate, pig and human. Preferably such polypeptides are obtained from a human source, and more preferably they are human proteins, recombinant of hybridoma cell lines.

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

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

Formulation compounds can be added to pharmaceutical compositions comprising an anti-CD40 antagonist antibody of the invention. These formulation compounds may include, but are limited to, oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants or fillers. Preferred carbohydrates include sugars or sugar alcohols such as mono-, di- or polysaccharides, or water soluble glucans. Saccharides or glucans may include fructose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulane, dextrin, cyclodextrin α and β, soluble starch, hydroxyethyl starch and carboxymethylcellulose, or mixtures thereof. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having a hydroxyl group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol and arabitol. These sugars or sugar alcohols can be used individually or in combination. The concentration of sugar or sugar alcohol is between 1.0% and 7% w / v, more preferably between 2.0% and 6.0% w / v. Preferred amino acids include levógira (L) forms of carnitine, arginine, and betaine; however, other amino acids can be added. Preferably the polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. The surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110.

In addition, the antibodies can be chemically modified by covalent conjugation to a polymer to, for example, increase their circulation half-life. Preferred polymers and methods for binding them to peptides are shown in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R (O-CH2 -CH2) n O-R where R can be hydrogen, or a protecting group such as an alkyl or alkanol group. Preferably, the protecting group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG preferably has an average molecular weight between

1,000 and 40,000, more preferably between 2,000 and 20,000, 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 hydroxy group that is preferably activated to react with a free amino group in the inhibitor. However, it will be understood that the type and amount of the reactive groups can be varied to obtain a covalently conjugated PEG / antibody of the present invention.

Water soluble polyoxyethylated polyols are also useful in the present invention. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and others. The POG is preferred. One reason is because the glycerol central structure of polyoxyethylated glycerol is the same central structure that occurs in nature in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching will not necessarily be considered as a foreign agent in the body. The POG has a molecular weight of preference in the same range as PEG. The structure for the POG is shown in Knauf et al. (1988)

J. Bio. Chem. 263: 15064-15070, and an analysis of POG / IL2 conjugates can be found in US Patent No. 4,766,106.

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

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

Procedures for controlling protein stability are well known in the art. See, for example, Jones (1993) Adv Drug Delivery Rev. 10: 2990; Lee, ed. (1991) Peptide and Protein Drug Delivery (Marcel Dekker, Inc., New York, New York); and the stability tests disclosed in this document below. In general, protein stability is measured at a chosen temperature for a specified period of time. In preferred embodiments, a stable antibody pharmaceutical formulation provides stability of the anti-CD40 antagonist antibody or its antigen binding fragment when stored at room temperature (approximately 25 ° C) for at least 1 month, at least 3 months, or at at least 6 months, and / or is stable at approximately 2-8 ° C for at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months.

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

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

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

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

Thus, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR-5.9 antibody, or its antigen binding fragment in a concentration of about 0.1 mg / ml to about 50, 0 mg / ml, approximately 0.5 mg / ml to approximately 40.0 mg / ml, approximately 1.0 mg / ml to approximately 30.0 mg / ml, approximately 5.0 mg / ml to approximately 25.0 mg / ml, approximately 5.0 mg / ml to approximately 20.0 mg / ml, or approximately 15.0 mg / ml to approximately 25.0 mg / ml. In some embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or its antigen binding fragment in a concentration of about 0.1 mg / ml to about 5.0 mg / ml, about 5.0 mg / ml to approximately 10.0 mg / ml, approximately 10.0 mg / ml to approximately 15.0 mg / ml, approximately 15.0 mg / ml to approximately 20.0 mg / ml, approximately 20.0 mg / ml to approximately 25.0 mg / ml, approximately 25.0 mg / ml to approximately 30.0 mg / ml, approximately 30.0 mg / ml to approximately 35.0 mg / ml, approximately 35.0 mg / ml to approximately 40, 0 mg / ml, approximately 40.0 mg / ml to approximately 45.0 mg / ml, or approximately 45.0 mg / ml to approximately 50.0 mg / ml. In other embodiments, the liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody or its antigen binding fragment in a concentration of about 15: 0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml , approximately 18.0 mg / ml, approximately 19.0 mg / ml, approximately 20.0 mg / ml, approximately 21.0 mg / ml, approximately 22.0 mg / ml, approximately 23.0 mg / ml, approximately 24.0 mg / ml, or 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 approximately pH 5.0 up to about pH 7.0, including about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and other 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 to about pH 6.5, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about 7.0, about pH 5.5 to about pH 6.5, or about pH 5.5 to about pH 6.0.

Any suitable buffer that maintains the pH of the liquid formulation of the anti CD40 antibody in the range of about pH 5.0 to about pH 7.0 can be used in the formulation, provided that the physicochemical stability and the desired biological activity of the antibody are maintained as noted earlier in this document. Buffers include but are not limited to conventional acids and their salts, where the counterion can be, for example, sodium, potassium, ammonium, calcium or magnesium. Examples of conventional acids and their salts that can be used to buffer the liquid pharmaceutical formulation include, but are not limited to, succinic acid or succinate, citric acid or citrate, acetic acid or acetate, tartaric acid buffers.

or tartrate, phosphoric acid or phosphate, gluconic acid or gluconate, glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate and malic or malate acid. The concentration of the buffer in the formulation can be from about 1 mM to about 50 mM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other similar values within the range of about 1 mM to about 50 mM. In some embodiments, the concentration of the buffer in the formulation is from about 5 mM to about 15 mM, including about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or other similar values within the range of about 5 mM to about 15 mM.

In some embodiments of the invention, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the CHIR 12,12 or CHIR-5.9 monoclonal antibody, 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 to about pH 7.0, preferably about pH 5.0 to about pH 6.5. By "succinate buffer" or "citrate buffer" is meant a buffer comprising a succinic acid salt or a citric acid salt, respectively. In a preferred embodiment, the succinate or citrate counterion is the sodium cation, therefore the buffer is sodium succinate or sodium citrate, respectively. However, any cation is expected to be effective. Other possible succinate or citrate cations include, but are not limited to, potassium, ammonium, calcium and magnesium. As noted above, the concentration of the succinate or citrate buffer in the formulation may be from about 1 mM to about 50 mM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other similar values within the range from about 1 mM to about 50 mM. In some embodiments, the concentration of buffer in the formulation ranges from about 5 mM to about 15 mM, including about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or about 15 mM. In other embodiments, the liquid pharmaceutical formulation comprises the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment at a concentration of about 0.1 mg / ml to about 50 , 0 mg / ml, or about 5.0 mg / ml to about 25.0 mg / ml, and succinate or citrate buffer, for example, sodium succinate buffer or sodium citrate, at a concentration of about 1 mM to about 20 mM, about 5 mM to about 15 mM, preferably about 10 mM.

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

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

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

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

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

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

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

Once the liquid pharmaceutical formulation or other pharmaceutical composition described herein is prepared, it can be lyophilized to prevent degradation. Procedures for lyophilizing liquid compositions are known to those skilled in the art. Just before using, the composition can be reconstituted with a sterile diluent (Ringer's solution, distilled water or saline, for example) that may include other components. After reconstitution, the composition is preferably administered to the subjects using procedures known to those skilled in the art. Use of anti-CD40 antagonist antibodies in the manufacture of medicines

The present invention also provides the use of an anti-CD40 antagonist antibody or its antigen binding fragment in the manufacture of a medicament for the treatment of CLL in a subject, wherein the medicament is coordinated with treatment with at least one other anti-therapy. Cancer. By "coordinated" is meant that the medication will be used before, during, or after the treatment of the subject with at least one other cancer therapy. Examples of other cancer therapies include, but are not limited to, those described earlier in this document, that is, surgery; radiation therapy; chemotherapy; when appropriate in combination with autologous bone marrow transplantation, where examples of other cancer therapies include, but are not limited to, surgery; radiation therapy; chemotherapy; when appropriate in combination with autologous bone marrow transplantation, where chemotherapeutic agents include, but are not limited to, fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-chlorodeoxydenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, their combinations, for example, anthracycline-containing regimens such as CAP (cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincritsine, doxorubicin, plus dexamethasone), MP (melphalan and more prednisone) 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 neutrophil; another anti-cancer therapy with monoclonal antibodies (e.g., alemtuzumab (Campath®) or other anti-CD52 antibody directed to the CD52 cell surface glycoprotein in malignant B cells; rituximab (Rituxan®), the fully human HuMaxCD20 antibody, R-1594, IMMU -106, TRU-015, AME-133, tositumomab / I-131 tositumomab (Bexxar®), ibritumomab tiuxetan (Zevalin®), or any other therapeutic anti-CD20 antibody directed at malignant B20 cells; anti CD19 antibody (for example, MT103, a bispecific antibody); anti-CD22 antibody (for example, humanized monoclonal antibody epratuzumab); bevacizumab (Avastin®) or other anti-cancer antibody directed to human endothelial vascular growth factor; anti-CD22 antibody directed to CD22 antigen in cells Malignant B (for example, monoclonal antibody BL-22, an alphaCD22 toxin); α-M-CSF antibody directed to the macrophage colony stimulating factor; antibodies directed to the nuclear kappaB factor receptor activator (RANK) and its ligand (RANKL); anti CD23 antibody directed to the CD23 antigen in malignant B cells (eg, IDEC-152); anti CD38 antibody directed to the CD38 antigen in malignant B cells; antibodies directed to the major histocompatibility class II complex antibodies (anti MHC antibodies) expressed in malignant B cells; other anti-CD40 antibodies (eg, SGN-40) directed to the CD40 antigen in malignant B cells; and antibodies directed to the receptor 1 of the apoptosis inducing ligand related to tumor necrosis factor (TRAIL-R1) (for example, the human agonist monoclonal antibody HGS-ETR1) expressed in a number of solid tumors and tumors of hematopoietic origin); cancer therapy based on small molecules, including, but not limited to, microtubule and / or topoisomerase inhibitors (for example, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin-binding agent T900607; XL119; and the inhibitor of Topoisomerase aminocamptothecin), SDX-105 (bendamustine hydrochloride), ixabepilone (an epothilone analogue, also called BMS-247550), protein kinase C inhibitors, for example, midostaurine ((PKC-412, CGP 41251, N-benzoylstaurosporine), Pixantrone, eloxathine (an antineoplastic agent), ganite (gallium nitrate), Thalomid® (thalidomide), immunomodulatory derivatives of thalidomide (e.g. revlimid (formerly revimid)), Affinitak ™ (C-alpha antisense proteinase inhibitor), SDX-101 (R-etodolac, which induces apoptosis of malignant lymphocytes), second generation purine nucleoside analogues such as clofarabine, inhibitors of the production of Bcl-2 protein by can cells waxy (for example, the antisense agents oblimersen and Genasense®), proteasome inhibitors (e.g., Velcade ™ (bortezomib)), small molecule kinase inhibitors (e.g., CHIR-258), small molecule VEGF inhibitors ( for example, ZD-6474), small molecule heat shock protein (HSP) 90 inhibitors (e.g., 17-AAG), small molecule histone deacetylase inhibitors (e.g., hybrid / polar HPC cytodifferentiation ) such as suberanylohydroxamic acid (SAHA), and FR-901228 and apoptotic agents such as Trisenox® (arsenic trioxide) and Xcytrin® (motexaphine gadolinium); Vaccine / immunotherapy-based cancer therapies, including, but not limited to, vaccine approaches (e.g., Id-KLH, oncophagus, vitaletin), personalized immunotherapy, or active idiotype immunotherapy (for example, MyVax® Personalized Immunotherapy, referred to as formally GTOP-99), Promune® (CpG 7909, a synthetic agonist for the toll 9 receptor (TLR9)), interferon alfa therapy, interleukin 2 (IL-2) therapy, IL-12 therapy, IL-12 therapy IL-15 and therapy with IL-21; steroid therapy; or other cancer therapy; in which the treatment with additional cancer therapy, or additional cancer therapies, is performed before, during, or after the treatment of the subject with the medicament comprising the anti-CD40 antagonist antibody or its antigen binding fragment, as noted earlier in this document. In such an embodiment, the present invention provides the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9 in the manufacture of a medicament for the treatment of CLL in a subject, in which the medicament is coordinated with treatment with at least another cancer therapy as indicated in this document above.

Therefore, for example, in some 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 medicament for the treatment of CLL in a subject, in that the medication is coordinated with chemotherapy treatment, chemotherapy being selected from the group consisting of fludarabine, chlorambucil, vincristine, pentostatin, 2-chlorodeoxydenosine (cladribine), cyclophosphamide, doxorubicin and prednisone, and anthracycline-containing regimens (CAP, cyclophosphine) doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin) and any combination thereof; in which the medication is to be used before, during, or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during, or after the treatment of the subject with the Other cancer therapies.

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 medicament for the treatment of multiple myeloma in a subject, in which the medicament is coordinated with treatment with at least one other anti-cancer antibody selected from the group consisting of alemtuzumab (Campath®) or another anti-CD52 antibody directed to the CD52 cell surface glycoprotein in malignant B cells; rituximab (Rituxan®), the fully human HuMax-CD20 antibody, R-1594, IMMU-106, TRU-015, AME-133, tositumomab / I-131 tositumomab (Bexxar®), ibritumomab tiuxetan (Zevalin®), or any another therapeutic anti CD20 antibody directed to the CD20 antigen in malignant B cells; anti CD23 antibody directed to the CD23 antigen in malignant B cells (eg, IDEC-152); and anti-CD22 antibody directed to the CD22 antigen in malignant B cells (eg, the BL-22 antibody, an alphaCD22 toxin); and any combination thereof; in which the medication is to be used before, during, or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during, or after the treatment of the subject with Other cancer therapies.

In still other embodiments the present 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 medicament for the treatment of CLL in a subject, in which the medicament is coordinated with treatment with at least one other small molecule-based teapia against cancer selected from the group consisting of SDX-101 (Retodolac, which induces apoptosis of malignant lymphocytes), second generation purine nucleoside analogues such as clofarabine, production inhibitors of Bcl-2 protein by cancer cells (for example, the antisense agents oblimersen and Genasense®), proteasome inhibitors (for example, Velcade ™ (bortezomib)), small molecule histone deacetylase inhibitors (for example, hybrid HPC / cytodifferentiation polar), agents such as suberanylohydroxamic acid (SAHA), and FR-901228) and any combination thereof; or with another cancer therapy, for example, immunization with the CD154 gene (for example ISF-154); in which the medication is to be used before, during, or after the treatment of the subject with the other cancer therapy or, in the case of multiple combination therapies, either before, during, or after the treatment of the subject with Other cancer therapies.

In some embodiments, the medicament comprising the anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR-5.9 monoclonal antibody described herein, or its antigen-binding fragment is coordinated with treatment with two other cancer therapies. Without intending to be limited, examples include coordination of the drug with treatment with two chemotherapeutic agents, for example, coordination with treatment with fludarabine and cyclophosphamide; and coordination of the medicament with treatment with a chemotherapeutic agent, for example, fludarabine and another anti-cancerous monoclonal antibody, for example, alemtuzurnab, rituximab or other anti-CD20 antibody including the fully human HuMaxCD20 antibody, R-1594, IMMU-106, TRU -015, AME-133, tositumomab / I-131 tositumomab (Bexxar®) and ibritumomab tiuxetan (Zevalin®), or anti-CD23 antibody. When the medicament comprising the anti-CD40 antagonist antibody is coordinated with two other cancer therapies, the use of the medication may be before, during, or after the treatment of the subject with one or both other cancer therapies.

The invention also provides the use of an anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR-5.9 monoclonal antibody described herein, or its antigen-binding fragment in the manufacture of a medicament for the treatment of CLL in a subject, in which the medication is used in a subject who has received prior treatment with at least one other cancer therapy. By "pretreated" or "pretreatment" is meant that the subject has been treated with one or more cancer therapies before receiving the medicine comprising the anti-CD40 antagonist antibody or its antigen binding fragment. "Pretreated" or "pretreatment" includes subjects who have been treated with at least one other cancer therapy, or therapies with cancer, within 2 years, within 18 months, within 1 year , within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or even within 1 day before the start of treatment with the medicament comprising the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 described herein, or its antigen binding fragment. It is not necessary that the subject has been a patient who responds to the previous treatment with the previous cancer therapy, or previous cancer therapies. Accordingly, the subject receiving the medicament comprising the anti-CD40 antagonist antibody or its antigen binding fragment may have responded, or may not have responded to prior treatment with the previous cancer therapy, or to one or more previous therapies against cancer where the previous treatment included multiple cancer therapies, for example, surgery and chemotherapy; surgery and other anti-cancer antibody therapy; chemotherapy and other anti-cancer antibody therapy; or surgery, chemotherapy, and other anti-cancer antibody therapy.

Accordingly, in some embodiments, the invention also provides the use of an anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9 described herein, or its antigen binding fragment in the manufacture of a medication used in a subject in need of CLL treatment, where the subject has been previously treated with one or more of the following cancer therapies: surgery; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplant; where suitable chemotherapeutic agents include, but are not limited to, fludarabine, chlorambucil, cladrabine, vincristine, pentostatin, 2-chlorodeoxydenosine, cyclophosphamide, doxorubicin, prednisone, and combinations thereof, for example, regimens containing anthracycline such as CAP (cycloxorbamide plus prednisone) and CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), another anti-cancer therapy with monoclonal antibodies (e.g. alemtuzumab (Campath®); rituximab (Rituxan®) or another therapeutic anti-CD50 antibody; or other targeted anti-CD23 antibody to the CD23 antigen in malignant B cells); interferon alfa therapy; interleukin-2 therapy (IL-2); therapy with IL-2, IL-5 or IL-21; or steroid therapy.

"Treatment" in the context of coordinated use of a medicament described herein with another or other cancer therapies is defined herein as the application or administration of the medicament or other cancer therapy to a subject, or the application or administration of the medicament or other cancer therapy to an isolated tissue or cell line of a subject, in which the subject has chronic lymphocytic leukemia, a symptom associated with such cancer, or a predisposition to develop such cancer, being the purpose of curing, healing, calming, relieving, altering, remedying, improving or affecting cancer, any associated symptoms of cancer, or the predisposition to develop cancer.

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

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

In summary, splenocytes from mice immunized with murine myeloma cells SP 2/0 or P 3 x 63 Agg 6,653 were fused 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) . The fused cells were then distributed between the wells of 96-well tissue culture plates, so that each contained 1 growing hybridoma on average.

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

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

Clones of another 7 hybridomas that had antagonistic activity were identified. Based on their relative antagonistic potency and ADCC activities, two 5 hybridoma clones were selected for further evaluation. They are called 131.2F8.5.9 (5.9) and

153.8E2.D10, D6.12.12 (12.12).

Table 1. Summary of the initial data series with anti CD40 CHIR IgG1 antibodies

5.9 and CHIR-12.12

Mother hybridoma

Hybridoma clones cell surface binding Antagonist ADCC CDC CMCC # DNA sequence of region V

131.2F5

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

153.8E2

158.8E2D10D6.12.12 +++ +++ ++++ - 12056 Yes

The hybridoma line 131.2F5.8.5.9 (CMCC # 12047) and the hybridoma line

10 153.8E2.D10.D6.12.12 have been deposited with the American Type Culture Collection [ATCC; 10801 University Blvd., Manassas, Virginia 20110-2209 (USA)] with Patent Deposit Number 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. Amino acid sequences 15 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 SEQ 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 in the

position 158 of SEQ ID No. 7.

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

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

The monoclonal antibodies CHIR-12.12 and CHIR-5.9 are strong antagonists and inhibit 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.

Chronic B-cell lymphocytic leukemia (CLL) is characterized by in vivo accumulation of long-lived CD5 + B cells. However, when grown in vitro, CLL cells die rapidly by apoptosis. Protection against apoptosis in vivo is believed to result from the supply of survival signals from the microenvironment. The CD40 stimulation of CLL cells by CD40 ligand is identified as being one of those survival signals.

The following studies were undertaken to determine whether mAbs CHIR-5.9 and CHIR-12.12 anti-CD40 antagonists show the following properties: (1) they bind to cells of patients with chronic lymphocytic leukemia (CLL); (2) promote cell death in cells of patients with CLL by blocking CD40-induced survival signals; (3) have some stimulatory / inhibitory activity on their own for chronic lymphocytic leukemia (CLL) cells; and / or (4) mediate the ADCC as a mode of action. Example 1: CHIR-5.9 and CHIR-12.12 can block CD40-mediated survival and proliferation of cancer cells from patients with CLL

Candidate antibodies can block CD40-mediated survival and proliferation of cancer cells of patients with CLL. CLL cells from suspension patients were cultured on CHO cells expressing formaldehyde-fixed CD40L under two different conditions: addition of human isotype IgG antibody (control); and addition of monoclonal antibody CHIR-5.9 or CHIR-12.12. All antibodies were added in concentrations of 1, 10 and 100 µg / ml in the absence of IL-4. Cell counts were performed at 24 and 48 hours by means of MTS assay. Reduced cell numbers were recovered from cultures treated with CHIR-5.9 (n = 6) and

5 CHIR-12.12 (n = 2) compared to the control group. The greatest differences in cell numbers between cultures treated with anti-CD40 mAb and treated with control antibody were observed at the time point of 48 hours. These data are summarized in Table 2.

10 Table 2. The effect of candidate antibodies on CD40-induced survival and proliferation of cancer cells of patients with CLL measured at 48 hours after starting culture

Patient No.

Conc. Ab µg / ml Relative number of cells % reduction in cell number *

IgG1

CHIR5.9 / 5.11 CHIR12.12 CHIR 5.95.11 CHIR12.12

one

1 10 100 269.31 101.58 130.71 25.27 33.07 40.16 ND ND ND 90.62 67.44 69.28 ND ND ND

2

1 10 100 265.55 227.57 265.99 75.8 128.5 6.4 ND ND ND 71.46 43.53 97.59 ND ND ND

3

1 10 100 85.9 70.44 77.65 35.39 39.51 20.95 ND ND ND 58.80 43.91 73.02 ND ND ND

4

1 10 100 80.48 63.01 55.69 15.03 19.51 3.65 ND ND ND 81.32 69.04 93.45 ND ND ND

5

1 10 100 90.63 78.13 63.53 91.66 82.28 86.47 89.59 60.41 39.59 -1.14 -5.31 -36.11 1.15 22.68 37.68

6

1 10 100 130.21 131.77 127.08 77.6 78.13 76.56 71.88 73.96 82.29 40.40 40.71 39.75 44.80 43.87 35.25

Patient No.

Conc. Ab µg / ml Relative number of cells % reduction in cell number *

IgG1

CHIR5.9 / 5.11 CHIR12.12 CHIR 5.95.11 CHIR12.12

*% reduction compared to Control Ab = 100 - (Test Ab / Control Ab) * 100

A second study revealed similar results. In this study they were grown

primary CLL cells of 9 suspension stokers on CHO cells fixed with

formaldehyde expressing CD40L in the presence in the absence of 1, 10 or 100 µg / ml of mAb

5 CHIR-12.12 anti-CD40 in the manner described above, using non-specific IgG as the control. After 48 and 72 hours, the proliferation of the cultures was measured as indicated above. In the absence of anti-CD40 CHIR-12.12 mAb, primary CLL cells either resisted spontaneous cell death or proliferated. This effect was inhibited in the presence of CHIR-12.12 mAb, which re-established the death of

10 CLL. Therefore, these results demonstrate the inhibition of CLL cell proliferation induced by CD40 with the anti-CD40 CHIR-12.12 mAb at 48 and 72 hours. See Figures 5A and 5B. Similar experiments were performed on unstimulated CLL cells in the presence of CHIR-12.12 mAb only. The CHIR-12.12 mAb (10 µg / ml) alone did not induce

CLL proliferation and therefore did not show a stimulatory effect on CLL cells compared to the control IgG (10 µg / ml) at 48 and 72 hours. See Figures 6A and 6B. Example 2: Ability of anti-CD40 CHIR-12.12 mAb to lyse chronic lymphocytic leukemia (CLL) cell lines by antibody-dependent cell cytotoxicity

20 (ADCC) The CLL EHEB cell line was cultured with anti-CD40 antagonist CHIR-12.12 mAb or the Rituxan® anti-CD20 antibody (IDEC Pharmaceuticals Corp., San Diego, California) and freshly isolated human NK cells from blood donors Normal volunteers as effector cells. Specific lysis was measured in percentage in

25 based on the release of target cell marker. The CHIR-12.12 anti-CD40 mAb showed lysis activity in a dose-dependent manner and reached maximum lysis levels at 0.1 µg / ml (Figure 7). As shown in Figure 7, CHIT-12.12 mAb induced higher ADCC-mediated cell lysis than Rituxan® (maximum specific lysis with CHIR-12.12 mAb = 27.2% versus maximum specific lysis

with Rituxan® = 16.2%; p = 0.007). Based on these results, approximately 10 times more binding sites were present for anti-CD20 antibody than for anti-CD40 antibody in target cells (see table 3), indicating that fewer CD40 molecules are expressed in the cell line of CLL EHEB when compared with the expression of CD20. In fact, the CLL target cell line expressed 509,053 + 13,560 CD20 molecules compared to 48,416 + 584 CD40 molecules. Therefore, the higher ADCC mediated by CHIR-12.12 mAb was not due to the higher density of CD40 molecules. Therefore, the higher ADCC mediated by CHIR-12.12 mAb was not due to the higher density of CD40 molecules in this CLL cell line in

10 comparison with CD20 molecules. Table 3: EHEB target cell line binding site ratio

Cellphone line

% of maximum lysis Maximum CD20 / CD40 site ratio from Union

mAb CHIR-12.12

Rituxan®

EHEB

27.19 16.21 10.51

Example 3: CHIR-5.9 and CHIR-12.12 bind to a different epitope than 15B8 in CD40 Candidate monoclonal antibodies CHIR-5.9 and CHIR-12.12 compete one

15 with the other by binding to CD40 but not with 15B8, an anti-CD40 IgG2 mAb (see International Publication Document No. WO 02/28904). Competitive antibody binding studies were designed using Biacore with CM5 biosensor chips with protein A immobilized by means of amine coupling, which was used to capture anti CD40, CHIR-12.12, or 15B8. The binding curves of

20 normal association / dissociation with varying concentrations of CD40-his (data not shown). For competition studies, CHIR-12.12 or 15B8 were captured on the surface of protein A. Subsequently, a CD40-his / Fab complex of CHIR-5.9 (100 nM CD40: CHIR-5.9 1 µM Fab) was flowed, in varying concentrations, across the modified surface. In the case of CHIR-12.12, no association of

Complex, which indicates that CHIR-5.9 blocks the binding of CHIR-12.12 to CD40-his. For 15B8, association of the Fab CHIR-5.9 complex was observed indicating that CHIR-5.9 does not block the binding of 15B8 to the CD40 binding site. However, the rate of dissociation of the complex increased dramatically (data not shown).

It was also determined that 15B8 and CHIR-12.12 do not compete for the union of CD4030 his. This experiment was carried out by capturing CHIR-12.12 on the protein A biosensor chip, blocking the residual sites of protein A with hIgG1 control, linking

CD40-his and then flowing 15B8 over the modified surface. 15B8 joined under these conditions indicating that CHIR-12.12 does not block the binding of 15B8 to CD40. Example 4: Binding properties of CHIR-12.12 and CHIR-5.9 mAb

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

10 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 20.005% P Surfactant (HBSEP). Kinetic and affinity constants were determined using the Biaevaluation software with a model / global interaction setting of 1: 1.

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 that results in a 24-fold higher affinity for CHIR-12.12. Table 4: Summary of binding properties of anti-CD40 antibodies CHIR-5.9 and CHIR

12.12

Antibody

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

Anti-CD40, CHIR-5.9

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

Anti-CD40, CHIR-12.12

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

To determine the location of the epitope on CD40 recognized by the

20 monoclonal antibodies CHIR-12.12 and CHIR-5.9, SDS-PAGE analysis and Western blotting were performed. Purified CD40 (0.5 µg) was separated on a 4-12% NUPAGE gel under reducing and non-reducing conditions, transferred to PVDF membranes, and probed with monoclonal antibodies at a concentration of 10 µg / ml. The transfers were probed with anti-human IgG conjugated to alkaline phosphatase and were

25 developed using the Western® Blue stabilized substrate for alkaline phosphatase (Promega). The results indicate that the anti-CD40 monoclonal antibody CHIR12.12 recognizes epitopes both in the non-reduced form and in the reduced form of CD40, exhibiting the non-reduced form of CD40 greater intensity than the reduced form of

30 CD40 (Table 15; transfers not shown). The fact that recognition was positive for both forms of CD40 indicates that this antibody interacts with a conformational epitope, part of which is a linear sequence. The CHIR-5.9 monoclonal antibody primarily recognizes the non-reduced form of CD40 by suggesting that this antibody interacts primarily with a conformational epitope (Table 5; transfers not shown).

Table 5. Domain identification.

Domain 1

Domain 2 Domain 3 Domain 4

mAb CHIR-12.12

- + - -

mAb CHIR-5.9

- + - -

mAb 15B8

+ - - -

5

To map the antigenic region on CD40, the four extracellular domains of CD40 were cloned and expressed in insect cells as GST fusion proteins. The secretion of the four domains was secured with a GP67 secretion signal. The supernatant of the insect cells was analyzed by SDS analysis

10 PAGE and Western blot to identify the domain that contained the epitope. The monoclonal antibody CHIR-12.12 recognizes an epitope in Domain 2 both under reducing and non-reducing conditions (Table 6; transfers not shown). In contrast, the CHIR-5.9 monoclonal antibody exhibits very weak recognition for Domain 2 (Table 6; transfers not shown). None of these antibodies

15 recognizes Domains 1, 3 or 4 in this analysis. Table 6. Domain Analysis2.

Reduced

Not reduced

mAb CHIR-12.12

++ +++

mAb CHIR-5.9

+ +

To more precisely define the epitope recognized by the CHIR-12.12 mAb, CD40 extracellular Domain 2 peptides were synthesized, corresponding to the sequence 20 PCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQ-KGTSETDTICT (residues 61-104 of the sequence shown in SEQ ID No. 10 or SEC 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 analysis 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. SPOTs analysis results with probing with anti-CD40 monoclonal antibody CHIR-12.12.

Transfer number

Sequence region

18

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

19

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

twenty

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

twenty-one

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

22

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

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

CD40

SPOTs with CHIR-5.9 mAbs showed a weak recognition of the

peptides represented by transfers 20-22 shown in Table 8, suggesting

involvement of the YCDPNLGL region (residues 82-89 of the sequence shown in SEC

15 ID No. 10 or SEQ ID No. 12) in connection with CD40. It should be noted that the CHIR-12.12 and CHR-5.9 mAbs compete with each other for binding to CD40 in the BIACORE analysis. Table 8. Results of SPOT analysis with probing with anti-CD40 monoclonal antibody CHIR-5.9.

Transfer number

Sequence region

twenty

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

twenty-one

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

22

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

The linear epitopes identified by the SPOTs analyzes are within the module

B1 of CD40. The sequence of module B1 of CD40 is:

HKYCDPNLGLRVQQKGTSETDTIC (residues 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 cysteine residue forms a disulfide bond with C103. It is likely that the conformational epitope of the CHIR-12.12 mAb contains this disulfide bond (C83-C103) and / or neighboring amino acids conformationally close to C103. Example 6: CHIR-12.12 blocks CD40L mediated survival and CD40 signal pathways in normal human B cells

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

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

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

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

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

In these experiments, 0.6 x 106 normal human B cells from healthy donors (percentage purity between 85 and 95%) were stimulated with 1 µg / ml sCD40L (Alexis Corp., Bingham, Nottinghamshire, UK). Then CHIR-12.12 (10 µg / ml) and control IgG were added. Cells were collected at 0 and 20 minutes, at 2 hours, 6 hours, 18 hours and 26 hours. Mcl-1, XIAP, CD40 and β actin controls were detected in cell lysates by Western blotting. In summary, stimulation with sCD40L resulted in sustained expression of Mcl-1 and XIAP over time. However, treatment of sCD40L stimulated cells with CHIR-12.12 resulted in a decrease in the expression of these proteins over time (data not shown). Since Mcl-1 and XIAP are "survival" signals capable of blocking the apoptotic pathway, these results demonstrate that treatment with CHIR-12.12 eliminates blockage against apoptosis in normal B cells stimulated with sCD40L. Treatment with CHIR-12.12 inhibited the phosphorylation of IKKα (Ser180) and IKKβ (Ser 181) in normal B cells.

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

In summary, stimulation by means of sCD40L resulted in phosphorylation of IKKα (Ser180) and IKKβ (Ser 181) over time; however, treatment with CHIR-12.12 nullified this response to stimulation of sCD40L in normal B cells (data not shown). Treatment with CHIR-12.12 inhibited CD40 ligand-mediated survival in a dose-dependent manner.

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

Briefly, treatment with CHIR-12.12 resulted in an increase in PARP cleavage in cells stimulated with sCD40L in a dose-dependent manner and, consequently, nullified the survival signal pathway in normal B cells stimulated with sCD40L ( data not revealed). CHIR-12.12 inhibited the expression of "survival" antiapoptotic proteins in a dose-dependent manner.

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

Briefly, treatment with CHIR-12.12 reduced the expression of Mcl-1 and XIAP and increased the expression of PARP in cells stimulated with sCD40L in a dose-dependent manner and, consequently, nullified these blockages to the apoptotic pathway in the cells. Normal B stimulated with sCD40L (data not shown). CHIR-12.12 does not affect the expression of antiapoptotic proteins, cleaved PARP and XIAP, in the absence of soluble CD40L signals.

In these experiments, 1.0 x 106 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 (i.e., non-cell were previously stimulated with sCD40L before adding the antibody). Cells were collected at 0, 4, 14 and 16 hours. XIAP, cleaved PARP and β actin controls were detected in cell lysates by Western blotting.

Briefly, the results show that without stimulation of sCD40L, cells expressed increased concentrations of cleaved PARP, while XIAP expression remained constant, both in cells treated with control IgG and in those treated with CHIR-12.12 (data not shown ). These data indicate that CHIR-12.12 does not cause apoptosis in normal human B cells without CD40L stimulation. CHIR-12.12 inhibits the phosphorylation of IKKα (Ser180) and IKKβ (Ser181), Akt, ERK and p38 in normal B cells.

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

In summary, stimulation with sCD40L resulted in increases in phosphorylation of IKKα / β, phosphorylation of ERK, phosphorylation of Akt and phosphorylation of p38, consequently resulting in cell survival and / or proliferation. Treatment of the cells with CHIR-12.12 nullified the effects of stimulation of sCD40L in these signal pathways in normal B cells (data not shown). CHIR 12.12 inhibits signal pathways such as PI3K and MEK / ERK in the CD40 signal cascade.

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

In summary, the results show that CHIR-12.12 annulled the phosphorylation of all these signal transduction molecules, while signal transduction inhibitors showed only specific signal cancellation, indicating that CHIR-12.12 probably inhibited at a stage prior to these signal transduction molecules mediated by CD40L stimulation (data not shown). CHIR-12.12 inhibits the binding of TRAF2 and TRAF3 signal molecules to the cytoplasmic domain of CD40 in normal B cells.

In these experiments, serum was deprived for four hours in medium containing 1% FBS 4.0 x 106 normal human B cells from healthy donors (purity percentage between 85 and 95%) and stimulated with sCD40L 1 µg / ml (Alexis Corp., Bingham, Nottinghamshire, UK) for 20 minutes. Cells were collected at 0 and 20 minutes. CD40 was immunoprecipitated using polyclonal anti CD40 (Santa Cruz Biotechnology, CA), and probed in a Western blot with anti TRAF2 mAb (Santa Cruz Biotechnology, CA), anti TRAF3 mAb (Santa Cruz Biotechnology, CA), and anti CD40 mAb ( Santa Cruz Biotechnology, CA).

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

Without being bound by theory, the results of these experiments, and the results in the examples summarized above, indicate that the CHIR antibody

12.12 is a dual-acting anti-CD40 monoclonal antibody that has a unique combination of attributes. This fully human monoclonal antibody blocks CD40L-mediated CD40 signal pathways for the survival and proliferation of B cells; This antagonism eventually results in cell death. CHIR-12.12 also mediates recognition and binding by effector cells, initiating antibody-dependent cellular cytotoxicity (ADCC). Once CHIR-12.12 is bound to effector cells, cytolytic enzymes are released, resulting in apoptosis and lysis of B cells. CHIR-12.12 is a more potent antitumor antibody than rituximab when compared in preclinical tumor models. . Example 7: Liquid pharmaceutical formulation for anti-CD40 antagonist antibodies

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

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

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

5 of purified anti CD40 (CHIR-12.12) obtained from CHO cells. The composition of the drug substance was CHIR-12.12 antibody 9.7 mg / ml in 10 mM sodium citrate, 150 mM sodium chloride, at pH 6.5. The control sample in the study was the substance of the drug received, followed by freezing at ≤ -60 ° C, thawing at RT and tested together with stability samples at predetermined time points.

10 Stability samples were prepared by dialysis of the drug substance against solutions of different pHs and the concentration of CHIR-12.12 in each of the samples was determined by UV 280 as presented in Table 9. Table 9. Formulations of CHIR-12.12.

Buffer composition

pH CHIR-12.12 concentration (mg / ml)

10 mM sodium citrate, 150 mM sodium chloride

4,5 9.0

10 mM sodium succinate, 150 mM sodium chloride

5.0 9.3

10 mM sodium succinate, 150 mM sodium chloride

5.5 9.2

10 mM sodium citrate, 150 mM sodium chloride

6.0 9.7

10 mM sodium citrate, 150 mM sodium chloride

6.5 9.4

10 mM sodium phosphate, 150 mM sodium chloride

7.0 9.4

10 mM sodium phosphate, 150 mM sodium chloride

7.5 9.5

10 mM glycine, 150 mM sodium chloride

9.0 9.5

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

Differential Scanning Calorimetry (DSC)

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

Fragmentation and aggregation were estimated using 4-20% Tris-glycine gel under non-reducing and reducing conditions. Proteins were detected by staining with Coomassie blue. Size exclusion chromatography (SEC-HPLC)

Protein fragmentation and aggregation was also measured by means of an HPLC Water Alliance system with a Tosohaas TSK-GEL 3000SWXL column, with 100 mM sodium phosphate, pH 7.0 as a mobile phase at a flow rate of 0.7 ml / min. Cation Exchange Chromatography (CEX-H PLC)

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

The thermal deployment of CHIR-12.12 revealed at least two thermal transitions, which probably represent the deployment and fusion of the Fab and Fc domains, respectively. At higher temperatures, the protein presumably added, resulting in loss of the DSC signal. In order to select the formulation, the lowest thermal transition temperature was defined as the melting temperature, Tf, in this study. Figure 8 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 the higher thermal fusion temperatures. SDS-PAGE analysis.

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

Under reducing conditions, CHIR-12.12 was reduced in heavy chains and free light chains with PM of 50 kDa and 24 kDa, respectively. The 100 kDa species appeared not to be fully reducible and increased with the increase in the pH of the solution, suggesting that there could be a different covalent association of disulfide in the molecules. As there were other species with unknown identities in SDS-PAGE, the stability comparison for each formulation is based on the remaining purity of

5 CHIR-12.12. Formulations at pH 5.0-6.0 provided a more stable environment for CHIR-12.12. Few aggregates were detected by means of SDS-PAGE (data not shown).

SEC-HPLC analysis.

SEC-HPLC analysis detected intact CHIR-12.12 as the peak species

10, a species of aggregation as a species of anterior peak separated from the species of the main peak, a species of large fragment such as a plateau peak in the back of the species of the main peak, and species of small fragments subsequent to the species of the main peak. After incubation at 5 ° C and 25 ° C for 3 months, insignificant amounts of fragments and aggregates of

15 proteins (<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 shown). However, fragments gradually developed after storage at 40 ° C and more fragments were formed at pH 4.5 and pH 6.5-9.0, as shown in Table 21. After incubating the CHIR-12.12 formulations at 40 ° C for 3 months, was detected

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

25 Table 10. SEC-HPLC results of CHIR-12.12 stability samples under real-time and accelerated storage conditions.

Sample

Main peak% Fragments%

t = 0

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

m

m

m

m

m

m

Control

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

pH 4.5

99.4 93.2 86.0 81.3 <1.0 6.4 13.2 18.1

pH 5.0

99.8 98.7 91.3 89.2 <1.0 <1.0 7.8 10.2

pH 5.5

99.8 98.9 91.4 90.6 <1.0 <1.0 7.6 8.8

pH 6.0

99.6 97.7 90.4 87.3 <1.0 1.9 8.2 11.7

pH 6.5

99.3 93.4 89.0 86.9 <1.0 5.6 9.9 12.4

pH 7.0

99.2 93.9 87.4 85.1 <1.0 5.5 11.1 13.5

Sample

Main peak% Fragments%

t = 0

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

m

m

m

m

m

m

pH 7.5

99.1 92.8 84.4 81.9 <1.0 6.4 12.9 16.2

pH 9.0

99.3 82.4 61.6 50.6 <1.0 15.4 36.2 47.6

CEX-FIPLC analysis.

The CEX-HPLC analysis detected the intact CHIR-12.12 as the species of the main peak, acid variants eluted earlier than the species of the main peak 5 and lysine addition variants at the C terminal end eluted after the peak species principal. Table 11 shows the dependence of the percentages of the remaining CHIR-12.12 species on the main peak and the acid variants on the pH of the solution. The control sample already contained a high degree of acidic species (approximately 33%), probably due to the early stage 10 fermentation and purification processes. The susceptibility of CHIR-12.12 to higher pH solutions is evidenced by two facts. First, the sample of the initial formulation at pH 9 (t = 0) already generated 12% more acidic species than the control. Second, the percentage of acidic species increased sharply with the increase in pH. The degradation related to the change of charges is probably due to deamidation. The

15 previous data indicate that this type of degradation of CHIR-12.12 was minimized at a pH of approximately 5.0-5.5.

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

Sample

Main peak% Acid variants%

t = 0

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

3 m

3 m

1 m 2 m  3 m  3 m 1 m 2 m

Control

49.2 49.8 49.8 49.2 50.3 32.0 33.7 33.7 32.0 33.6

pH 4.5

48.5 49.7 43.7 39.7 30.0 32.5 32.6 38.0 44.2 56.4

pH 5.0

49.6 49.8 48.3 40.6 31.4 32.7 31.8 35.0 44.3 57.1

pH 5.5

50.7 50.3 48.1 40.0 30.2 32.6 31.8 37.8 48.9 63.3

pH 6.0

50.2 49.9 47.9 37.4 23.9 33.1 33.6 38.5 54.9 72.7

pH 6.5

49.4 49.9 42.3 29.7 14.6 33.3 33.6 47.7 65.2 84.6

pH 7.0

49.7 49.9 21.9 - - 34.4 36.4 64.4 - -

pH 7.5

49.3 48.3 12.7 - - 35.5 40.1 79.2 - -

pH 9.0

41.3 31.8 - - - 44.7 59.9 - - -

conclusion

The pH has a significant effect on the conformational and physicochemical stability of CHIR-12.12. Degradation related to charge change was determined to be the main degradation pathway for CHIR-12.12, which was minimized to pH 5.0-5.5. Based on the general stability data, a recommended liquid pharmaceutical formulation comprising this antibody is a formulation comprising CHIR

12.12 at a concentration of approximately 20 mg / ml formulated in approximately 10 mM sodium succinate, approximately 150 mM sodium chloride, and having a pH of approximately 5.5. Example 8: Clinical studies with CHIR-5.9 and CHIR-12.12

Clinical objectives

The general objective is to provide an effective therapy for chronic lymphocytic leukemia (CLL) by converting these cancers into targets with an anti-CD40 IgG1. The signal for these diseases is determined in phase I although some measurement of activity can be obtained in phase I. Initially, the agent is studied as a single agent, but will be combined with other agents, chemotherapeutics, and other antibodies as development proceeds. . Phase I

•

To assess the safety and pharmacokinetics - dose increase in subjects with chronic lymphocytic leukemia (CLL).

•

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

•

Consider more than one dose when it may be necessary to find a phase II dose.

•

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

•

This trial (s) is open to subjects with CLL.

•

The decision to interrupt or continue the studies is based on safety, doses and preliminary tests of antitumor activity.

•

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

II.

• Identify dose (s) for Phase II.

Phase II

Several trials will be initiated in subjects with CLL. More than one dose and more than one program can be tested in a randomized phase II configuration. Go to a CLL population that has failed with current 5 conventional care (chemotherapy failures)

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

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

clinical efficacy 10 image 1 Identify the doses for Phase III

Phase III

Phase III will depend on when the signal is detected in phase II and on which competing therapies are considered the reference standard. If the signal is detected at a stage of the disease in which there is no therapy reference pattern, then it may serve

15 a study of a single arm, well controlled, as a fundamental essay. If there are competing agents that are considered reference standards, then direct studies are performed.

Many modifications and other embodiments of the inventions described in this document will occur to the person skilled in the art to whom these inventions belong to the benefit of the findings presented in the preceding descriptions and the associated drawings. In any case it should be understood that the inventions are not limited to the specific embodiments described and such modifications and other embodiments are intended to be included within the scope of the appended claims and list of embodiments described in this document. Although specific terms are used

25 in it, these are used in a generic and descriptive sense only and not for limiting purposes.

SEQUENCE LIST

30 <110> Aukeman, Lea Long, Li Luqman, Mohammad Yabannavar, Asha Zaror, Isabel

35

<120> Use of antagonist monoclonal anti-CD40 antibodies for the treatment of chronic lymphocytic leukemia

<130> PP22708.002 (284267)

<150> 60 / 611,794

<151> 2004-09-21

<150> 60 / 565,710

<151> 2004-04-27

<150> 60 / 525,579

<151> 2003-11-26

<150> 60 / 517,337

<151> 2003-11-04

<160> 12

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<223> Light chain coding sequence of human anti CD40 12.12 antibody

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image2

image2

<210> 2

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> Human anti-CD40 12.12 antibody light chain

<400> 2 <210> 3

image2

<211> 2016

<212> DNA

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<220>

<223> Heavy chain coding sequence of human anti CD40 12.12 antibody (with introns)

<400> 3

image2

<210> 4 5 <211> 469

<212> PRT

<213> Artificial sequence

<220> 10 <223> Heavy chain of human anti CD40 12.12 antibody

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<223> Heavy chain of human anti-CD40 12.12 antibody variant

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<223> Human anti-CD40 5.9 antibody heavy chain

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image2

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<220>

<223> Heavy chain of human anti-CD40 5.9 antibody variant

10

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5

10

fifteen

20 5

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image2

Claims (12)

1. A human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40, said monoclonal antibody being free of significant agonist activity, whereby when said antibody binds monoclonal to the CD40 antigen expressed on the surface of said cell, the growth or differentiation of said cell is inhibited, for use in a method of treating a human subject with chronic lymphocytic leukemia (CLL), the method comprising the administration to said subject of an effective amount of the human anti-CD-40 monoclonal antibody, said human monoclonal anti-CD40 antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding the antibody monoclonal CHIR-5.9 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. 2. Use of an effective amount of a human anti-CD40 monoclonal antibody that is capable of specifically binding to a human CD40 antigen expressed on the surface of a cell expressing human CD40, said monoclonal antibody being free of significant agonist activity, in the manufacture of a medicament for the treatment of a human subject with chronic lymphocytic leukemia (CLL), so that when said monoclonal antibody is bound to the CD40 antigen expressed on the surface of said cell, the growth or differentiation of said cell, said human monoclonal anti-CD40 antibody being selected from the group Constituted by: a) a monoclonal antibody that binds to an epitope capable of binding to the CHIR-5.9 monoclonal antibody that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the CHIR-12.12 monoclonal antibody that can Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. 3. A human anti-CD40 monoclonal antibody that is capable of specifically binding to a CD40 antigen, said monoclonal antibody being free of significant agonist activity when bound to the CD40 antigen, for use in a method of inhibiting lymphocytic leukemia cell growth. chronic (CLL) expressing CD40 antigen, the method comprising contacting said cells with an effective amount of the monoclonal anti-CD40 antibody, said antibody being selected from the group consisting of: a) a monoclonal antibody that binds to a capable epitope of binding to the antibody monoclonal CHIR-5.9 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. 4. Use of an effective amount of a human anti-CD40 monoclonal antibody that is capable of specifically binding to CD40 antigen in the manufacture of a medicament for growth inhibition of chronic lymphocytic leukemia (CLL) cells expressing CD40 antigen, being said monoclonal antibody free of significant agonist activity when bound to CD40 antigen, said antibody being selected from the group consisting of: a) a monoclonal antibody that binds to an epitope capable of binding the antibody monoclonal CHIR-5.9 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or to the monoclonal antibody CHIR-12.12 that can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. 5. An in vitro method for the inhibition of the growth of chronic lymphocytic leukemia (CLL) cells expressing CD40 antigen, said method comprising contacting said cells with an effective amount of a human monoclonal anti-CD40 antibody that is capable to specifically join said CD40 antigen, said monoclonal antibody being free of agonist activity significant 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 Obtained from the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543; b) a monoclonal antibody that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; c) a monoclonal antibody that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or SEQ ID No. 12; 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. 6. The antibody, use or method of any preceding claim, wherein said human monoclonal anti-CD40 antibody is selected from the group consisting of:

(i)

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

(ii)

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

(iii) an antibody comprising a light chain variable domain containing residues 46-52, 70-72 and 111-116 of SEQ ID No. 2 or SEQ ID No. 6; Y (iv) an antibody comprising a heavy chain variable domain containing residues 45-51, 72-74 and 115-120 of SEQ ID No. 4 or SEQ ID No. 7. 7. The antibody, use or method of claim 6, wherein said human monoclonal anti-CD40 antibody comprises an amino acid sequence selected from the group consisting of:

(i)

residues 21-132 of SEQ ID NO: 2;

(ii)

residues 21-239 of SEQ ID NO: 2;

(iii) SEQ ID NO: 2;

(iv)

residues 20-139 of SEQ ID NO: 4;

(v)

residues 20-469 of SEQ ID NO: 4;

(saw)

SEQ ID NO. 4;

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

(ix)

waste 21-132 of SEQ ID No. 2 and waste 20-139 of SEQ ID No. 4;

(x)

residues 21-239 of SEQ ID No. 2 and residues 20-469 of SEQ ID No. 4;

(xi)

waste 21-239 of SEQ ID No. 2 and waste 20-469 of SEQ ID No. 5;

(xii) SEQ ID No. 2 and SEC ID No. 4; Y (xiii) SEQ ID No. 2 and SECID No. 5. 8. The antibody, use or method of claim 6, wherein said human monoclonal anti-CD40 antibody comprises an amino acid sequence selected from the group consisting of:

(i)

residues 21-132 of SEQ ID NO: 6;

(ii)

residues 21-239 of SEQ ID NO: 6;

(iii) SEQ ID No. 6;

(iv)

residues 20-144 of SEQ ID NO: 7;

(v)

residues 20-474 of SEQ ID NO: 7;

(saw)

SEQ ID NO. 7;

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

(ix)

waste 21-132 of SEQ ID No. 6 and waste 20-144 of SEQ ID No. 7;

(x)

waste 21-239 of SEQ ID No. 6 and waste 20-474 of SEQ ID No. 7;

(xi)

waste 21-239 of SEQ ID No. 6 and waste 20-474 of SEQ ID No. 8

(xii) SEQ ID No. 6 and SEC ID No. 7; Y (xiii) SEQ ID No. 6 and SECID No. 8. 9. The antibody, use or method of any of claims 1 to 8, wherein said antibody is selected from the group consisting of the CHIR-5.9 antibody produced by the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA -5542 or CHIR-12.12 produced by the hybridoma cell line 5 deposited with ATCC as Patent Deposit No. PTA-5543.

10.

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

eleven.

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

10 12. The antibody, use or method of any of claims 1 to 8, wherein said monoclonal antibody is an antigen binding fragment of said anti-CD40 monoclonal antibody, wherein said fragment retains the ability to specifically bind to said human CD40 antigen. 13. The antibody, use or method of claim 12, wherein said fragment is selected from the group consisting of a Fab fragment, an F (ab ’) 2 fragment, an Fv fragment and a single chain Fv fragment. 14. The antibody, use or method of any preceding claim, wherein said monoclonal antibody is produced in a CHO cell line.