ES2367892T3 - THERAPY PROCEDURES FOR CANCERS RELATED TO CELLS B. - Google Patents

Abstract

An anti-CD40 antagonist antibody or an antigen-binding fragment thereof for use in a method of treating a human subject of a cancer characterized by a neoplastic B-cell growth by combining antibody therapy with an anti-CD20 antibody or a antigen-binding fragment thereof, wherein said anti-CD40 antagonistic antibody or said antigen-binding fragment thereof are selected from the group consisting of: (a) an antibody or an antigen-binding fragment thereof that binds with an epitope capable of binding with the monoclonal antibody CHIR-5.9, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542, or with the monoclonal antibody CHIR-12.12, which can be obtained from the line hybridoma cell deposited in the ATCC as Patent Deposit No. PTA-5543; (b) an antibody or an antigen binding fragment thereof that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12; (c) an antibody or an antigen-binding fragment thereof 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) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay.

Description

Field of the Invention

The present invention relates to methods of combining antibody therapy for B cell-related cancers, particularly cancers comprising neoplastic cells that express the CD40 and CD20 cell surface antigens.

Background of the invention

Leukemia, lymphoma and myeloma affect more than 100,000 individuals every year in the US alone. UU. A large percentage of these cases are characterized by hypertrophy of neoplastic B cells that express the CD40 and CD20 antigens. CD40 is a 55 kDa antigen from the cell surface present on the surface of both normal and neoplastic human B cells, dendritic cells, other antigen presenting cells (APC), endothelial cells, monocytic cells and epithelial cells . The binding of the CD40 ligand with the CD40 in the B cell membrane provides a positive costimulatory signal that stimulates the activation and proliferation of the B cells, which results in the maturation of the B cell in a plasma cell that secretes high levels of soluble immunoglobulin. Transformed cells of patients with low-grade and high-grade B-cell lymphomas, acute B-cell lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia and Hodgkin's disease express CD40. CD40 expression is also detected in two out of three cases of acute myeloblastic leukemia and in 50% of AIDS-related lymphomas. Malignant B cells of several tumors with B-cell lineage express an elevated level of CD40 and appear to depend on CD40 signaling for survival and proliferation. This makes the CD40 antigen a potential target for anticancer therapy.

CD20 is expressed early in the differentiation of B cells and remains on the cell surface throughout the development of cell B. CD20 is involved in the activation of B cells, it is expressed with very high levels in B cells. neoplastic and is a clinically recognized therapeutic target (see, for example, Hooijberg et al. (1995) Cancer Research 55: 2627). The U.S. The Food and Drug Administration has authorized the use of antibodies, such as Rituxan®, that target CD20 for the treatment of non-Hodgkin's lymphoma (see, for example, Boye et al. (2003) Ann. Oncol. 14: 520) . Rituxan® has been shown to be an effective treatment for low, intermediate and high grade non-Hodgkin's lymphoma (NHL) (see, for example, Maloney et al. (1994) Blood 84: 2457-2466; McLaughlin et al. (1998) J. Clin. Oncol. 16: 2825-2833; Maloney et al. (1997) Blood 90: 2188-2195; Hainsworth et. Al. (2000) Blood 95: 3052-3056; Colombat et al. (2001 ) Blood 97: 101-106; Coiffier et al. (1998) Blood 92: 1927-1932; Foran et al. (2000) J. Clin. Oncol. 18: 317-324; Anderson et al. (1997) Biochem. Soc. Trans. 25: 705-708; Vose et al. (1999) Ann. Oncol. 10: 58a).

Although the exact mechanism of action is unknown, the evidence indicates that the anti-lymphoma effects of Rituxan® are partly due to complement-mediated cytotoxicity (CMC), antibody-dependent cell-mediated cytotoxicity (ADCC), inhibition of cell proliferation and, finally, direct the induction of apoptosis. However, some patients become resistant to treatment with Rituxan® (Witzig et al. (2002) J. Clin. Oncol. 20: 3262; Grillo-López et al. (1998) J. Clin. Oncol. 16: 2825; Jazirehi et al. (2003) Mol. Cancer Ther. 2: 1183-1193). For example, some patients lose CD20 expression in malignant B cells after anti-CD20 antibody therapy (Davis et al. (1999) Clin. Cancer Res. 5: 611). In addition, 30% to 50% of patients with low-grade NHL show no clinical response to this monoclonal antibody (Hainsworth et. Al. (2000) Blood 95: 3052-3056; Colombat et al. (2001) Blood 97 : 101-106). Alternative forms of therapeutic intervention are required for patients who develop resistance to this monoclonal antibody or who have a B-cell lymphoma that is resistant to initial therapy with this antibody.

Thus, there is a need for treatment regimes for B-cell-related cancers that do not create resistance to antibodies and that can provide effective therapy in the event of resistance to antibodies. Consequently, the discovery of an antibody combination therapy with an antitumor activity superior to that of the single agent Rituxan® could drastically improve cancer therapy procedures in individuals with myelomas, leukemias and lymphomas, particularly B-cell lymphomas.

WO 01/83755 and WO 02/88186 describe antibodies capable of binding with CD40, procedures for producing these antibodies and methods for their use.

WO 02/28480 describes the use of anti-CD40 antibodies in combination with anti-CD20 antibodies for cancer treatment in which malignant B cells are involved.

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

Brief Summary of the Invention

The invention provides an anti-CD40 antagonist antibody or an antigen-binding fragment thereof for use in a method of treating a human subject of a cancer characterized by a neoplastic B-cell growth by combining antibody therapy with an anti-antibody. CD20 or a

5 antigen-binding fragment thereof in which said anti-CD40 antagonistic antibody or said antigen-binding fragment thereof are selected from the group consisting of:

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

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

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

d) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR- monoclonal antibody 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 anti-CD40 antagonist antibody or an antigen binding fragment thereof in the manufacture of a medicament to be used to treat a human subject of a cancer characterized by a neoplastic cell growth. B by combination antibody therapy with an anti-CD20 antibody or an antigen-binding fragment thereof in which

Said anti-CD40 antagonist antibody or said antigen binding fragment thereof are selected from the group consisting of:

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

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

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

d) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR- monoclonal antibody 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 an anti-CD40 antagonist antibody or an antigen-binding fragment thereof for use in a method of inhibiting the growth of a tumor comprising neoplastic B cells by combining antibody therapy with an anti-CD20 antibody or a fragment. thereof antigen binding in which said anti CD40 antagonistic antibody or said fragment thereof binding to the

45 antigens are selected from the group consisting of:

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

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

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

d) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR- monoclonal antibody 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 anti-CD40 antagonist antibody or an antigen-binding fragment thereof in the manufacture of a medicament to be used to inhibit the growth of a tumor comprising neoplastic B cells by combination therapy. of antibodies with an anti-CD20 antibody or a fragment thereof to the antigen in which said anti-CD40 antagonist antibody or said fragment thereof to the antigen is selected from the group consisting of:

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

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

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

d) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR- monoclonal antibody 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 an in vitro method for inhibiting the growth of a tumor comprising neoplastic B cells comprising contacting said cells with an effective amount of an anti-CD40 antagonistic antibody or a fragment thereof antigen binding in combination with a anti CD20 antibody

or an antigen binding fragment thereof, wherein said antibody or said antigen binding fragment thereof is selected from the group consisting of:

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

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

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

d) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR- monoclonal antibody 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.

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

Brief summary of the disclosure

Methods for treating a cancer subject characterized by a neoplastic growth of B cells are provided. The methods comprise the administration of a combination of antibodies that have a therapeutic effect against neoplastic B cells expressing the CD40 and CD20 antigens of the cell surface. In some embodiments, a synergistic therapeutic effect occurs, which makes the disclosure especially useful in the treatment of cancers that are refractory to antibody therapy that has a single antigen on the surface of the B cell.

According to the methods disclosed herein, a combination of an anti-CD40 antagonist antibody (or a fragment thereof antigen-binding) and an anti-CD20 antibody (or a fragment of the antigen) is administered to an individual in need thereof. same antigen binding). Anti-CD40 antagonist antibodies suitable for use in the methods disclosed herein include monoclonal antibodies or antigen-binding fragments thereof that are capable of specifically binding with the human CD40 antigen expressed on the surface of a human cell. They are free of significant agonist activity, but exhibit antagonistic activity when they bind with the CD40 antigen on human cells, particularly when they bind with the CD40 antigen in human neoplastic B cells. Suitable anti-CD40 monoclonal antibodies have constant human regions; preferably, they also have partially humanized framework regions; and most preferably they are fully human antibodies or fragments thereof antigen binding). Examples of such anti-CD40 monoclonal antibodies are the antibodies designated herein as CHIR-5.9 and CHIR-12.12, which can be produced recombinantly; the monoclonal antibodies produced by the hybridoma cell lines designated 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.D10.D6.12.12 (referred to herein 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 sequence shown in both SEQ ID No. 6 as in SEQ ID No. 7, and the sequence shown in both SEQ ID No. 6 and SEQ ID No. 8; a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID No. 2, the sequence shown in SEQ ID No. 4, the sequence shown in SEQ ID No. 5, the sequence shown in both SEQ ID No. 2 as in SEQ ID No. 4, and the sequence shown in both SEQ ID No. 2 and SEQ ID No. 5; a monoclonal antibody comprising an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID No. 1, the sequence shown in SEQ ID No. 3, and the sequence shown in both SEQ ID No. 1 and SEQ ID No. 3; and fragments of these monoclonal antigen-binding antibodies that retain the ability to specifically bind with human CD40 and that are free of significant agonist activity, but exhibit antagonistic activity when they bind with the CD40 antigen on human cells. Examples of such anti-CD40 monoclonal antibodies also include a monoclonal antibody that binds with an epitope capable of binding with the monoclonal antibody produced by the cell line.

12.12 of hybridoma or that produced by the hybridoma cell line 5.9; a monoclonal antibody that binds to an epitope comprising residues 82-87 of the amino acid sequence shown in SEQ ID No. 10 or in 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 a fragment of the CHIR monoclonal antibody

12.12 or CHIR-5.9 antigen binding or any of the preceding monoclonal antibodies in which the fragment retains the ability to specifically bind with the human CD40 antigen.

Anti-CD20 antibodies suitable for practicing the disclosure include, without limitation, the chimeric monoclonal antibody IDEC-C2B8 (Rituxan® or rituximab); anti-CD20 antibodies having the binding characteristics of IDEC-C2B8, in which anti-CD20 antibodies compete with the IDEC-C2B8 antibody in a competitive binding assay or bind with an epitope capable of binding with the IDEC-C2B8 antibody. The methods disclosed herein are particularly effective when administering anti-CD40 antagonist antibodies produced by a hybridoma such as 5.9 or 12.12 in combination with an anti-CD20 antibody such as IDEC-C2B8. The disclosure further includes pharmaceutical compositions comprising such combinations of antibodies in a pharmaceutically acceptable carrier.

The methods disclosed herein are useful for treating individuals with B-cell lymphomas such as non-Hodgkin's lymphomas (high grade lymphomas, intermediate grade lymphomas and low grade lymphomas), Hodgkin's disease, acute lymphoblastic leukemia, Myelomas, chronic lymphocytic leukemias and myeloblastic leukemias, and are particularly useful for the treatment of B-cell-related cancers that are refractory to treatment with a single antibody therapy that targets the CD20 cell surface antigen.

Brief description of the drawings

Figure 1 shows the effect, over time, of the combined administration of CHIR-12.12 mAb and IDEC-C2B8 mAb on tumor volume in a murine model of Rituxan® resistant tumor.

Figure 2 shows the amino acid sequences of the light and heavy chains of the CHIR-12.12 mAb. Figure 2A shows the forward 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 Figure 2B shows the forward 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 heavy chain The alternative constant region of the CHIR-12.12 mAb heavy chain shown in Figure 2B reflects a substitution of an alanine residue with the serine residue at position 153 of SEQ ID NO. 4. The complete sequence of this variant of the CHIR-12.12 mAb heavy chain is presented in SEQ ID NO: 5.

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

Figure 4 shows the amino acid sequences for the light and heavy chains of the CHIR-5.9 mAb. Figure 4A shows the front 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 Figure 4B shows the forward 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 heavy chain The alternative constant region of the CHIR-5.9 mAb heavy chain shown in Figure 4B reflects a substitution of an alanine residue with the serine residue at position 158 of SEQ ID NO. 7. The complete sequence of this variant of the heavy chain of the CHIR-5.9 mAb is presented in SEQ ID NO.

8.

Figure 5 shows the coding sequence (Figure 5A; SEQ ID No. 9) of the short isoform of human CD40 (amino acid sequence shown in Figure 5B; SEQ ID No. 10) and the coding sequence (Figure 5C; SEQ ID No. 11) of the long isoform of human CD40 (amino acid sequence shown in Figure 5D; SEQ ID No. 12).

Figure 6 shows the thermal 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, either malignant or benign, and to all cells and all precancerous and cancerous tissues. "Neoplastic," as used herein, refers to any form of deregulated or unregulated cell growth, whether malignant or benign, that results in abnormal tissue growth.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is normally characterized by unregulated cell growth. Examples of cancer include, without limitation, lymphoma and leukemia. By "B cell-related cancer" is meant any type of cancer in which deregulated or unregulated cell growth is associated with B cells.

By "refractory", in the context of a cancer, it is meant that the particular cancer is resistant to therapy with a particular therapeutic agent, or that does not respond to it. A cancer may be refractory to a therapy with a particular therapeutic agent either from the start of treatment with the particular therapeutic agent (i.e., does not respond to initial exposure to the therapeutic agent), or as a consequence of the development of resistance to the therapeutic agent , either during the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.

"Antibodies" and "immunoglobulins" (Ig) are glycoproteins that have the same structural characteristics. The terms are used synonymously. In some cases, the specificity of the immunoglobulin antigen may be known.

The term "antibody" is used in the broadest sense and encompasses fully assembled antibodies, antibody fragments that can bind with an antigen (eg, Fab, F (ab ') 2, Fv, single chain antibodies, diabodies, antibody chimeras , hybrid antibodies, bispecific antibodies, humanized antibodies and the like) and recombinant peptides comprising the above.

The terms "monoclonal antibody" and "mAb", as used herein, refer to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical, except in possible mutations that occur naturally that may be present in minor amounts. As used herein, "anti CD40 antibody" encompasses any antibody that specifically recognizes the cell surface CD40 antigen, including polyclonal antibodies, monoclonal antibodies, single chain antibodies and fragments thereof, such as Fab, F (ab ' ) 2, Fv, and other fragments that retain the antigen binding function of the anti-CD40 parent antibody. Of particular interest to implement the methods disclosed herein are anti-CD40 antibodies or fragments thereof antigen-binding that have the binding properties of human CH40-5.9 and CHIR anti-CD40 monoclonal antibodies.

12.12 described below in this document.

As used herein, "anti CD20 antibody" encompasses any antibody that specifically recognizes the cell surface CD20 antigen, including polyclonal antibodies, monoclonal antibodies, single chain antibodies and fragments thereof, such as Fab, F (ab ') 2, Fv, and other fragments that retain the antigen binding function of the anti-CD20 parent antibody. Of particular interest to implement the methods disclosed herein are anti-CD20 antibodies or fragments thereof antigen-binding having the binding properties of the monoclonal antibody IDEC-C2B8 described below in the present document

"Native antibodies" and "native immunoglobulins" are typically 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 means of a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly separated intracatenary disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the variable domains of the light and heavy chains.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence between antibodies. Variable regions confer specificity on binding with antigens. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called complementarity determining regions (CDR)

or hypervariable regions in both the light chain and heavy chain variable domains. The most highly conserved portions of the variable domains are boxed in the framework regions (FR). The variable domains of the native light and heavy chains each comprise four FR regions, which largely adopt a folded sheet configuration β, connected by three CDRs, which form loops that connect the structure of the folded sheet β, and in some cases are part of it. The CDRs in each chain are held together in close proximity through 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 Pub. No. 91-3242, Vol. I, pages 647-669).

Constant domains do not directly participate in the binding of an antibody with an antigen, but exhibit various effector functions, such as the binding of the Fc receptor, the participation of the antibody in antibody-dependent cellular toxicity, the initiation of cytotoxicity dependent on the complement 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 2434 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th ed., Public Health Service, National Institute of Health, Bethesda, Maryland) and / or waste from a “hypervariable loop” (ie, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Clothia and Lesk (1987) J. Mol. Biol.

196: 901-917). The residues of the "framework" or "FR" are the residues of the variable domain other than the residues of the hypervariable region, as estimated herein.

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

"Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain domain in close association, not covalent. Precisely in this configuration the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to 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 entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab ’fragments by the addition of some residues at the carboxy terminus of the CH1 domain of the heavy chain that includes one or more cysteines of the hinge region of the antibody. Fab'-SH is the designation herein for Fab 'in which the cysteine residue or residues of the constant domains have a free thiol group. Fab ’fragments are produced by reducing the disulfide bridge of the heavy chain of fragment F (ab’) 2. Other chemical pairings 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, IgA1 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 subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, the IgG1 and IgG3 isotypes have ADCC activity (antibody-dependent cell-mediated cytotoxicity).

The word "label," when used herein, refers to a detectable compound or composition that conjugates directly or indirectly with the antibody to generate a "labeled" antibody. The label can be detectable on its own (for example, radioisotopic markers or fluorescent markers) or, in the case of an enzymatic label, it can catalyze the chemical alteration of a compound or a 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 that are non-toxic to the cell or mammal that are exposed thereto at the doses and concentrations used. Often, the physiologically acceptable carrier is an aqueous solution of buffered pH. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate 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 poloxamers (Pluronic). Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) administration and consecutive (ie, sequential) administration in any order.

A "host cell," as used herein, refers to a microorganism or a eukaryotic cell or cell line grown as a unicellular entity that can be used, or has been used, as a receptor for a recombinant vector or others. transfer polynucleotides, and include 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 killer cells (NK), monocytes, macrophages, eosinophils and neutrophils, with PBMC and NK cells being preferred. Antibodies that have ADCC activity are normally 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 designing a variable region from an antibody that does not mediate ADCC or from a variable region fragment over 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 FcγRI, FcγRII and FcγRIII subclasses, 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 reason for activation of tyrosine-based immunoreceptors (ITAM) in its cytoplasmic domain. The FcγRIIB inhibitor receptor contains a reason for inhibition of tyrosine-based immunoreceptors (ITIM) in its cytoplasmic domain (see Daeron (1997) Annu. Rev. Immunol. 15: 203-234). FcRs are reviewed in Ravetch and Kinet (1991) Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al. (1994) Immunomethods 4: 25-34; and de 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).

The term "synergy" is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, when the combined effect of two or more agents results in a "synergistic inhibition" of an activity or a process, for example tumor growth, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with a combination of two or more therapies in which the therapeutic effect (as measured by any of several parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.

The terms "therapeutically effective dose", "therapeutically effective amount" or "effective amount" are intended to mean an amount of the anti-CD40 antagonist antibody (or the antigen-binding fragment thereof) that, when administered in combination with an amount of the anti-CD20 antibody (or the antigen binding fragment thereof), produces a positive therapeutic response in a subject suffering from cancer comprising neoplastic B cells.

Combination therapy with anti CD40 and anti CD20 antibodies

The present disclosure is about methods of treating a subject who has a cancer characterized by a growth of neoplastic B cells. Such neoplastic B cells include, without limitation, neoplastic B cells derived from lymphomas that include low, intermediate and high grade B cell lymphomas, immunoblastic lymphomas, non-Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr virus-induced lymphomas ( EBV), AIDS-related lymphomas, as well as acute lymphoblastic leukemias, myelomas, chronic lymphocytic leukemias, acute myeloblastic leukemias and the like.

The methods disclosed herein encompass a combination antibody therapy with an anti-CD40 antagonist antibody, or an antigen-binding fragment thereof, and an anti-CD20 antibody, or an antigen-binding fragment thereof. The methods disclosed herein are especially useful for the treatment of cancers comprising neoplastic B cells that express both CD40 and CD20 cell surface antigens, such as B-cell lymphomas. Examples of lymphomas that can express CD40 and CD20 antigens include, without limitation, acute B-cell lymphoblastic leukemia, Hodgkin's disease, diffuse small cell lymphocytic lymphoma, prolymphocytic leukemia, lymphoid tissue lymphoma associated with mucous membranes, B-cell monocytoid lymphoma, splenic lymphoma, lymphomatoid granulomatosis , intravascular lymphomatosis, immunoblastic lymphoma, AIDS-related lymphoma and the like.

Thus, the procedures disclosed herein find use in the treatment of non-Hodgkin's lymphomas related to abnormal and uncontrollable accumulation of B cells. For the purposes of disclosure, reference will be made to such lymphomas according to the model. of working formulation classification, that is, those B-cell lymphomas classified as low grade, intermediate grade and high grade (see “The Non-Hodgkin's Lymphoma Pathologic Classification Project”, Cancer 49 (1982): 2112-2135) . Thus, low-grade B-cell lymphomas include small cell lymphocytic lymphomas, small follicular follicular lymphomas, and mixed follicular small-cell follicles and large cells; intermediate-grade B-cell lymphomas include follicular large cell lymphomas, diffuse cleft small cells, mixed diffuse small and large cells, and diffuse large cell; and high-grade B-cell lymphomas include immunoblastic large cell, lymphoblastic and non-cleft small cell lymphomas of the Burkitt type and of which it is not.

It is recognized that the procedures disclosed herein are useful in the therapeutic treatment of B-cell lymphomas that are classified according to the revised European and North American lymphoma classification system (REAL). Such B cell lymphomas include, without limitation, lymphomas classified as precursor B cell neoplasms, such as leukemia or lymphoblastic B lymphoma; peripheral B-cell neoplasms, including chronic B-cell leukemia / small cell lymphocytic lymphoma, lymphoma / lymphoplasmacyte immunocytoma, mantle cell lymphoma (MCL), central follicular (follicular) lymphoma (including diffuse lymphomas of small cells, mixed diffuse small and large cells and diffuse large cells), B-cell lymphoma of the marginal zone (including extranodal, nodal and splenic types), hairy cell leukemia, plasmacytoma / myeloma, diffuse lymphoma of large cell B cells of the primary mediastinal (thymic) subtype, Burkitt lymphoma and high-grade B-cell lymphoma similar to Burkitt; acute leukemia; acute lymphocytic leukemias; myeloblastic leukemias; acute myelocytic leukemias; promyelocytic leukemia; myelomonocytic leukemia; monocytic leukemia; erythroleukemia; granulocytic leukemia (chronic meilocytic leukemia); Chronic lymphocytic leukemia; polycythemia vera; multiple myeloma; Waldenstrom macroglobulinemia; Heavy chain disease and unclassifiable low-grade or high-grade B-cell lymphomas.

In particular, the methods disclosed herein are useful for the treatment of B-cell lymphomas, including those listed above, which are refractory (i.e., resistant or have become resistant) to first-rate oncotherapeutic treatments. line. The term "oncotherapeutic" is intended to mean a treatment for cancer such as chemotherapy, surgery, radiation therapy, simple anti-cancer antibody therapy and combinations thereof.

"Treatment" is defined herein as the application or administration of an anti-CD40 antagonist antibody or a fragment thereof to the antigen binding to a subject, or the application or administration of an anti-CD40 antagonist antibody or a fragment of the same to an isolated tissue or cell line from a subject, in combination with the application or administration of an anti-CD20 antibody or a fragment thereof antigen binding to a subject, or to an isolated tissue or cell line from a subject, in which the subject has a disease, a symptom of a disease or a predisposition to the disease, in which the purpose is to cure, heal, calm, mitigate, alter, remedy, relieve, improve or affect the disease , the symptoms of the disease or the predisposition to the disease. By "treatment" it is also meant that the combination of these antibodies or the antigen binding fragments thereof can be applied or administered to the subject, or to the isolated tissue or cell line from the subject, as part of a composition. single pharmaceutical or, alternatively, as part of individual pharmaceutical compositions, each of which comprises either the anti-CD40 antibody (or the antigen binding fragment thereof) or the anti CD20 antibody (or a binding fragment thereof to the antigen), in which the subject has a disease, a symptom of a disease or a predisposition to a disease, in which the purpose is to cure, heal, calm, mitigate, alter, remedy, relieve, improve or affect the disease, disease symptoms or predisposition to disease.

Anti-CD40 antibodies suitable for use in the methods disclosed herein specifically bind to a human CD40 antigen expressed on the surface of a human cell and are free of significant agonist activity when bound to the CD40 antigen in a human cell expressing CD40, including normal and neoplastic human B cells (either malignant or benign). In some embodiments, their binding to the CD40 shown on the surface of human cells results in the inhibition of proliferation and differentiation of these human cells. Thus, anti-CD40 antagonist antibodies suitable for use in the methods disclosed herein include those monoclonal antibodies that can exhibit antagonistic activity towards normal and neoplastic human cells that express the CD40 antigen on the cell surface. These anti-CD40 antibodies and the antigen-binding fragments thereof are referred to herein as "anti-CD40 antagonistic antibodies." Such antibodies include, without limitation, the fully human monoclonal antibodies CHIR-5.9 and CHIR-12.12, described below, and the monoclonal antibodies having the binding characteristics of the monoclonal antibodies CHIR-5.9 and CHIR-12.12. These monoclonal antibodies, which can be produced recombinantly, are described below.

In addition to the monoclonal antibodies CHIR-5.9 and CHIR.12.12, other anti-CD40 antibodies that would be useful in the implementation of the procedures described herein include, without limitation: (1) the monoclonal antibodies produced by the cell lines of designated hybridoma 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 in 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 sequence shown both in SEQ ID No. 2 and in SEQ ID No. 4, and the sequence shown in both SEQ ID No. 2 and SEQ ID No. 5; (3) a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID No. 6, the sequence shown in SEQ ID No. 7, the sequence shown in SEQ ID No. 8, the sequence shown both in SEQ ID No. 6 and in SEQ ID No. 7, and the sequence shown in both SEQ ID No. 6 and SEQ ID No. 8; (4) 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 nucleotide sequence shown in SEQ ID NO. 3, and the sequence shown in both SEQ ID No. 1 and SEQ ID No. 3; (5) a monoclonal antibody that binds with an epitope capable of binding with the monoclonal antibody produced by hybridoma cell line 5.9 or 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 in 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; Y

(8) a monoclonal antibody that is a fragment of the CHIR-12.12 or CHIR-5.9 monoclonal antibody to the antigen or the above monoclonal antibodies in the preceding elements (1) - (7), in which the fragment retains the ability of specifically joining the human CD40. Those skilled in the art admit that the antibodies and fragments of these antigen-binding antibodies, suitable for use in the methods disclosed herein, include antibodies and antigen-binding fragments thereof that are produced from recombinant form using methods well known in the art and described below and include, for example, monoclonal antibodies CHIR-5.9 and CHIR

12.12 that have been produced recombinantly.

Anti-CD20 antibodies suitable for use in the methods disclosed herein specifically bind to the human CD20 antigen expressed on the surface of a human cell. Anti-CD20 antibodies useful in the implementation of this disclosure may have one or many mechanisms of action. Although the methods disclosed herein are not limited by any particular mechanism of action, it has been shown that anti-CD20 antibodies induce at least antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxity. (CDC), the regulation of downward proliferation and apoptosis in target cells. Such antibodies include, without limitation, the IDEC-C2B8 antibody (Biogen Idec Pharmaceuticals Corp., Cambridge, Massachusetts; commercially available under the trade name Rituxan®, also referred to as rituximab) which is a chimeric anti-CD20 monoclonal antibody that contains human IgG1 and kappa constant regions with isolated murine variable regions from an anti-CD20 murine monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood 83: 435-445; see also US Patent No. 5,736,137); the radiolabelled anti-CD20 Zevali® antibody (Ibritumomab tiuxetan), manufactured by Biogen IDEC Pharmaceuticals Corp. (Cambridge, Massachusetts); Bexxar® (Tositumomab, which is the murine version of rituximab, combined with iodine-labeled Tositumomab (I131), manufactured by Corixa Corp. (Seattle, Washington); the fully human HuMax-CD20 antibody; R-1594; IMMU-106; TRU-015; AME-133; and monoclonal antibodies that have the binding characteristics of IDEC-C2B8,

that is, the binding specificity of IDEC-C2B8 and the ability to induce one or more of the following activities when bound to the CD20 antigen or to B cells expressing CD20: (1) antibody-dependent cell-mediated cytotoxicity (ADCC); (2) complement dependent cytotoxity (CDC); (3) downregulation of B cell proliferation; and (4) apoptosis in the target cells. In vitro and in vivo assays to measure the ability of anti CD20 antibodies to induce these activities are well known in the art. See, for example, the tests disclosed in US Patent No. 5,736,137. Other antibodies useful for the implementation of the methods disclosed herein are murine and human anti-CD20 antibodies conjugated to radiolabels, such as In-111 and Y-90, and other therapeutic agents, such as toxins.

In addition to using the anti-CD40 anti-gonist antibodies and the anti-CD20 antibodies mentioned above, and more fully described herein below, the procedures disclosed herein can be practiced using antibodies having the binding characteristics. of the monoclonal antibodies CHIR-5.9, CHIR-12.12 or IDEC-C2B8 and that competitively interfere with the binding of these antibodies to their respective antigens or that bind with the same epitopes. One skilled in the art could determine whether an antibody interferes competitively with the binding of CHIR-5.9, CHIR-12.12 or IDEC-C2B8 using standard procedures.

Combination therapy with anti CD20 antibodies (or with antigen binding fragments thereof) and anti CD40 antagonist antibodies (or with antigen binding fragments thereof) provides a therapeutic benefit that is greater than that provided by use. of any of these anticancer agents alone. In addition, these two types of antibodies can be used in combination to treat tumors that are refractory to treatment with a simple antibody therapy, particularly anti-CD20 antibody therapy, either as a result of an initial resistance to simple antibody therapy or as a result of a resistance that develops during one or more sessions of therapy with the single antibody. In other additional embodiments, the combination therapy with these two antibodies has a synergistic therapeutic effect against tumors that are refractory or non-refractory (i.e., sensitive) to therapy with the single antibody. In some embodiments, the methods disclosed herein comprise a combination therapy with the anti-CD20 monoclonal antibody IDEC-C2B8 and the anti-CD40 monoclonal antibody CHIR-12.12. In other embodiments, the methods disclosed herein comprise a combination therapy with the anti-CD20 monoclonal antibody IDEC-C2B8 and the anti-CD40 monoclonal antibody CHIR-5.9. In other further embodiments, the methods disclosed herein comprise a combination therapy with an antigen-binding fragment of the anti-CD20 monoclonal antibody IDEC-C2B8 and an antigen-binding fragment of the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9. In alternative embodiments, the methods disclosed herein comprise a combination therapy with the anti-CD20 monoclonal antibody IDEC-C2B8 and an antigen-binding fragment of the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9. In other embodiments, the methods disclosed herein comprise a combination therapy with an antigen-binding fragment of the anti-CD20 monoclonal antibody IDEC-C2B8 and the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9. The combination therapy described herein may comprise other variations, provided that both the CD20 and the CD40 antigen are target of the treatment procedure.

Multiple parameters may be indicative of the effectiveness of the treatment. These include, without limitation, a reduction in the size of the tumor mass; a reduction in metastatic invasiveness of the tumor; a reduction in the growth rate of the tumor; a decrease in the severity or incidence of sequelae related to tumors, such as cachexia and ascites production; a decrease and / or prevention of complications related to tumors, such as bone pathological fractures, autoimmune hemolytic anemia, prolymphocytic transformation, Richter's syndrome and the like; tumor sensitization to chemotherapy and other treatments; an increase in the patient's survival rate; an increase in observed clinical correlates of improved prognosis, such as an increase in lymphocytes infiltrating the tumor and a decrease in vascularization of the tumor; and the like Thus, in some embodiments, administration of the combination of these two types of antibodies will result in an improvement of one or more of these parameters in a patient (i.e., a subject) who is undergoing treatment. In other embodiments, the improvements in the patient will be synergistic with respect to some parameters, but additive with respect to others.

By "positive therapeutic response" with respect to cancer treatment is meant an improvement in treatment in association with the antitumor activity of these antibodies or fragments thereof, and / or an improvement in the symptoms associated with the disease. That is, an antiproliferative effect, the prevention of more tumor hypertrophy, a reduction in tumor size, a reduction in the number of cancer cells and / or a decrease in one or more neoplastic B-mediated symptoms can be observed. Thus, for example, an improvement in the disease can be characterized as a complete response. By "complete response" is meant an absence of clinically detectable disease, with the normalization of any radiographic, bone marrow and cerebrospinal fluid (CSF) studies previously abnormal. Such response must persist for at least one month after the treatment according to the methods of the invention. Alternatively, an improvement in the disease can be categorized as a partial response. By "partial response" is meant at least about a 50% decrease in the entire measurable burden of the tumor (ie, the number 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 measurable tumors.

Tumor response can be evaluated by looking for changes in tumor morphology (i.e., overall tumor burden, tumor size and the like) using detection techniques such as magnetic resonance imaging (MRI) scanning, formation of X-ray imaging, computed tomographic scanning (CT), flow cytometry or fluorescence-activated cell sorting (FACS) analysis, bioluminescent imaging, for example luciferase imaging, imaging Bone scanning, tumor biopsy sampling, including bone marrow aspiration (BMA). In addition to these positive therapeutic responses, the subject undergoing therapy may experience the beneficial effect of an improvement in the symptoms associated with the disease. Thus, for B-cell tumors, the subject may experience a decrease in what has been called B-symptoms, that is, night sweats, fever, weight loss and / or hives.

By "therapeutically effective dose", "therapeutically effective amount" or "effective amount" is meant an amount of the anti-CD40 antagonist antibody (or antigen-binding fragment thereof) that, when administered in combination with an amount of the antibody anti CD20 (or the antigen binding fragment thereof), produces a positive therapeutic response with respect to the treatment of a subject for a cancer comprising neoplastic B cells. In some embodiments of the disclosure, a therapeutically effective dose of either the anti-CD20 antibody (or the antigen-binding fragment thereof) or the anti-CD40 antagonist antibody (or the antigen-binding fragment thereof), 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 admitted that the treatment method may comprise a single administration of a therapeutically effective dose of the antibody combination useful in the implementation of this disclosure, or multiple administrations of a therapeutically effective dose of the antibody combination.

A procedure for predicting clinical efficacy is to measure the effects of combination therapy with these antibodies in a suitable model; for example, the use of the combination of an anti-CD20 antibody and an anti-CD40 antagonist antibody in murine cancer models. These models include xenograft tumor models in hairless mice, such as those used by Burkitt's lymphoma human cell lines known as Namalwa and Daudi. In some embodiments, antitumor activity is evaluated in a phase model of xenograft tumor in hairless mice using Daudi's human lymphoma cell line. A phase model cell line of xenograft tumor in hairless mice is generally more effective in distinguishing the therapeutic efficacy of an antibody given a phaseless model, since in the phase model the antibody dosing starts only after The tumor has reached a measurable size. In the phaseless model, antibody dosing is generally initiated in less than about 1 day after tumor inoculation and before a palpable tumor is present. The ability of an antibody to exhibit increased antitumor activity in a phased model is a clear indication that the antibody will be therapeutically effective.

The procedures disclosed herein comprise the use of a combination therapy. The term "combination" is used in its broadest sense and means that a subject is treated with at least two therapeutic regimens. Thus, "antibody combination therapy" means that a subject is treated with at least two antibody regimens; more particularly, with at least one anti-CD20 antibody (or an antigen binding fragment thereof) in combination with at least one anti-CD40 antibody (or an antigen binding fragment thereof), but the timing of administration of the Different antibody regimens can be varied, as long as the beneficial effects of the combination of these antibodies are achieved. Treatment with an anti CD20 antibody (or a fragment thereof antigen binding) in combination with an anti CD40 antagonist antibody (or a fragment thereof antigen binding) may be simultaneous (concurrent), consecutive (sequential) or a combination of both. Therefore, a subject undergoing antibody combination therapy can receive both antibodies at the same time (i.e., simultaneously) or at different times (i.e. sequentially, in any order, on the same day or on days different), provided that the therapeutic effect of the combination of both substances is provoked in the subject who is undergoing therapy. In some embodiments, the combination of antibodies will be given simultaneously for a dosage, but other dosages will include sequential administration, in either order, on the same day or on different days. Sequential administration can be carried out regardless of whether the subject responds to the administration of the first monoclonal antibody. When both antibodies are administered simultaneously, they can be administered as separate pharmaceutical compositions, each comprising either the anti-CD20 antibody (or the antigen binding fragment thereof) or the anti-CD40 antagonist antibody (or the binding fragment thereof to the antigen), or they can be administered as a single pharmaceutical composition comprising these two anticancer agents.

In addition, treatment can be achieved with varying doses, as well as with dosing regimens. In some embodiments, the dose of a monoclonal antibody will differ from the dose administered for the other monoclonal antibody, provided that the combination of these doses is effective in the treatment of any one.

or more than several therapeutic parameters. These treatment regimens are based on doses and dosing programs that maximize therapeutic effects, such as those described above. Those skilled in the art admit that a dose of any one monoclonal antibody may not be therapeutically effective when administered individually, but will be therapeutically effective when administered in combination with the other antibody. See, for example, Figure 1, in which an anti-CD20 antibody administered alone was therapeutically ineffective, while the anti-CD40 antagonist antibody administered alone significantly inhibited the growth of human xenograft from rituximab-resistant lymphoma. When these two antibodies were administered in combination, synergistic antitumor activity was observed. Thus, in some embodiments, the therapeutically effective dose of a combination of an anti-CD20 antibody and an anti-CD40 antagonist antibody may comprise doses of individual active agents that, when administered alone, would not be therapeutically effective or would be less therapeutically effective than when They are administered in mutual combination.

In some embodiments, the antibodies can be administered in equivalent amounts. Thus, when an equivalent dosage regimen is contemplated, the anti-CD40 antagonist antibody, for example the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9, is dosed at about 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 or 10 mg / kg, and the anti-CD20 antibody, for example IDEC-C2B8 (Rituxan®) is also dosed in the equivalent dose of approximately 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 and 10 mg / kg, respectively. In other embodiments, these antibodies can be administered in non-equivalent amounts.

Those skilled in the art admit that the antibody combination therapy methods disclosed herein can be used before, after or concurrently with other forms of oncotherapy. Such oncotherapy may include chemotherapy regimens, such as treatment with CVP (cyclophosphamide, vincristine, and prednisone), CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), ICE (ifosfamide, carboplatin and etoposide), Mitoxantrone, Cytarabine, dana, cystarabine and vincristine), ATRA (holo-trans-retinoic acid), Idarrubicin, Hoelzer chemotherapy regimen, La La chemotherapy regimen, ABVD (adriamycin, bleomycin, vinblastine and dacarbazine), CEOP (cyclophosphamide, epirubicin, vincristine and prednisone) , CEOP-BE (cyclophosphamide, epirubicin, vincristine, prednisone, bleomycin and etoposide), 2-CdA (2-chlorodeoxyadenosine, 2-CDA), FLAG and IDA (fludarabine, cytarabine and idarubicin; with or without subsequent treatment with G-CSF), VAD (vincristine, doxorubicin and dexamethasone), M&P (melphalan and prednisone), weekly Q (cyclophosphamide and prednisone), ABCM (adriamycin (doxorubicin), BCNU, cyclophosphamide and melphalan), MOPP (nitro mustard geno, oncovin, procarbazine and prednisone) and DHAP (dexamethasone, ara-C in high doses and platinol). Alternatively, such oncoterapies may include radiation treatment, including myeloablative therapies. Thus, the procedures disclosed herein find use as a concurrent treatment to kill residual tumor cells, either in vivo or ex vivo, after such oncotherapies.

Anti CD40 Antagonist Antibodies

The monoclonal antibodies CHIR-5.9 and CHIR 12.12 represent anti-CD40 antagonist antibodies suitable for use in the methods disclosed herein. CHIR-5.9 and CHIR antibodies

12.12 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 that contain the human IgG1 heavy chain locus and the human κ chain locus (Abgenix). 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 forward, variable and constant regions for the light chain and heavy chain of the CHIR-12.12 mAb are presented herein in Figures 2A and 2B, respectively. See also SEQ ID No. 2 (complete sequence of the CHIR-12.12 mAb light chain), SEQ ID No. 4 (complete sequence of the CHIR-12.12 mAb heavy chain) and SEQ ID No. 5 (complete sequence of a variant of the heavy chain of the CHIR-12.12 mAb presented in SEQ ID No. 4 in which the variant comprises a replacement of the alanine residue with serine at position 153 of SEQ ID No. 4). Nucleotide sequences encoding the light chain and heavy chain of the CHIR-12.12 mAb are presented herein in Figures 3A and 3B, respectively. See also SEQ ID No. 1 (coding sequence of the CHIR-12.12 mAb light chain), and SEQ ID No. 3 (CHIR 12.12 mAb heavy chain coding sequence). The amino acid sequences for the forward, variable and constant regions for the light chain and heavy chain of the CHIR-5.9 mAb are presented herein in Figures 4A and 4B, respectively. See also SEQ ID No. 6 (complete sequence of the CHIR-5.9 mAb light chain), SEQ ID No. 7 (complete sequence of the CHIR-5.9 mAb heavy chain) and SEQ ID No. 8 (complete sequence of a variant of the heavy chain of the CHIR-5.9 mAb presented in SEQ ID No. 7 in which the variant comprises a replacement of the alanine residue by serine at position 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 designations of Patent Deposit PTA-5542 and PTA-5543, respectively.

In addition to antagonistic activity, anti-CD40 antibodies may have another mechanism of action against a tumor cell. For example, the native antibodies CHIR-5.9 and CHIR-12.12 have ADCC activity. Alternatively, the variable regions of the CHIR-5.9 and CHIR-12.12 antibodies can be expressed in another 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 binding to the antigen with a cytotoxin, a therapeutic agent, or a radioisotope.

The monoclonal antibodies CHIR-5.9 and CHIR-12.12 bind to soluble CD40 in ELISA type assays, prevent binding of the CD40 ligand to the CD40 of the cell surface, and displace the previously bound CD40 ligand, as determined by assays of flow cytometry. The CHIR-5.9 and CHIR-12.12 antibodies compete with each other for binding to CD40 but not with 15B8, the anti-CD40 monoclonal antibody described in WO 02/28904, International Application No. PCT / US01 / 30857, published as WO 02 / 28904, also entitled "Human Anti CD40 Antibodies", filed on October 2, 2001 (Agent File No. PP16092.003). When the effects on B-cell proliferation of normal human subjects are tested in vitro, CHIR-5.9 and CHIR-12.12 act as anti-CD40 antagonistic antibodies. In addition, CHIR-5.9 and CHIR-12.12 do not induce an intense 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 × 10-8 M and the binding affinity of CHIR-12.12 is 5 × 10-10 M, as determined by the Biacore ™ assay.

Anti-CD40 antagonist antibodies suitable for use in the methods disclosed herein have a strong single site binding affinity for the CD40 cell surface antigen. The monoclonal antibodies disclosed herein have a dissociation equilibrium constant (KD) for the CD40 of at least 10-5 M, at least 3 × 10-5 M, preferably at least 10-6 M up to 10- 7 M, more preferably at least 10-8 M to about 10-12 M, measured using a standard 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 U.S. 5,674,492 and 4,708,871; Stamenkovic et al. (1989) EMBO 8: 1403; Clark (1990) Tissue Antigens 36:33; Barclay et al. (1997) The Leucocyte Antigen Facts Book (2nd ed .; Academic Press, San Diego)). Two isoforms of human CD40 have been identified, encoded by transcription variants with alternative splices of this gene. The first isoform (also known as the "long isoform" or "isoform 1") is expressed as a 277 amino acid precursor polypeptide (SEQ ID No. 12 (first documented in GenBank with accession no. CAA43045, and identified as isoform 1 in GenBank with accession number NP_001241), encoded by SEQ ID No. 11 (see GenBank accession numbers 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 access 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 a coding segment, which results in a change of the translation framework; The resulting CD40 isoform contains a shorter and more differentiated C-terminal end (residues 166-203 of SEQ ID No. 10) than is contained in the long isoform of CD40 (C-terminal end shown in residues 166-277 of SEQ ID NO. 12). For the purposes of the present disclosure, the terms "CD40 antigen", "cell surface CD40 antigen", "CD40 receptor" or "CD40" encompass both the short and the long isoform of CD40. The anti-CD40 antibodies disclosed herein 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 below in This document.

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" means that everything

or a portion 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 an analog receptor in a cell and initiates a reaction or activity that is similar to that which is initiated by the natural ligand of the receptor; for example, it transduces a signal to the cell. A CD40 agonist induces, without limitation, any or all of the following responses: B cell proliferation and differentiation, antibody production, intercellular adhesion, B cell memory generation, isotype change, upregulation 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 can 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 even 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, without limitation, standard competitive binding assays, assays to monitor immunoglobulin secretion by B cells, B cell proliferation assays, Banchereau type B cell proliferation assays, cooperative T cell assays for antibody production, costimulation B cell proliferation assays and assays for ascending regulation of cell activation markers B. See, for example, the tests disclosed in WO 00/75348 and in US Patent No. 6,087,329.

By "significant" agonist 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. Thus, for example, when the B cell response of interest is B cell proliferation , the "significant" agonist activity would be the induction of a level of B cell proliferation that is at least 2 times greater or at least 3 times greater than the level of B cell proliferation induced by a neutral substance or a negative control. In one embodiment, a non-specific immunoglobulin, for example IgG1, which does not bind with CD40, serves as a negative control. A substance "free of significant agonist activity" would have an agonist activity no more than 25% greater than the agonist activity induced by a neutral substance or a negative control, preferably no more than about 25% greater than the agonist activity induced by a substance neutral or a negative control, preferably no more than about 20% greater, 15% greater, 10% greater, 5% greater, 1% greater, 0.5% greater or even no more than approximately 0.1% greater than agonist activity induced by a neutral substance or a negative control as measured in the assay of a B-cell response. Anti-CD40 antagonist antibodies useful in the methods disclosed herein are free of significant agonist activity, as It has been noted above, when they are bound to a CD40 antigen in a human cell. In one embodiment of the disclosure, the anti-CD40 antagonist antibody is free of significant agonist activity in a B-cell response. In another embodiment of the disclosure, the anti-CD40 antagonistic 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).

Monoclonal antibodies to CD40 are known in the art. See, for example, sections dedicated to the B-cell antigen in McMichael, ed. (1987; 1989) Leukocyte Typing III and IV (Oxford University Press, New York); U.S. Patent Nos. 5,674,492, 5,874,082, 5,677,165, 6,456,959; WO 00/63395; Nos of International Publication 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. 14.6: 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 for the present disclosure are the anti-CD40 antagonist antibodies disclosed herein that share the binding characteristics of the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described above. Such antibodies include, without limitation: (1) the monoclonal antibodies produced by the hybridoma cell lines designated 131.2F8.5.9 (referred to herein as cell line 5.9) and 153.8E2.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 sequence shown both in SEQ ID No. 2 and in SEQ ID No. 4, and the sequence shown in both SEQ ID No. 2 and SEQ ID No. 5; (3) a monoclonal antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID No. 6, the sequence shown in SEQ ID No. 7, the sequence shown in SEQ ID No. 8, the sequence shown both in SEQ ID No. 6 and in SEQ ID No. 7, and the sequence shown in both SEQ ID No. 6 and SEQ ID No. 8; (4) 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 nucleotide sequence shown in SEQ ID NO. 3, and the sequence shown in both SEQ ID No. 1 and SEQ ID No. 3; (5) a monoclonal antibody that binds with an epitope capable of binding with the monoclonal antibody produced by hybridoma cell line 5.9 or 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 in SEQ ID No. 12; (7) a monoclonal antibody that competes with the CHIR-5.9 or CHIR monoclonal antibody

12.12 in a competitive union trial; and (8) a monoclonal antibody that is a fragment of the CHIR-12.12 or CHIR-5.9 monoclonal antibody to the antigen or the above monoclonal antibodies in the preceding elements (1) - (7), in which the fragment retains the ability to specifically bind with human CD40 antigen.

Anti CD20 antibodies

By "CD20 antigen" is meant a 33-37 kDa non-glycosylated transmembrane protein that is expressed in B cells committed to the lineage from the pre-B cell phase to the B cell lymphoblastic phase (GenBank accession number X12530; Barclay et al. (1997) The Leucocyte Antigen Facts Book (2nd ed .; Academic Press, San Diego)). The CD20 receptor is exposed on the surface of B cell types, as described elsewhere in this document. By "exposed on the surface" and "expressed on the surface" is meant that all or a portion of the CD20 antigen is exposed to the outside of the cell.

Anti-CD20 antibodies are known in the art. See, for example, U.S. Patent Nos. 5,595,721,

6,399,061 and 6,455,043. Human and chimeric anti CD20 antibodies are particularly useful in the practice of the methods of the invention. Examples of chimeric anti-CD20 antibodies include, without limitation, IDEC-C2B8, commercially available under the name of Rituxan® (IDEC Pharmaceuticals Corp., San Diego, California) and described in U.S. Patent Nos. 5,736,137, 5,776,456 and 5,843,439; the chimeric antibodies described in U.S. Patent No. 5,750,105; and the antibodies described in U.S. Patent Nos. 5,500,362, 5,677,180, 5,721,108 and 5,843,685. Anti-CD20 antibodies of murine origin are also suitable for use in the methods disclosed herein. Examples of such murine anti-CD20 antibodies include, without limitation, the B 1 antibody (described in US Patent No. 6,015,542); IF5 antibody (see Press et al. (1989) J. Clin. Oncol. 7: 1027); anti-CD20 antibodies NKIB20 and BCA-B20 (described in Hooijberg et al. (1995) Cancer Research 55: 840-846); and EDEC-2BB (commercially available from IDEC Pharmaceuticals Corp., San Diego, California); 2H7 antibody (described in Clark et al. (1985) Proc. Natl. Acad. Sci. USA 82: 1766-1770); and others described in Clark et al. (1985) supra and Stashenko et al. (1980) J. Immunol. 125: 1618-1685.

Production of anti CD20 and anti CD40 antibodies

Anti-CD40 antibodies and anti-CD20 antibodies for use in the methods disclosed herein can be produced using any of the procedures well known to those skilled in the art. Polyclonal sera can be prepared by conventional procedures. In general, a solution containing the CD40 or CD20 antigen is first used to immunize a suitable animal, preferably a mouse, a rat, a rabbit or a goat. Rabbits or goats are preferred for the preparation of polyclonal sera due to the volume of serum that can be obtained and the availability of labeled anti-rabbit and anti-goat antibodies.

Polyclonal sera can be prepared in a transgenic animal, preferably a mouse that carries human immunoglobulin loci. In a preferred embodiment, Sf 9 cells expressing CD40 are used as an immunogen

or CD20. Immunization can also be carried out by mixing or emulsifying the solution containing the physiological serum antigen, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). Normally, a dose of 50-200 μg / injection is sufficient. Generally, it is reinforced 2-6 weeks later with one or more injections of the physiological whey protein, preferably using Freund's incomplete adjuvant. Alternatively, antibodies can be generated by in vitro immunization using methods known in the art, which, for the purposes of this disclosure, is considered equivalent to in vivo immunization. Polyclonal antisera are obtained by bleeding the immunized animal and collecting the blood 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, at 1,000 × g for 10 minutes). From rabbits approximately 20-50 ml can be obtained by bleeding.

The production of Sf 9 cells (Spodoptera frugiperda) is disclosed in U.S. Patent No.

6,004,552. Briefly, sequences encoding human CD40 in a baculovirus were recombined using transfer vectors. Plasmids were cotransfected with wild-type baculovirus DNA in Sf 9 cells. Recombinant Sf 9 cells infected with baculovirus were identified and purified by cloning.

Preferably, the antibody is monoclonal in nature. Monoclonal antibodies are highly specific, being directed against a single antigenic site; that is, the CD40 or CD20 antigen on the cell surface. In addition, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The "monoclonal" modifier indicates the character of the antibody that is obtained from a substantially homogeneous population of antibodies, such as those produced by a clonal population of B cells, and should not be construed as requiring the production of the antibody by any particular procedure. For example, monoclonal antibodies to be used according to the present disclosure can be created by the hybridoma method, first described by Kohler et al. (1975) Nature 256: 495, or they can be created by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in 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 an antigenic molecule for which an antibody is produced and to which the antibody will bind. The epitopes may comprise linear amino acid residues (that is, residues within the epitope are sequentially arranged one after another in a linear manner), non-linear amino acid residues (referred to as "non-linear epitopes" herein; these epitopes are not arranged sequentially), or both linear and non-linear amino acid residues.

Monoclonal antibodies can be prepared using the method of Kohler et al. (1975) Nature 256: 495496 or a modification thereof. Normally, a mouse is immunized with a solution containing an antigen. Immunization can be carried out by mixing or emulsifying the solution containing the antigen in physiological serum, preferably in an adjuvant such as Freund's complete adjuvant, and parenterally injecting the mixture or emulsion. Any immunization procedure known in the art can be used to obtain the monoclonal antibodies of the disclosure. After immunization of the animal, the spleen (and, optionally, several lymph nodes) is removed and dissociated into individual cells. Spleen cells can be investigated by applying a cell suspension to a plate or well coated with the antigen of interest. B cells that express an antigen-specific membrane-bound immunoglobulin bind to the plaque and do not disappear when rinsing. The resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas and are cultured in a selective medium. The resulting cells are plated by serial dilution and tested for the production of antibodies that specifically bind to the antigen of interest (and that do not bind unrelated antigens). Selected hybridomas that secrete the monoclonal antibody (mAb) are then cultured, either in vitro (e.g., tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

When the anti-CD40 antagonistic antibodies of the disclosure must be prepared using recombinant DNA procedures, the DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are capable of specifically binding to genes that encode the heavy and light chains of murine antibodies). The hybridoma cells described herein serve as a preferred source of such DNA. Once isolated, the DNA can be put into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that otherwise do not they produce the hemoglobin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Articles with reviews on recombinant expression in DNA bacteria encoding the antibody include Skerra et al. (1993) Curr. Opinion in Immunol. 5: 256 and Phickthun (1992) Immunol. Revs 130: 151. As an alternative to the use of hybridomas, the antibody can be produced in a cell line such as a CHO cell line, as disclosed in US Patent Nos. 5,545,403, 5,545,405 and 5,998,144. In summary, the cell line is transfected with vectors capable of expressing a light chain and a heavy chain, respectively. By transfecting the two proteins into separate vectors, chimeric antibodies can be produced. Another advantage is the correct glycosylation of the antibody.

In some embodiments, the anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR-5.9 antibody, or the antigen-binding fragment thereof is produced in CHO cells using the GS gene expression system (Lonza Biologics, Portsmouth , New Hampshire), which uses glutamine synthetase as a marker. See also U.S. Patent Nos. 5,122,464, 5,591,639, 5,658,759, 5,770,359, 5,827,739, 5,879,936,

5,891,693 and 5,981,216.

The 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 an anti-CD40 monoclonal antibody or an anti-CD20 monoclonal antibody. Antigen epitopes may comprise proteins, protein fragments, peptides, carbohydrates, lipids and other molecules, but, for the purposes of the present invention, the most common is that they are proteins, short oligopeptides, oligopeptide mimetics (i.e. , organic compounds that mimic the antibody binding properties of the CD40 or CD20 antigen), or combinations thereof. Suitable oligopeptide mimetics are described, among others, in PCT application US 91/04282.

In addition, the term "antibody", as used herein, encompasses anti-CD40 chimeric antibodies or anti-CD20 antibodies. The anti-CD40 chimeric antibodies for use in the methods disclosed herein have the binding characteristics of the anti-CD40 monoclonal antibodies CHIR-5.9 or CHIR-12.12, while the anti-CD20 chimeric antibodies for use in the procedures given to known herein have the binding characteristics of the anti-CD20 monoclonal antibody IDECC2B8. By "chimeric" antibodies are meant antibodies that, more preferably, are obtained using recombinant deoxyribonucleic acid techniques and comprising both human components (including immunologically "related" species, for example, the chimpanzee) and nonhuman. Thus, most preferably, the constant region of the chimeric antibody is substantially identical to the constant region of a natural human antibody; Most preferably, the variable region of the chimeric antibody is obtained from a non-human source and has the desired antigen specificity for the CD40 or CD20 cell surface antigen. The non-human source can be any vertebrate source that can be used to generate antibodies against a human CD40 or CD20 cell surface antigen or material comprising a human CD40 or CD20 cell surface antigen. Such non-human sources include, without limitation, rodents (e.g., rabbit, rat, mouse, etc .; see, for example, U.S. Patent No. 4,816,567) and non-human primates (e.g., Old World monkey, ape , etc .; see, for example, U.S. 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 or, when used in reference to chimeric anti-CD20 antibodies, means a chimeric antibody that binds to human CD20.

The anti-CD40 antibodies and the humanized anti-CD 20 antibodies represent additional anti-CD40 antibodies and anti-CD20 antibodies suitable for use in the methods disclosed herein. By "humanized" are meant forms of anti-CD40 antibodies or anti-CD20 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 portion 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 present 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 methods disclosed herein have binding characteristics similar to those exhibited by the CHIR-5.9 and CHIR-12.12 monoclonal antibodies described herein. Humanized anti-CD20 antibodies for use in the methods disclosed herein have binding characteristics similar to those exhibited by the IDECC2B8 monoclonal antibody 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 Nos. 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, U.S. Patent 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, the humanized antibody will comprise substantially all of at least one variable domain, and usually two, 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. Optionally, the humanized antibody will also comprise at least a part of a constant region (Fc) of immunoglobulin, usually 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 normally 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, U.S. 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 having better affinity for a predetermined antigen are disclosed.

Also encompassed by the terms "anti-CD40 antibodies" or "anti-CD20 antibodies" are anti-CD40 antibodies or xenogenic or modified anti-CD20 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 the host immunoglobulins become nonfunctional and are replaced with the human immunoglobulin analog loci. These transgenic animals produce human antibodies in 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 and CD20 are obtained by immunizing transgenic mice. Such a 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 can be immunized for the human IgG1 heavy chain locus and the human κ light chain locus with Sf9 cells expressing human CD40 or human CD20. Mice can also be transgenic for other isotypes. Fully human antibodies useful in the methods disclosed herein are characterized by having binding properties similar to those exhibited by the monoclonal antibodies CHIR-5.9, CHIR-12.12 and IDEC-C2B8.

Fragments of anti CD40 antibodies or anti CD20 antibodies are suitable for use in the methods disclosed herein 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 CD40 antigen on the surface of B cells, and a fragment of an anti CD20 antibody will retain the ability to bind to the CD20 antigen on the surface of B cells, respectively. Such fragments are characterized by having properties similar to the corresponding full length antibody. For example, anti-CD40 antagonist antibody fragments will specifically bind to a human CD40 antigen expressed on the surface of a human cell, and are free of significant agonist activity, but exhibit antagonistic activity when bound to a CD40 antigen in a human cell. which expresses CD40; while anti-CD20 antibody fragments will specifically bind to CD20. These fragments are referred to herein as "antigen binding" fragments.

Suitable fragments of an antigen-binding antibody comprise a part of a full-length antibody, usually the antigen binding region or one of its variable regions. Examples of antibody fragments include, without limitation, 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" antibody fragments are 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 the 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.

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 U.S. Patent No. 5,514,548. Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 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 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 phage antibody libraries presented 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: 163167). According to another approach, F (ab ’) 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

Combinations of antibodies useful in the methods disclosed herein include anti-CD 40 antagonist monoclonal antibodies CHIR-5.9 and CHIR 12.12, disclosed herein, as well as anti-CD20 antibodies, such as IDEC-C2B8, which they differ in regions that are not CDR; and antibodies with one or more additions, deletions or substitutions. The disclosure also encompasses anti-CD20 antibodies and anti-CD40 antagonistic anti-immunized antibodies, which can be produced as described, for example, in International Publication Nos. WO 98/52976 and WO 0034317. Thus, residues within useful antibodies to implement the procedures disclosed herein, they are modified to make antibodies non-immunogenic or less immunogenic for humans while maintaining their binding specificity and biological activity, in which such activity is measured. through the tests indicated elsewhere in this document. Also included in the disclosure are fusion proteins comprising anti-CD20 antibodies or anti-CD40 antagonist antibodies, or a fragment thereof, fusion proteins that can be synthesized or expressed from the corresponding polynucleotide vectors, as known in the technique. Such fusion proteins are described with reference to antibody conjugation as noted below.

Antibodies useful in the implementation of the methods disclosed herein may have sequence variations produced using the procedures described, for example, in Patent Publication Nos. 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 MHC Class II and trigger an unwanted response from helper T cells. A conservative substitution may allow the antibody to retain binding activity and, however, lose its ability to trigger an unwanted T-cell response. Any such conservative or non-conservative substitutions may be performed using methods recognized in the art, such as those indicated elsewhere in this document. Variant antibodies can be routinely tested to verify their antagonistic activity, their affinity and their specificity using the procedures described herein.

An anti-CD40 antagonist antibody produced by any of the methods described above, or any other procedure not disclosed herein, will be useful according to the present disclosure if it possesses at least one of the following biological activities: inhibition of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells; inhibition of proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of proliferation of normal human peripheral B cells stimulated by cells expressing soluble CD40L or CD40; and inhibition of the proliferation of human malignant B cells as noted below.

See also the trials 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 US patents we

5,674,492 and 5,847,082.

A representative assay for detecting anti-CD40 antagonistic antibodies specific for the CD40 antigen epitopes identified herein is a "competitive binding assay." Competitive binding assays are serological assays in which the unknown is detected and quantified by its ability to inhibit the binding of a known labeled ligand to its specific antibody. This is also known as a competitive inhibition assay. In a representative competitive binding assay, labeled CD40 polypeptide is precipitated by means of candidate antibodies in a sample; for example, in combination with monoclonal antibodies raised against one or more epitopes of the monoclonal antibodies disclosed herein. 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 a fragment of the protein 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 5B (SEQ ID NO. 10), encoded by the sequence presented in Figure 5A (SEQ ID No. 9; see also GenBank accession number NM_152854), or the long isoform of human CD40 (see GenBank accession numbers CAA43045 and NP_001241) presented in Figure 5D (SEQ ID No. 12), encoded by the sequence presented in Figure 5C (SEQ ID No. 11; see GenBank Access Nos N X60592 and NM_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.

An anti-CD20 antibody produced by any of the methods described above, or any other procedure not disclosed herein, will be useful according to the present disclosure if it possesses at least one of the following biological activities: initiation of cell-mediated cytotoxicity antibody dependent against a cell expressing CD20; initiation of complement-mediated cytotoxicity against a cell expressing CD20; delivery of a cytotoxin or a radionuclide to the cell expressing CD20; inhibition of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells; inhibition of proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of proliferation of normal human peripheral B cells stimulated by cells expressing soluble CD40L or CD40; and inhibition of the proliferation of human malignant B cells as noted below. Assays for detecting these activities are well known in the art, including those disclosed in US Patent No. 5,736,137.

Any of the anti-CD40 antagonist antibodies (or their antigen binding fragments) or the anti CD20 antibodies (or their antigen binding fragments) previously described may be conjugated before use in the methods disclosed herein. document. Methods for producing conjugated antibodies are known in the art. Accordingly, the anti CD40 antibody or the anti CD20 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, for example, in the case of an antigen and a specific monoclonal antibody thereto. 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 differentiated 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 to achieve the desired effect. For example, mAbs and avidin also require markers in the implementation of this invention: thus, a mAb can be labeled with biotin, and its presence detected with avidin labeled with 121I, or with an antibiotic mAb labeled with HRP. Other permutations and possibilities will be immediately apparent to people with a normal mastery of the technique, and are considered as equivalent to what is described in the disclosure.

Alternatively, the anti CD40 antibody or the anti CD20 antibody can be labeled using "direct labeling" or a "direct labeling approach", in which a radionuclide is covalently fixed to an antibody (usually 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 Nos 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 antibodies described in U.S. Patent No. 6,015,542.

In addition, an antibody (or a fragment thereof) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, a radioactive metal ion or a radioisotope. 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, glutathrinthroid, actinomycortin, 1-dihydrocortin, glutathin-acetonic acid procaine, tetracaine, lidocaine, propranolol and puromycin, and their analogues or homologues. Therapeutic agents include, without limitation, antimetabolites (e.g., fludarabine, 2-chlorodeoxyadenosine, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, moth carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines (for example, daunorubicin (formerly daunomycin) and doxorubin (doxorubin), doxorubin) for example, dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)) and antimitotic agents (for example, vincristine and vinblastine) Radioisotopes include, without limitation, I-131, I-123, I-125, Y- 90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111 and the like. Revelation conjugates can be used to modify a biological response given; it should not be construed that the 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 macrophage colony stimulating factor ("GM-CSF ”), Granulocyte colony stimulating factor (“ 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 U.S. Patent No. 4,676,980. In addition, connectors between the markers and the antibodies of the disclosure 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 sequential, in any order, the same day or on different days (documents WO 00/52031 and WO 00/52473). In some embodiments, the anti CD20 antibody is conjugated to the anti CD40 antibody. In other additional embodiments, a single antibody comprises a dual specificity towards both CD20 and CD40. Such bispecific antibodies are known in the art. See, for example, U.S. Patent No. 5,959,084.

Variants of anti-CD40 antagonist antibodies and anti-CD20 antibodies

In the methods disclosed herein, variants of the antagonist anti-CD40 antibodies or suitable biologically active anti-CD20 antibodies can be used. Such variants will retain the desired binding properties of the parent antibody, that is, the original anti-CD40 antagonist antibody or the original anti-CD20 antibody. Generally, methods for producing antibody variants are available in the art.

For example, amino acid sequence variants of an anti-CD20 antibody, for example the IDEC-C2B8 described herein, or of an anti-CD40 antagonist antibody, for example, the CHIR-5.9 or CHIR-12.12 monoclonal antibodies described can be prepared herein, by means of 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 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.) substitution guidance can be found

suitable amino acids that do not affect the biological activity of the polypeptide of interest. Conservative substitutions, such as the exchange of one amino acid with another having properties, may be preferred.

Glu,
֞ Leu, Asp
֞ Ile
֞ Ala, Val
֞ similar. Examples of conservative substitutions include, without limitation, Gly Tyr.
֞ Trp
֞ Gln, yPhe
֞ Arg, Asn
֞ Lys

In the construction of polypeptide variants of interest of the anti-CD20 antibody or anti-CD40 antagonist antibody, the modifications are made such that the variants continue to have the desired activity, i.e. similar binding affinity, and are capable of specifically binding to a human CD20 or CD40 antigen expressed on the surface of a human cell, respectively, and free of significant agonist activity but exhibiting antagonistic activity when they bind to a CD40 antigen in a human cell expressing 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 a 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 U.S. Patent No. 6,737,056B1 and Patent Application Publication No. 2004 / 0132101A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.

Preferably, the variants of an anti-CD20 antibody or a reference anti-CD40 antagonist antibody have amino acid sequences having at least 70% or 75% sequence identity, preferably at least 80% or 85% identity of sequence, more preferably at least 90%, 91%, 92%, 93%, 94% or 95% sequence identity with respect to the amino acid sequence for the reference anti CD20 antibody, for example IDEC-C2B8, as described herein, or to the reference anti-CD40 antagonist antibody molecule, for example the CHIR-5.9 monoclonal antibodies

or CHIR-12.12 described herein, or with respect to a shorter portion of the reference antibody molecule. More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence identity. For the purposes of the present disclosure, the percent sequence identity 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 anti-CD40 antagonist antibody in only 1 to 15 amino acid residues, only 1 to 10 amino acid residues, such as 6-10, only 5, only 4, 3, 2 , or even in 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 for sequence identity associated with gaps or conservative substitutions of residues can be made (see Smith-Waterman's homology 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 CD20 antigen, particularly when it binds to the antigen in malignant B cells, depends on a number of factors. Since ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide can be obtained as an acidic or basic salt, or in a neutral form. All preparations that retain their biological activity when placed under suitable environmental conditions are included in the definition of anti-CD40 antagonist antibodies or anti-CD20 antibodies as used herein. In addition, the main amino acid sequence of the polypeptide can be increased by derivatization with sugar moieties (glycosylation) or by means of other complementary molecules such as lipids, phosphate, acetyl groups and the like. It can also be increased by conjugation with saccharides. Certain aspects of 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 or an anti CD20 antibody used herein as long as the properties of the anti CD40 antibody (including antagonistic activity) or anti CD20 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 desirable activity of the unmodified antibody do not remove the polypeptide sequence from the definition of anti CD40 and anti CD20 antibodies of interest as used herein.

The technique provides substantial guidance regarding the preparation and use of polypeptide variants. In the preparation of 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 pharmaceutical composition. used in the procedures described herein.

Pharmaceutical formulations and modes of administration

The combination of the anti CD20 antibody (or the antigen binding fragment thereof) and the anti CD40 antagonist antibody (or the antigen binding fragment thereof) is administered in a concentration that is therapeutically effective for preventing or treating a characterized cancer. by a growth of neoplastic B cells, particularly cancers comprising neoplastic B cells expressing both CD40 and CD20 antigens. To achieve this goal, antibodies can be formulated using a variety of acceptable excipients known in the art. Normally, antibodies are administered by injection, either intravenously, intraperitoneally or subcutaneously. The procedures for carrying out this administration are known to people with a normal skill 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 for about 4 to about 6 hours, depending on the 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 disclosure 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.

Antibodies are normally provided by conventional techniques in a pharmaceutically acceptable buffer, for example sterile saline, sterile buffered water, propylene glycol, combinations of the above, etc. The anti-CD40 antagonist antibody (or the antigen binding fragment thereof) and the anti CD20 antibody (or the antigen binding fragment thereof) can be formulated in separate pharmaceutical compositions, or can be formulated within a single pharmaceutical composition. for simultaneous administration. 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.

A person with a normal domain of the art can easily determine without undue experimentation the amount of a combination of at least one anti-CD40 antagonist antibody or a fragment thereof antigen-binding and at least one anti-CD20 antibody or a fragment thereof of binding to the antigen to be administered. Factors that influence the mode of administration and the respective amount of the combination of the antibodies disclosed herein include, without limitation, the severity of the disease, the history of the disease, and the age, height, weight. , health and physical condition of the individual undergoing therapy. Similarly, the amount of the combination of antibodies disclosed herein to be administered will depend on the mode of administration and whether the subject will undergo a single dose or multiple doses of these antitumor agents. Generally, by increasing the weight of the subject undergoing therapy, a higher dose of the antibody combination disclosed herein preferably results. The dose of either the anti-CD20 antibody (or the antigen-binding fragment thereof) or the anti-CD40 antagonist antibody (or the antigen-binding fragment thereof) 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. Thus, for example, the dose of any antibody of the combination 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 disclosure comprises the administration of multiple doses of anti-CD20 antibody (or a fragment thereof binding with the antigen) in combination with multiple doses of anti-CD40 antagonistic antibody (or a fragment thereof binding with the antigen). 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 that already comprises either an anti-CD20 antibody (or a fragment thereof binding with the antigen) or an anti-CD40 antagonistic antibody (or a fragment thereof binding with the antigen), or both. The frequency and duration of administration of multiple doses of the pharmaceutical compositions 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 a combination of antibodies may include a single treatment or, preferably, may include a series of treatments. In a preferred example, the subject is treated with the combination of an anti-CD20 antibody (or a fragment thereof binding with the antigen) and an anti-CD40 antagonistic antibody (or a fragment thereof binding with the antigen) in which both are administered with a dose in the range of between about 0.1 and 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 understood that the effective dose of the antibodies or their antigen binding fragments used for the treatment may increase or decrease in the course of a particular treatment. Changes in the dose may be a consequence and may be apparent from the results of the diagnostic tests as described herein. Thus, in one embodiment, the dosage regimen includes a first administration of a therapeutically effective dose of the anti CD20 antibody (or the fragment thereof binding with the antigen) in combination with a therapeutically effective dose of the anti CD40 antagonist antibody ( or of the antigen binding fragment thereof) in which the combination is administered on days 1, 8, 15 and 22 of a treatment period. In another embodiment, the dosage regimen includes a first administration of a therapeutically effective dose of the anti-CD20 antibody (or the fragment thereof binding with the antigen) in combination with a therapeutically effective dose of the anti-CD40 antagonist antibody (or of the fragment thereof binding with the antigen) in which the combination is administered on days 1, 2, 3, 4, 5, 6 and 7 of a week in a treatment period. Other additional embodiments include a dosage regimen in which a therapeutically effective dose of anti-CD20 antibody (or a fragment thereof antigen binding) is administered in combination with a therapeutically effective dose of anti-CD40 antagonist antibody (or a fragment antigen binding agent) in which the combination is administered 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 the anti CD20 antibody (or a fragment thereof antigen binding) in combination with a therapeutically effective dose of the anti CD40 antagonist antibody (or a fragment thereof binding to the antigen) in which the combination is administered 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 the anti-CD20 antibody (or a fragment thereof antigen binding) in combination with a therapeutically effective dose of the anti-CD40 antagonist antibody (or a fragment of the same antigen binding) in which the combination is administered on day 1 of any given 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. The treatment using a combination of the anti-CD40 antagonist antibody (or the antigen binding fragment thereof) and the anti CD20 antibody (or an antigen binding fragment thereof) may comprise administration of one or both antibodies simultaneously or in a manner concurrent, provided that the treatment includes the combination of the anti-CD20 antibody (or the antigen-binding fragment thereof) and the anti-CD40 antagonist antibody (or the antigen-binding fragment thereof) at some point during the treatment. The effect of combination therapy can also be optimized by varying the timing of administration, either of the anti-CD20 antibody treatment and / or of the anti-CD40 antagonist antibody. Treatment with an anti-CD20 antibody or a fragment thereof antigen-binding in combination with an anti-CD40 antagonistic antibody or a fragment thereof antigen-binding may be simultaneous (concurrent), consecutive (sequential) or a combination thereof. . Therefore, a subject undergoing antibody combination therapy can receive both the anti-CD20 antibody (or the antigen-binding fragment thereof) and the anti-CD40 antagonist (or the antigen-binding fragment thereof) at the same time. (that is, simultaneously) or at different times (that is, sequentially, in any order, on the same day or on different days). Thus, in some embodiments, the anti-CD20 antibody, for example Rituxan® (or a fragment thereof antigen binding) is administered simultaneously with the anti-CD40 antagonistic antibody, such as the monoclonal antibody CHIR-12.12 or the CHIR

5.9 (or a fragment thereof antigen binding). In other embodiments, the anti-CD20 antibody, such as Rituxan® (or an antigen binding fragment thereof) is first administered and then the anti-CD40 antagonistic antibody, such as monoclonal antibody CHIR-12.12 or CHIR-5.9 ( or a fragment thereof antigen binding). In other additional embodiments, the anti-CD40 antagonist antibody is first administered, such as the monoclonal antibody CHIR-12.12 or the CHIR-5.9 (or a fragment thereof to the antigen) and then the anti-CD20 antibody, such as Rituxan®, is administered. (or a fragment thereof antigen binding). In some embodiments, the combination of anti-CD20 antibodies and anti-CD40 antagonist antibodies, such as Rituxan® and monoclonal antibodies CHIR-12.12 or CHIR-5.9, is given concurrently for one dose, but other doses include sequential administration, in any order, on the same day or on different days. When the anti-CD20 antibody, such as Rituxan®, and the anti-CD40 antagonist antibody, such as the monoclonal antibody CHIR-12.12 or CHIR-5.9, are simultaneously administered, they can be administered as separate pharmaceutical compositions, each comprising either the antibody. anti CD20 (or the antigen binding fragment thereof) or the anti CD40 antagonist antibody (or the antigen binding fragment thereof),

or they can be administered as a single pharmaceutical composition comprising both anticancer agents.

In some embodiments, the therapeutically effective dose of the anti-CD40 antagonist antibody or of an antigen binding fragment thereof 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. Thus, for example, the dose of any anti-CD40 antagonist antibody of a fragment thereof to the antigen, for example the anti-CD40 monoclonal antibody CHIR-12.12 or CHIR-5.9 or a fragment thereof of the antigen-binding, 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 of an antigen binding fragment thereof can be administered throughout each week of antibody dosing. Alternatively, different therapeutically effective doses of an anti-CD40 antagonist antibody or of an antigen binding fragment thereof can be used during the course of a treatment period.

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

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

Alternatively, in some embodiments, after the "loading dose", subsequent therapeutically effective doses of the anti-CD40 antagonist antibody or its antigen binding fragment are administered according to a "maintenance schedule", in which the Therapeutically effective dose of the antibody or its antigen binding fragment is administered once a month, once every 6 weeks, once every two months, once every 10 weeks, once every three months, once every 14 weeks, a 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.

Pharmaceutical compositions useful in the methods disclosed herein may comprise biologically active variants of either anti-CD20 antibodies (or antigen binding fragments thereof) or anti-CD40 antagonist antibodies (or fragments of the same antigen binding), or both. Such variants must 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 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 the monoclonal antibodies CHIR-5.9 or CHIR-12.12, as expressed by the hybridoma cell line 5.9 or 12.12, and IDEC-C2B8, respectively. Methods are available in the art to determine if a variant 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-CD20 antibody that has the binding properties described herein or an anti-CD40 antagonistic antibody that has the binding properties described herein as the therapeutically active component can be used in the methods disclosed. in the present document. Accordingly, liquid, lyophilized compositions

or spray dried comprising one or more of the antibodies useful in the implementation of the disclosure may be prepared as an aqueous or non-aqueous solution or suspension for subsequent administration to a subject according to the procedures disclosed herein. document. Each of these compositions will comprise at least one of the anti-CD20 antibodies (or a fragment thereof antigen binding) or anti-CD40 antagonist antibodies (or a fragment thereof antigen binding) as a therapeutically or prophylactically active component. By "therapeutically or prophylactically active component" is meant that the antibody or antigen binding fragment thereof 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 pharmaceutical composition is administered to that subject. Preferably the pharmaceutical composition comprises stabilizing agents, suitable loading agents, or both, to minimize the problems associated with the loss of stability and biological activity of the proteins during preparation and storage.

Formulation compounds may be added to pharmaceutical compositions comprising antibodies useful in the practice of the disclosure. These formulation compounds may include, without limitation, oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants or fillers. Carbohydrates preferably 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, pululane, 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 sugar or sugar alcohol concentration is between 1.0% and 7% w / v, more preferably between 2.0% and 6.0% w / v. The amino acids, preferably, 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. Preferably, polymers and methods for binding them to peptides are shown in US Patent Nos. 4,766,106, 4,179,337, 4,495,285 and 4,609,546. The polymers, preferably, 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 protective group has between 1 and 8 carbons; more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG preferably has an average molecular weight between 1,000 and 40,000, more preferably between 2,000 and 20,000, more preferably even 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 PEG / antibody of the present invention covalently conjugated.

Water soluble polyoxyethylated polyols are also useful in the present invention. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and others. 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, preferably, in the same range as the 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 analyzed in Gabizon et al. (1982) Cancer Research 42: 4734; Cafiso (1981) Biochem Biophys Acta 649: 129; and Szoka (1980) Ann. Rev. Biophys. Eng.

9: 467. Other drug delivery systems are known in the art and are described, for example, in Poznansky et al. (1980) Drug Delivery Systems (R. L. Juliano, ed., Oxford, New York) 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 antagonistic 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, without limitation, inhibition of immunoglobulin secretion by normal human peripheral B cells stimulated by T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by Jurkat T cells; inhibition of survival and / or proliferation of normal human peripheral B cells stimulated by cells expressing CD40L or soluble CD40 ligand (sCD40L); inhibition of "survival" intracellular antiapoptotic signals in any cell stimulated by solid phase sCD40L or CD40L; inhibition of CD40 signal transduction in any cell after binding with solid phase sCD40L or CD40L; and inhibition of the proliferation of malignant human B cells, as noted elsewhere herein.

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

A protein such as an antibody, when formulated in a pharmaceutical composition, is considered to maintain its physical stability at a given time if it shows no 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) retains its biological activity of interest in that pharmaceutical composition. Methods for controlling changes in chemical stability are well known in the art and include, without limitation, methods for detecting chemically altered forms of the protein, such as those resulting from cuts using, for example, SDS-PAGE, SEC, and / or mass spectrometry by matrix-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.

It is considered that an anti-CD40 antagonist antibody or its antigen binding fragment, when formulated in a pharmaceutical composition, maintains a desired biological activity at a given time 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 trials 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 recombinantly produced 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 about 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, about 18.0 mg / ml, approximately 19.0 mg / ml, approximately 20.0 mg / ml, approximately 21.0 mg / ml, approximately 22.0 mg / ml, approximately 23.0 mg / ml, approximately 24, 0 mg / ml, or about 25.0 mg / ml. The liquid pharmaceutical composition comprises the anti-CD40 antagonist antibody, for example, the CHIR-12.12 or CHIR5.9 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, without limitation, 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, without limitation, succinic acid or succinate buffers, citric acid or citrate, acetic acid or acetate, tartaric acid or tartrate, phosphoric acid or phosphate, acid gluconic 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 monoclonal antibody CHIR 12.12 or CHIR-5.9, or its antigen-binding fragment and succinate buffer or citrate buffer at a concentration that maintains the pH of the formulation in the range of about pH 5.0 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; thus, the buffer is sodium succinate or sodium citrate, respectively. However, any cation is expected to be effective. Other possible succinate or citrate cations include, without limitation, 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, without limitation, 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.

Preferably, in some 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 an isotonicity agent. The concentration of sodium chloride in the formulation will depend on the contribution of other components to the tonicity. In some embodiments, the concentration of sodium chloride is 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, thawing or mechanical shearing during the processing of a liquid pharmaceutical formulation of the disclosure can be inhibited by incorporating surfactants in the formulation to decrease surface tension on the surface of the solution-air contact. Thus, in some embodiments, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR 5.9, or its antigen binding fragment, a buffer to maintain the pH of the formulation within the range of about pH 5.0 to about pH 7.0, and further comprises a surfactant. In other embodiments, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, a buffer to maintain the pH of the formulation. within the range of about pH 5.0 to about pH 7.0, an isotonicity agent such as sodium chloride in a concentration of about 50 mM to about 300 mM, and further comprises a surfactant.

Typical surfactants used are nonionic surfactants, including polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); polyoxypropylene-polyoxyethylene esters such as Pluronic F68; polyoxyethylene alcohols such as Brij 35; simethicone; polyethylene glycol such as PEG400; lysophosphatidylcholine; and polyoxyethylene-p-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 surfactant preferably used in the practice of the present invention is polysorbate 80. When a surfactant is included, it is usually added in an amount from about 0.001% to about 1.0% (w / v), about 0.001% to about 0.5%, about 0.001% to about 0.4%, about 0.001% to about 0.3%, about 0.001% to about 0.2%, about 0.005% to about 0.5%, approximately 0.005% to approximately 0.2%, approximately 0.01% to approximately 0.5%, approximately 0.01% to approximately rl 0.2%, approximately rl 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 %.

Thus, in some embodiments, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antagonist antibody, for example, the monoclonal antibody CHIR-12.12 or CHIR-5.9, or its antigen binding fragment, the buffer is sodium succinate buffer. or sodium citrate in a concentration of about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 5 mM to about 15 mM; the formulation has a pH of 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 about 0.1 mg / ml to about 50.0 mg / ml or approximately 5.0 mg / ml to approximately 25.0 mg / ml, including approximately 20.0 mg / ml; 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%; the liquid pharmaceutical formulation having 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 approximately pH 6.5, or approximately pH 5.5 to approximately pH 6.0.

The liquid pharmaceutical formulation may be essentially free of preservatives and other vehicles, excipients,

or stabilizers noted above in this document. Alternatively, the formulation may include one or more preservatives, for example, pharmaceutically acceptable antibacterial agents, carriers, excipients or stabilizers described hereinbefore with the proviso that they do not adversely affect the physicochemical stability of the anti-CD40 antagonist antibody or its antigen binding fragment. Examples of acceptable carriers, excipients or stabilizers include, without limitation, 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 exposure 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 are prepared, they can be lyophilized to prevent degradation. Procedures for lyophilizing liquid compositions are known to those skilled in the art. Immediately before use, 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 disclosure also provides the use of an anti-CD40 antagonistic antibody or its antigen-binding fragment in the manufacture of a medicament for treating a cancer subject characterized by a growth of neoplastic B cells, in which the medicament is coordinated. with treatment using an anti-CD20 antibody or fragment thereof antigen binding. Such cancers include, without limitation, the B-cell related cancers presented herein above, for example non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, B-cell lymphoma, high-grade B-cell lymphoma, cell lymphoma. Intermediate grade B, low grade B cell lymphoma, acute B-cell lymphoblastic leukemia, myeloblastic leukemia, Hodgkin's disease, plasmacytoma, follicular lymphoma, folded small cell follicular lymphoma, large cell follicular lymphoma, mixed follicular cell lymphoma small clefts, diffuse small cell cleft lymphoma, diffuse small cell lymphocytic lymphoma, prolymphocytic leukemia, lymphoplasmacytic lymphoma, marginal area lymphoma, lymphoma of lymphoid tissue associated with mucous membranes, B-cell monocytoid lymphoma, splenic lymphoma, hairy leukemia, diffuse large cell lymphoma, mediastinal lymphoma ico large B cell, lymphomatoid granulomatosis, intravascular lymphomatosis, diffuse mixed cell lymphoma, diffuse large cell lymphoma, immunoblastic lymphoma, Burkitt lymphoma, AIDS-related lymphoma and mantle cell lymphoma.

By "coordinated" is meant that the medicament comprising the anti-CD40 antagonist antibody or the antigen binding fragment thereof must be used before, during or after the treatment of the subject using an anti CD20 antibody or a fragment thereof binding to the antigen. In such an embodiment, the present disclosure also relates to the use of the monoclonal antibody CHIR-12.12 or CHIR-5.9 in the manufacture of a medicament for treating a cancer related to B cells in a subject, in which the medicament is coordinated with a treatment using an anti-CD20 antibody, for example rituximab (Rituxan®), or an antigen-binding fragment thereof, in which the medicament has to be used before, during or after the treatment of the subject using an anti-antibody CD20 or an antigen binding fragment thereof.

In some embodiments, the medicament comprising the anti-CD40 antagonist antibody, for example the CHIR-12.12 monoclonal antibody or the CHIR-5.9 disclosed herein, or the antigen binding fragment thereof is coordinated with a treatment that use an anti-CD20 antibody or a fragment thereof antigen-binding and at least one other type of cancer therapy. Examples of other cancer therapies include, without limitation, those described earlier in this document, that is, surgery; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplantation; including suitable chemotherapeutic agents, without limitation, fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-chlorodeoxydenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations thereof, for example, anthracycline-containing regimes (CAP) doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincristine, doxorubicin plus dexamethasone), MP (melphalan plus prednisone) and other cytotoxic and / or therapeutic agents used in chemotherapy, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, dauncin asparaginase and antimetabolites, including, without limitation, cytarabine, methotrexate, 5-fluorouracil decarbacin, 6-thioguanine, 6-mercaptopurine and nelarabine; 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; anti-CD19 antibody (e.g., MT103, a bispecific antibody); anti-CD22 antibody (for example, the humanized monoclonal antibody epratuzumab); bevacizumab (Avastin®) or another anti-cancer antibody directed to human endothelial vascular growth factor; anti-CD22 antibody directed to the CD22 antigen in malignant B cells (e.g., the monoclonal antibody BL-22 , an alphaCD22 toxin); α-M-CSF antibody directed to the macrophage colony stimulating factor; antibodies directed to the nuclear kappaB receptor activator (RANK) and its ligand (RANKL), which are overexpressed in multiple myeloma; antibody anti CD23 directed to the CD23 antigen in malignant B cells (for example, IDEC-152); anti CD80 antibody directed to the CD80 antigen (by e example, IDEC-114); 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) and TRAIL-R2 expressed in several solid tumors and tumors of hematopoietic origin ); cancer therapy based on small molecules, including, without limitation, microtubule and / or topoisomerase inhibitors (for example, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin-binding agent T900607; XL119; and the aminocamptothecin topoisomerase inhibitor ), 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, eloxatin (an antineoplastic agent), ganite (gallium nitrate), Thalomid® (thalidomide), immunomodulatory derivatives of thalidomide (e.g. revlimid (formerly revimid)), Affinitak ™ (antisense protein kinase C-alpha 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 cancer cells (for example, the antisense agents oblimersen and Genasense®), proteasome inhibitors (for example, Velcade ™ (bortezomib)), small molecule kinase inhibitors (for example, CHIR-258), small molecule VEGF inhibitors (e.g., ZD-6474), small molecule heat shock protein (HSP) 90 inhibitors (e.g., 17-AAG), small molecule histone deacetylase inhibitor agents (e.g., hybrid / polar HPC of 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, without limitation, vaccine approaches (e.g., Id-KLH, oncophagus, vitaletin), personalized immunotherapy or active idiotype immunotherapy (for example, MyVax® Personalized Immunotherapy, formally referred to as 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- therapy 15 and therapy with IL-21; steroid therapy; or other cancer therapy; in which treatment with the anti-CD20 antibody or the antigen-binding fragment thereof and additional cancer therapy or additional cancer therapies occur before, during or after treatment of the subject with the medicament comprising the antibody anti CD40 antagonist or the antigen binding fragment thereof, as noted above herein. When the medicament comprising the anti-CD40 antagonist antibody or the antigen binding fragment thereof is coordinated with the treatment using an anti CD20 antibody or a fragment thereof antigen binding and at least one other cancer therapy, the use The medication may be before, during or after the treatment of the subject with either or both of the additional cancer therapies.

Also disclosed herein is the use of a synergistic combination of an anti-CD40 antagonist antibody or a fragment thereof binding to the antigen in the manufacture of a medicament for treating a subject of a cancer characterized by a growth of Neoplastic B cells, including the B-cell-related cancers described herein above, in which the medicament is coordinated with the treatment using an anti-CD20 antibody or a fragment thereof antigen-binding. By "synergistic combination" is meant that the medicament comprises an amount of the anti-CD40 antagonist antibody or the antigen-binding fragment thereof that allows a synergistic therapeutic effect when the medication is coordinated with a treatment using an anti-CD20 antibody or a fragment. thereof binding to the antigen in the manner set forth above herein. "Synergistic therapeutic effect" refers to a therapeutic effect observed with a combination of two or more therapies (in this case, anti-CD40 antagonist antibody therapy and anti-CD20 antibody therapy) in which the therapeutic effect (as se measured by any of several parameters, including the efficacy measures described above herein) is greater than the sum of the respective individual therapeutic effects observed with the respective individual therapies.

In such an embodiment, the disclosure relates to the use of a synergistic combination of the monoclonal antibody CHIR-12.12 or CHIR-5.9 in the manufacture of a medicament for treating a B-cell related cancer in a subject, in which the medicament is coordinated with a treatment using an anti-CD20 antibody, for example rituximab (Rituxan®), or an antigen-binding fragment thereof, in which the medicament has to be used before, during or after the treatment of the subject using the anti antibody CD20 or the antigen binding fragment thereof. In some embodiments, the medicament comprising the synergistic combination of the anti-CD40 antagonist antibody, for example the CHIR-12.12 monoclonal antibody or the CHIR-5.9 disclosed herein, or the antigen binding fragment thereof is coordinated with a treatment using an anti-CD20 antibody, for example rituximab (Rituxan®), or a fragment thereof antigen-binding and at least one other type of cancer therapy, as noted hereinbefore.

The disclosure also relates to the use of an anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9, disclosed herein, or the antigen binding fragment thereof in the manufacture of a medicament for treating a subject of a cancer characterized by a growth of neoplastic B cells, including cancers related to B cells, described above herein, in which the medicament is used in a subject that has been pretreated with an anti CD20 antibody, for example rituximab (Rituxan®), or an antigen binding fragment thereof. By "pretreated" or "pretreatment" is meant that the subject has received therapy with anti CD20 antibodies (ie, has been treated using an anti CD20 antibody or an antigen binding fragment thereof) before receiving the medicament comprising the anti-CD40 antagonist antibody or the antigen binding fragment thereof. "Pretreated" or "pretreatment" includes subjects who have been treated using an anti-CD20 antibody or a fragment thereof binding to the variant antigen thereof, alone or in combination with other cancer therapies, within 2 years. , within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within within 2 days, or even within 1 day before the start of treatment with the medicament comprising the anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9

disclosed herein, or its antigen binding fragment. It is not necessary that the subject has been a patient who responded to the previous treatment with the previous anti-CD20 antibody therapy, or to the previous anti-CD20 antibody therapy and other cancer therapies. Thus, the subject receiving the medicine comprising the anti-CD40 antagonist antibody or its antigen binding fragment could have responded, or could not have responded (i.e., the cancer was refractory) to the previous treatment with the previous anti-CD20 antibody therapy. , or to one or more previous cancer therapies in which the previous treatment comprised multiple cancer therapies, one of which was an anti-CD20 antibody therapy, for example an anti-CD20 antibody therapy and surgery; anti-CD20 antibody therapy and chemotherapy; anti-CD20 antibody therapy and IL-2 therapy; or anti-CD20 antibody therapy, chemotherapy and IL-2 therapy.

Thus, in some embodiments, the disclosure relates to the use of an anti-CD40 antagonist antibody, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9 disclosed herein, or the antigen-binding fragment thereof. in the manufacture of a medicament to be used in a subject in need of treatment of a cancer characterized by a growth of neoplastic B cells, as described above herein, in which the subject has previously been treated with antibody therapy anti CD20 and one or more of the following additional cancer therapies: surgery; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplantation; including suitable chemotherapeutic agents, without limitation, fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-chlorodeoxydenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations thereof, for example, anthracycline-containing regimes (CAP) doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincristine, doxorubicin plus dexamethasone), MP (melphalan plus prednisone) and other cytotoxic and / or therapeutic agents used in chemotherapy, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, dauncin asparaginase and antimetabolites, including, without limitation, cytarabine, methotrexate, 5-fluorouracil decarbacin, 6-thioguanine, 6-mercaptopurine and nelarabine; 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; anti-CD19 antibody (e.g., MT103, a bispecific antibody); anti-CD22 antibody (for example, the humanized monoclonal antibody epratuzumab); bevacizumab (Avastin®) or another anti-cancer antibody directed to human endothelial vascular growth factor; anti-CD22 antibody directed to the CD22 antigen in malignant B cells (e.g., the monoclonal antibody BL-22 , an alphaCD22 toxin); α-M-CSF antibody directed to the macrophage colony stimulating factor; antibodies directed to the nuclear kappaB receptor activator (RANK) and its ligand (RANKL), which are overexpressed in multiple myeloma; antibody anti CD23 directed to the CD23 antigen in malignant B cells (for example, IDEC-152); anti CD80 antibody directed to the CD80 antigen (by e example, IDEC-114); 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) and TRAIL-R2 expressed in several solid tumors and tumors of hematopoietic origin ); cancer therapy based on small molecules, including, without limitation, microtubule and / or topoisomerase inhibitors (for example, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin-binding agent T900607; XL119; and the aminocamptothecin topoisomerase inhibitor ), 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, eloxatin (an antineoplastic agent), ganite (gallium nitrate), Thalomid® (thalidomide), immunomodulatory derivatives of thalidomide (e.g. revlimid (formerly revimid)), Affinitak ™ (antisense protein kinase C-alpha 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 cancer cells (for example, the antisense agents oblimersen and Genasense®), proteasome inhibitors (for example, Velcade ™ (bortezomib)), small molecule kinase inhibitors (for example, CHIR-258), small molecule VEGF inhibitors (e.g., ZD-6474), small molecule heat shock protein (HSP) 90 inhibitors (e.g., 17-AAG), small molecule histone deacetylase inhibitor agents (e.g., hybrid / polar HPC of 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, without limitation, vaccine approaches (e.g., Id-KLH, oncophagus, vitaletin), personalized immunotherapy or active idiotype immunotherapy (for example, MyVax® Personalized Immunotherapy, formally referred to as 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- therapy 15 and therapy with IL-21; steroid therapy; or other cancer therapy.

The disclosure also relates to the use of an anti-CD20 antibody or an antigen-binding fragment thereof in the manufacture of a medicament for treating a subject of a cancer characterized by a growth of neoplastic B cells, including cancer related to B cells, in which the drug is coordinated with the treatment using an anti-CD40 antagonist antibody or a fragment thereof antigen binding. In these embodiments, "coordinated" is intended to mean that the medicament comprising the anti-CD20 antibody or the antigen-binding fragment thereof must be used before, during or after the treatment of the subject using the anti-CD40 antagonist antibody or the fragment. thereof antigen binding. In such an embodiment, the disclosure relates to the use of an anti-CD20 antibody, for example rituximab (Rituxan®), or an antigen-binding fragment thereof in the manufacture of a medicament for treating a subject of a cancer characterized by a growth of neoplastic B cells, such as a cancer related to B cells, in which the drug is coordinated with the treatment using the monoclonal antibody CHIR 12.12 or CHIR5.9, in which the drug has to be used before, during or after treatment of the subject with the monoclonal antibody CHIR-12.12 or CHIR-5.9.

In some embodiments, the medicament comprising the anti CD20 antibody, for example rituximab (Rituxan®), or a fragment thereof antigen binding, is coordinated with a treatment using an anti CD40 antagonistic antibody or a fragment thereof binding. to the antigen, for example the monoclonal antibody CHIR-12.12 or CHIR-5.9, or the antigen binding fragment thereof, and at least one other type of cancer therapy. Examples of other cancer therapies include, without limitation, those described earlier in this document, that is, surgery; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplantation; including suitable chemotherapeutic agents, without limitation, fludarabine or fludarabine phosphate, chlorambucil, vincristine, pentostatin, 2-chlorodeoxydenosine (cladribine), cyclophosphamide, doxorubicin, prednisone, and combinations thereof, for example, anthracycline-containing regimes (CAP) doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), VAD (vincristine, doxorubicin plus dexamethasone), MP (melphalan plus prednisone) and other cytotoxic and / or therapeutic agents used in chemotherapy, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, daubicin, dauncin asparaginase and antimetabolites, including, without limitation, cytarabine, methotrexate, 5-fluorouracil decarbacin, 6-thioguanine, 6-mercaptopurine and nelarabine; 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; anti-CD19 antibody (e.g., MT103, a bispecific antibody); anti-CD22 antibody (for example, the humanized monoclonal antibody epratuzumab); bevacizumab (Avastin®) or another anti-cancer antibody directed to human endothelial vascular growth factor; anti-CD22 antibody directed to the CD22 antigen in malignant B cells (e.g., the monoclonal antibody BL-22 , an alphaCD22 toxin); α-MCSF antibody directed to the macrophage colony stimulating factor; antibodies directed to the nuclear kappaB factor receptor activator (RANK) and its ligand (RANKL), which are overexpressed in multiple myeloma; anti CD23 antibody directed to the CD23 antigen in malignant B cells (for example, IDEC-152); anti-CD80 antibody directed to the CD80 antigen (e.g. emplo, IDEC-114); 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) and TRAIL-R2 expressed in several solid tumors and tumors of hematopoietic origin ); cancer therapy based on small molecules, including, without limitation, microtubule and / or topoisomerase inhibitors (for example, the mitotic inhibitor dolastatin and dolastatin analogues; the tubulin-binding agent T900607; XL119; and the aminocamptothecin topoisomerase inhibitor ), 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, eloxatin (an antineoplastic agent), ganite (gallium nitrate), Thalomid® (thalidomide), immunomodulatory derivatives of thalidomide (e.g. revlimid (formerly revimid)), Affinitak ™ (antisense protein kinase C-alpha 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 cancer cells (for example, the antisense agents oblimersen and Genasense®), proteasome inhibitors (for example, Velcade ™ (bortezomib)), small molecule kinase inhibitors (for example, CHIR-258), small molecule VEGF inhibitors (e.g., ZD-6474), small molecule heat shock protein (HSP) 90 inhibitors (e.g., 17-AAG), small molecule histone deacetylase inhibitor agents (e.g., hybrid / polar HPC of 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, without limitation, vaccine approaches (e.g., Id-KLH, oncophagus, vitaletin), personalized immunotherapy or active idiotype immunotherapy (for example, MyVax® Personalized Immunotherapy, formally referred to as 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- therapy 15 and therapy with IL-21; steroid therapy; or other cancer therapy; wherein treatment with the anti-CD40 antagonist antibody or the antigen-binding fragment thereof and additional cancer therapy or additional cancer therapies occur before, during or after treatment of the subject with the medicament comprising the anti CD20 antibody or the antigen binding fragment thereof, as noted herein above. When the medicament comprising the anti CD20 antibody or a fragment thereof antigen binding is coordinated with the treatment using an anti CD40 antagonist antibody or the fragment thereof antigen binding and at least one other cancer therapy, the use The medication may be before, during or after the treatment of the subject with either or both of the additional cancer therapies.

"Treatment" in the context of the 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 to a cell line of a subject, in which the subject has a cancer

5 characterized by a growth of neoplastic B cells, a symptom associated with such cancer, or a predisposition to develop such cancer, the purpose being to heal, soothe, mitigate, alter, remedy, relieve, improve or affect 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). They were used

15 as immunogen SF9 insect cells expressing the CD40 extracellular domain.

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 at 0.5 ng / ml (Genzyme, Cambridge, Massachusetts) . Then, the fused cells were distributed between the wells of 96-well tissue culture plates, so that each contained on average 1 growing hybridoma.

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, they were combined

25 supernatants from each well and were tested for specificity of anti-CD 40 activity by ELISA first. The positives for cell fluorescent staining of B cells transformed with EBV were then used using a standard FACS assay. Positive hybridoma cells were cloned twice by limiting dilution in IMDM / FBS containing hIL-6 at 0.5 ng / ml.

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, approximately 10% of hybridomas produced using Abgenix XenoMouse® technology (Abgenix; Fremont, California) 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

35 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 their ADCC activities, two hybridoma clones were selected for further evaluation (Table 1 below). They are called 131.2F8.5.9 (5.9) and 153.8E2.D10.D6.12.12 (12.12).

Table 1. Summary of the initial data set with anti-CD40 IgG1 antibodies CHIR-5.9 and CHIR-12.12.

Mother hybridoma

Hybridoma clones Binding to the cell surface  Antagonist ADCC CDC CMCC No. DNA sequence of region V

131.2F5

 131.2F5.8.5.9 +++ +++ ++ - 12047 Yes

153.8E2

 153.8E2D10D6.12.12 +++  +++ ++++ - 12056 Yes

The mouse hybridoma line 131.2F8.5.9 (CMCC No. 12047) and the hybridoma line 153.8E2.D10.D6.12.12 (CMCC No. 12056) have been deposited in the American Type Culture Collection [ATCC; 10801 University Blvd., Manassas, Virginia 2011.0-2209 (USA)] with the Patent Deposit numbers PTA-5542 and PTA-5543, respectively.

The cDNAs encoding the variable regions of the candidate antibodies were amplified by PCR, cloned and sequenced. The amino acid sequences for the light chain and heavy chain of the CHIR antibody

12.12 are presented in Figures 2A and 2B, respectively. See also SEQ ID No. 2 (light chain for CHIR-12.12 mAb) and SEQ ID No. 4 (heavy CHIR-12.12 mAb chain). A heavy chain variant for CHIR-12.12 mAb is shown in Figure 2B (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. Nucleotide sequences encoding the light chain and heavy chain of the CHIR-12.12 antibody are presented in Figures 3A and 3B, 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 4A and 4B, respectively. See also SEQ ID No. 6 (light chain for CHIR-5.9 mAb) and SEQ ID No. 7 (heavy CHIR-5.9 mAb chain). A heavy chain variant for CHIR-5.9 mAb is shown in Figure 3B (see also SEQ ID No. 8), which differs from SEQ ID No. 7 because it has an alanine residue substituted by a serine residue at position 158 of SEQ ID No. 7.

As expected for antibodies derived from independent hybridomas, there is 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 prebinding CD40 ligand to CD40 on the cell surface. The binding affinity of CHIR-5.9 to human CD40 is 1.2 × 10-8 M and the binding affinity of CHIR-12.12 to human CD40 is 5 × 10-10 M.

The monoclonal antibodies CHIR-12.12 and CHIR-5.9 are strong antagonists and inhibit ligand-mediated proliferation of normal B-cell CD40 in vitro; they also inhibit CD40-mediated proliferation in vitro of cancer cells in NHL and CLL patients. In vitro, both antibodies kill primary cancer cells from NHL patients ADCC. Dose-dependent antitumor activity was observed in a model of human xenograft lymphoma.

Example 1: The combination of CHIR-12.12 mAb and Rituxan® shows anti-tumor activity against Rituxan®-resistant Burkitt lymphoma in a xenograft model

Combinations of the rituximab anti-CD20 chimeric monoclonal antibody (Rituxan®; IDEC-C2B8; IDEC Pharmaceuticals Corp., San Diego, California) and the anti-CD40 antagonist monoclonal antibody CHIR-12.12 were tested in a murine model. Specifically, 120 weeks of female nu / nu mice (Charles River Laboratories, Wilmington, Massachusetts) underwent an acclimatization period of at least 7 days. One day before inoculation of tumor cells, mice received a 3 Gy irradiation using a Gammacell 40 cesium 137 irradiation unit manufactured by Atomic Energy of Canada. Namalwa cells (ATCC, Manassas, Virginia), Rituxan®-resistant aggressive Burkitt lymphoma cell, were grown in RPMI 1640 medium with 15% bovine fetal serum. On the day of inoculation, the cells were harvested, counted and resuspended in 50% HBSS + 50% matrigel with a density of 5 × 10 7 cells / mL. Tumor cells were inoculated subcutaneously in the right ijar at 5 × 10 6 cells / 100μl / mouse.

One day after tumor inoculation, the mice were randomly selected and injected intraperitoneally (i.p.) once every 7 days (q7d) with CD40 CHIR-12.12 mAb and Rituxan® as follows:

to.

 IgG1, 10 mg / kg, i.p., q7d, x up to 5 doses.

b.

 Rituxan 10 mg / kg, i.p., q7d, x up to 5 doses.

C.

 Rituxan 20 mg / kg, i.p., q7d, x up to 5 doses.

d.

 CHIR-12.12, 5 mg / kg, i.p., q7d, x up to 5 doses.

and.

 CHk-12.12, 10 mg / kg, i.p., q7d, x up to 5 doses.

F.

 Rituxan®, 10 mg / kg + IgG1, 5 mg / kg, i.p., q7d, × up to 5 doses.

g.

 Rituxan®, 10 mg / kg + CHIR-12.12, 5 mg / kg, i.p., q7d, × up to 5 doses.

h.

 Rituxan®, 10 mg / kg + IgG1, 10 mg / kg, i.p., q7d, × up to 5 doses.

i.

 Rituxan®, 10 mg / kg + CHIR-12.12, 10 mg / kg, i.p., q7d, × up to 5 doses.

Tumor volume was measured twice a week using an electronic caliber. When the mean tumor volume in a group reached 2000 mm3, mice from that group were sacrificed. If the tumor in the treatment group responded, the mice were kept until the average tumor volume reached 2000 mm3.

ANOVA was used to analyze the difference in mean tumor volume between all groups. Multiple Tuckey comparison of least squares means was used to compare the difference in mean tumor volume between two specific groups.

As shown in Figure 1, the primary tumor growth was significantly inhibited by the i.p. CHIR-12.12 alone at a rate of 5 mg / kg once a week for up to 5 weeks (60%, P = 0.02). CHIR-12.12 administered alone at 10 mg / kg showed a tendency towards a significant inhibition of tumor volume (39%, P = 0.22). Rituxan® alone at 10 mg / kg and 20 mg / kg no

5 inhibited tumor growth at all. The combination of CHIR-2.12 and Rituxan® resulted in a synergistic and dose-dependent tumor growth inhibition of CHIR-12.12, with a tumor volume inhibition of 77% (P = 0.001) and 83% (P = 0.003) for CHIR-12.12 at 5 mg / kg plus Rituxan® at 10 mg / kg, and CHIR-12.12 at 10 mg / kg and Rituxan® at 10 mg / kg, respectively. No signs of toxicity were observed among any of the treated animals subjected to current doses and regimens. These data suggest that CHIR-12.12 mAb alone is a therapeutic agent for aggressive and Rituxan® resistant lymphoma. However, when used in combination with Rituxan®, the CHIR-12.12 mAb was more effective than the CHIR-12.12 mAb alone, the Rituxan® alone or the sum of the efficiencies of these two mAbs used alone.

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

The candidate monoclonal antibodies CHIR-5.9 and CHIR-12.12 compete with each other for binding to CD40 but not

15 with 15B8, an anti CD40 IgG2 mAb (see International Publication 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 any anti CD40, either CHIR

12.12 or 15B8. Normal association / dissociation binding curves were observed with varying concentrations of CD40-his (data not shown). For competition studies, CHIR-12.12 or 15B8 were captured 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 CHIR12.12, no association of the complex was observed, indicating that CHIR-5.9 blocks the binding of CHIR-12.12 to CD40his. 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

25 not shown).

It was also determined that 15B8 and CHIR-12.12 do not compete for the binding of CD40-his. This experiment was prepared by capturing CHIR-12.12 on the protein A biosensor chip, blocking the residual protein A sites with control hIgG1, binding CD40-his and then flowing 15B8 over the modified surface. Indeed, 15B8 was bound under these conditions, indicating that CHIR-12.12 does not block the binding of 15B8 to CD40.

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

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. Was flowed recombinant soluble CD40-his over the biosensor surface at varying concentrations. The antibody and antigen were diluted in 0.01 M HEPES pH 7.4, 0.15 M NaCl, EDTA 3

35 mM, 0.005% P20 surfactant (HBS-EP). Kinetic and affinity constants were determined using Biaevaluation software with a model / global interaction setting of 1: 1.

As shown in Table 2 below, there is a 121-fold difference in the CHIR dissociation rate

5.9 and CHIR-12.12 resulting in a 24-fold affinity for CHIR-12.12.

Table 2. Summary of the binding properties of the 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) × 105  (15.0 ± 1.3) × 10-3  12.15 ± 0.35

Anti CD40, CHIR-12.12

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

Example 4: Epitope characterization for monoclonal antibodies CHIR-12.12 and CHIR-5.9

To determine the location of the epitope on CD40 recognized by the monoclonal antibodies CHIR-12.12 and CHIR-5.9, SDS-PAGE analysis and Western blotting were performed. Purified CD40 (0.5 µg) was separated on a 4-12% NUPAGE gel under reducing and non-reducing conditions, transferred to PVDF membranes, and probed with monoclonal antibodies at a concentration of 10 µg / ml. The transfers were probed with IgG

Anti-human conjugated with alkaline phosphatase and developed using the Western® Blue stabilized substrate for alkaline phosphatase (Promega).

The results indicate that the anti-CD40 monoclonal antibody CHIR12.12 recognizes epitopes 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 CD40 (Table 3; transfers not shown ). The fact that recognition was positive for both forms of CD40 indicates that this antibody interacts with a conformational epitope, part of which is a linear sequence. The monoclonal antibody CHIR-5.9 primarily recognizes the non-reduced form of CD40, suggesting that this antibody interacts primarily with a conformational epitope (Table 3; transfers not shown).

Table 3. Domain identification.

Domain 1

Domain 2 Domain 3 Domain 4

mAb CHIR-12.12

- + - -

mAb CHIR-5.9

- + - -

mAb 15B8

+ - - -

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

5 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 4; transfers not shown). In contrast, the CHIR-5.9 monoclonal antibody exhibits very weak recognition for Domain 2 (Table 4; transfers not shown). None of these antibodies recognize Domains 1, 3 or 4 in this analysis.

10 Table 4. Domain analysis 2.

 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 PCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICT (residues 61-104 of the sequence shown in SEQ ID No. 10 or SEQ ID No. 12). SPOT synthesis membranes (Sigma) containing thirty-five were generated

15 10-mer peptides with a 1 amino acid shift. Western blot analysis was performed with the CHIR-12.12 mAb and the human anti-IgG labeled with beta galactosidase as secondary antibody. The transfer was dismantled and re-probed with the CHIR-5.9 mAb to determine the region recognized by this antibody.

SPOT assays probing with anti-CD40 monoclonal antibody CHIR-12.12 at a concentration of 10 µg / ml

20 gave positive reactions with transfers 18 to 22. The region of the sequence covered by these peptides is shown in Table 5.

Table 5. Results of SPOT analytical probing with anti-CD40 monoclonal antibody CHIR-12.12.

Transfer number

Sequence region

18

HQHKYCDPNL (residues 78-87 of SEQ ID 10 or 12)

19

   QHKYCDPNLG (residues 79-88 of SEQ ID No. 10 or 12)

twenty

  HKYCDPNLGL (residues 80-89 of SEQ ID 10 or 12)

twenty-one

  KYCDPNLGLR (residues 81-90 of SEQ ID 10 or 12)

22

  YCDPNLGLRV (residues 82-91 of SEQ ID 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, D84 and N86, which have been predicted to be involved in the

25 CD40-CD40 ligand interaction.

SPOT analyzes with CHIR-5.9 mAb showed a weak recognition of the peptides represented by the 20-22 transfers shown in Table 6, suggesting involvement of the YCDPNLGL region (residues 82-89 of the sequence shown in SEQ ID No. 10 or SEQ ID No. 12) in its binding to CD40. It should be noted that the CHIR mAbs

12.12 and CHR-5.9 compete with each other for binding to CD40 in the BIACORE analysis.

30 Table 6. Results of SPOT analytical 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 SPOT analyzes are within the B1 module of CD40. The sequence of module B1 of CD40 is:

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

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 5: 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, PBK / 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 the 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 trigger 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 × 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, United Kingdom). 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 sCD40L, restoring apoptosis of B cells (data not shown).

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

In these experiments, 0.6 × 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, United Kingdom). 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 (Ser 180) and IKKβ (Ser 181) in normal B cells.

In these experiments, 1.0 × 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, United Kingdom). Then CHIR-12.12 (10 µg / ml) and control IgG were added. Cells were collected at 0 and 20 minutes. IKK α (Ser 180) and IKK β (Ser 181) phosphorylated and total IKK β controls were detected in cell lysates by Western blotting.

In summary, stimulation by means of sCD40L resulted in the phosphorylation of IKKα (Ser 180) 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 × 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, United Kingdom). 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. Excised PARP and β actin controls were detected in cell lysates by Western blotting.

Briefly, treatment with CHIR-12.12 resulted in increased 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 × 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, United Kingdom). Then CHIR-12.12 (0.5, 2 and 10 µg / ml) and control IgG were added. Cells were collected at 22 hours. 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 cleaved PARP in cells stimulated with sCD40L in a dose-dependent manner and, consequently, nullified these blockages to the apoptotic pathway in the Normal B cells stimulated with sCD40L (data not shown).

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

In these experiments, 1.0 × 10 6 normal human B cells from healthy donors (purity percentage between 85 and 95%) were stimulated with CHIR-12.12 (10 µg / ml) and only control IgG (i.e. non-cell were previously stimulated with sCD40L before adding the antibody). Cells were collected at 0, 4, 14 and 16 hours. Controls of XIAP, cleaved PARP and β actin 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 those treated with CHIR-12.12 (data not shown). These data indicate that CHIR-12.12 does not trigger apoptosis in normal human B cells without CD40L stimulation.

CHIR-12.12 inhibits the phosphorylation of IKKα (Ser 180) and IKKβ (Ser 181), Akt, ERK and p38 in normal B cells.

In these experiments, serum was deprived in a medium containing 1% FBS at 1.0 × 10 6 normal human B cells from healthy donors (purity percentage between 85 and 95%) and stimulated with sCD40L 1 µg / ml ( Alexis Corp., Bingham, Nottinghamshire, United Kingdom). Cultures were treated with CHIR-12.12 (1 and 10 µg / ml) and control IgG. Cells were collected at 0 and 20 minutes. Phosphorus IKKα, phosphorus IKKβ, total IKKβ, phosphorus ERK, total ERK, phosphorus Akt, total Akt, phospho p38 and total p38 were detected in cell lysates by Western blotting.

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

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

In these experiments, serum was deprived in a medium containing 1% FBS at 1.0 × 10 6 normal human B cells from healthy donors (purity percentage between 85 and 95%) and stimulated with sCD40L 1 µg / ml ( Alexis Corp., Bingham, Nottinghamshire, United Kingdom). 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. Phosphorus ERK, phospho Akt, total Akt, phosphorus 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 inhibits at one 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 at 4.0 × 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, United Kingdom) for 20 minutes. Cells were collected at 0 and 20 minutes. CD40 was immunoprecipitated using polyclonal anti CD40 (Santa Cruz Biotechnology, California), and probed in a Western blot with anti TRAF2 mAb (Santa Cruz Biotechnology, California), anti TRAF3 mAb (Santa Cruz Biotechnology, California), and anti CD40 mAb ( Santa Cruz Biotechnology, California).

In summary, the results show that TRAF2 and TRAF3 precipitated together with CD40 after stimulation with 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).

5 Without being bound by theory, the results of these experiments, and the results in the examples summarized above, indicate that the CHIR 12.12 antibody is a dual-acting anti-CD40 antagonist monoclonal antibody that has an exceptional combination of attributes. This fully human monoclonal antibody blocks CD40L-mediated CD40 signal pathways for B cell survival and proliferation; This antagonism eventually results in cell death. CHIR-12.12 also mediates recognition and union by

10 effector cells, initiating antibody-dependent cellular cytotoxicity (ADCC). Once the 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 6: Liquid pharmaceutical formulation for anti-CD40 antagonist antibodies

15 It is known that protein stability is sensitive to the pH of the solution, since the surface charge of a protein varies with the pH of the solution, which results in stabilization or destabilization. Thus, the non-optimal pH of a formulation can alter the electrostatic interaction, which leads to the physical degradation of an anti-CD40 antagonistic antibody, such as aggregation, precipitation and the like. A change in pH conditions could also cause the rupture of covalent bonds, which leads to chemical degradation, such as

20 fragmentation, deamidation and the like. Therefore, this study was designed to identify an optimal pH of the solution to minimize degradation of the anti-CD40 antagonistic antibody due to aggregation, fragmentation or deamidation.

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 a pH of 5.5-6.5. Based on a combination of SDS-PAGE analysis, size exclusion HPLC (SEC-HPLC) and cation exchange HPLC (CEX-HPLC), the physicochemical stability of CHIR-12.12 is optimal at a pH of approximately 5.0- 5.5. In view of these results, a recommended liquid pharmaceutical formulation comprising this antibody is

A formulation comprising CHIR-12.12 in a concentration of approximately 20 mg / ml formulated in approximately 10 mM sodium succinate, in 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

35 a CHO cell culture process. This mAb has a molecular weight of 150 kDa and consists of two light chains and two heavy chains linked together by disulfide bonds. It is directed against the CD40 cell surface receptor in cells expressing CD40, including normal and malignant B cells, for the treatment of various types of cancer and autoimmune / inflammatory diseases.

The substance of the anti-CD40 drug used for this study was a bulk batch of anti-CD40 (CHIR-12.12)

Purified 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 testing together with stability samples at predetermined time points. Stability samples were prepared by dialysis of the drug substance against solutions of different pH and the concentration of CHIR-12.12 in each

One of the samples was determined by UV 280, as presented in Table 7.

Table 7. CHIR-12.12 formulations.

Buffer Composition

pH CHIR-12.12 concentration (mg / ml)

10 mM sodium citrate, 150 mM sodium chloride

4,5 9.0

10 mM sodium succinate, 150 mM sodium chloride

5.0 9.3

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, 150mM sodium chloride

7.0 9.4

10 mM sodium phosphate, 150mM 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 means of staining with Coomassie blue.

Size exclusion chromatography (SEC-HPLC)

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

Cation Exchange Chromatography (CEX-HPLC)

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 was presumably added, resulting in the loss of the DSC signal. In order to select the formulation, the lowest thermal transition temperature was defined as the melting temperature, Tm, in this study. Figure 6 shows the thermal melting temperature as a function of the pH of the formulations. Formulations at pH 5.5-6.5 provided an 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 totally reducible and increased with the increase in the pH of the solution, suggesting that there could be a different covalent association of disulfide in the molecules. As there were other species with unknown identities in SDS-PAGE, the stability comparison for each formulation is based on the remaining purity of CHIR-12.12. 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.

The SEC-HPLC analysis detected the CHIR-12.12 intact as the main peak species, an aggregation species as a species of anterior peak separated from the main peak species, a large fragment species such as a plateau peak at the back of the species of the main peak, and small fragment species subsequent to the species of the main peak were detected. After incubation at 5 ° C and 25 ° C for 3 months, insignificant amounts of protein fragments and aggregates (<1.0%) were detected in the above formulations and the species of the main peak of CHIR-12.12 continued to have more than 99% of purity (data not shown). However, protein fragments gradually developed after storage at 40 ° C and more fragments were formed at pH 4.5 and pH 6.5-9.0, as shown in Table 8. After incubating the CHIR- formulations 12.12 at 40 ° C for 3 months, approximately 2-3% of aggregates were detected at pH 7.5 and pH 9.0, while less than 1% of aggregates were detected in formulations at other pHs (data not shown) . The results of SEC-HPLC indicate that CHIR-12.12 is more stable at a pH of approximately 5.0-6.0.

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

Sample

% Main peak % Fragments

t = 0

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

Control

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

pH 4.5

99.4 93.2  86.0  81.3  <1.0 6.4 13.2  18.1

pH 5.0

99.8 98.7  91.3  89.2 <1.0 <1.0 7.8 10.2

pH 5.5

99.8 98.9  91.4  90.6 <1.0 <1.0 7.6 8.8

pH 6.0

99.6 97.7  90.4  87.3 <1.0 1.9 8.2 11.7

pH 6.5

99.3 93.4  89.0  86.9 <1.0 5.6 9.9 12.4

pH 7.0

99.2 93.9  87.4  85.1  <1.0 5.5 11.1  13.5

pH 7.5

99.1 92.8  84.4  81.9  <1.0 6.4 12.9  16.2

pH 9.0

99.3 82.4  61.6  50.6  <1.0 15.4  36.2  47.6

CEX-HPLC analysis.

The CEX-HPLC analysis detected the intact CHIR-12.12 as the main peak species, acid variants eluted

5 earlier than the main peak species and lysine addition variants at the C-terminal end eluted after the main peak species. Table 9 shows the dependence of the percentages of the remaining CHIR-12.12 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 (~ 33%), probably due to the early stage fermentation and purification processes. The susceptibility of CHIR-12.12 to higher pH solutions is evidenced

10 for two facts. First, the sample of the initial formulation at pH 9 (t = 0) already generated 12% more acidic species than the control. Secondly, the percentage of acidic species increased sharply with the increase in pH. The degradation related to the change of charges is probably due to deamidation. The above data indicates that this type of degradation of CHIR-12.12 was minimized at a pH of approximately 5.0-5.5.

Table 9. Percentage of the 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 3 m 25 ° C 3 m 40 ° C 1 m 40 ° C 2 m  t = 0 5 ° C 3 m 25 ° C 3 m 40 ° C 1 m 40 ° C 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 general stability data, a liquid pharmaceutical formulation

20 recommended 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 7: Clinical studies with CHIR-5.9 and CHIR-12.12

Clinical objectives

The general objective is to provide an effective therapy for B cell tumors making them targets of a combination of an anti-CD40 antagonist antibody and an anti-CD20 antibody. These tumors include B-cell lymphoma, chronic lymphocytic lymphoma (CLL), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), Waldenstrom macroglobulinemia, and Castleman's systemic disease. The signal for these diseases is determined in phase II although some activity measurement can be obtained in phase I. The antagonist antibody

Initial anti CD40 is the CHIR-12.12 mAb, and the initial anti CD20 antibody is rituximab (Rituxan®). Further research studies the combined effects of CHIR-12.12 mAb or CHIR-5.9 with other anti-CD20 antibodies that have the binding characteristics of rituximab.

Phase I

 Assess the safety and pharmacokinetics: increasing the dose of these two antibodies in subjects with malignant B-cell processes.

 Choose the dose of each antibody based on safety, tolerability and change in the serum markers of the respective targets, that is, CD40 or CD20. In general, a DMT is sought for each of these antibodies when used in combination, but other indications of efficacy (CD40 + and / or CD20 + B cell depletion, etc.) may be appropriate for the dose finding.

 Consider more than one dose combination specifically for different indications; for example, the combination dose for CLL may be different from that of NHL. Thus, in phase II some dose finding may be necessary.

 Patients are dosed weekly with a sample of pharmacokinetics (Pk) in real time. Initially, the maximum dose allowed is a 4-week cycle. Pk can be very variable depending on the disease studied, the density of CD40 and / or CD20, etc.

 This or these trials are open to subjects with B-cell lymphoma, CLL and, potentially, other malignant B-cell processes.

 The decision to interrupt or continue studies is based on safety, dose and preliminary tests of antitumor activity, particularly of a synergistic nature.

 The activity of antibody combination therapy, determined by the response rate, is determined in phase II.

 Identify the dose (s) for phase II.

Phase II

Several trials will be initiated on the aforementioned tumor types, with concentration in B-cell lymphoma, CLL and multiple myeloma (MM). Separate trials may be required in low and medium / high grade NHL, since CD40 and / or CD20 may have a different function, depending on the lymphoma grade. More than one combination of antibody doses and more than one program can be tested in a randomized phase II configuration.

In each disease, address a population that has not responded to current standard care:

 CLL: patients who have been resistant to Campath® and chemotherapy.

 Low grade NHL: Rituxan® or CHOP-R failures.

 Intermediate NHL: CHOP-R failures.

 Multiple myeloma: chemotherapy failures.

 The decision to interrupt or continue the study is based on the proof of the therapeutic concept in phase II

 Determine if a substitute marker can be used as an early indication of clinical efficacy

 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 as the standard. If the signal is at a stage of the disease in which there is no standard of therapy, then a well-controlled single-arm study could serve as a fundamental trial. If there are competing agents that are considered standards, then direct studies are conducted.

SEQUENCE LIST

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

<120> Therapy procedures for B-cell-related cancers

<130> PP22244.002 (284270)

<150> 60 / 613,885 <151> 2004-09-28

5 10

<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 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 720 <212> DNA <213> Artificial sequence <220> <223> Coding sequence for the light chain of the human anti-CD40 antibody CHIR-12.12 <221> CDS <222> ( 1)… (720) <400> 1 atg gcg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48

Met

To Leu Pro To Gln Leu Leu Gly Leu Leu  Met Leu Trp Val Be

one

5 10 fifteen

gga

tcc agt ggg gat att gtg atg act cag tct cca ctc tcc ctg acc 96

Gly

Be Be  Gly Asp Ile Val Met Thr Gln Be Pro Leu Be Leu Thr

twenty

25 30

gtc

acc cct gga gag ccg gcc tcc atc tcc tgc agg tcc agt cag agc 144

Val

Thr Pro Gly Glu Pro To Be Ile Be Cys Arg Be Be Gln Be

35

40 Four. Five

ctc

ctg tat agt aat gga tac aac tat ttg gat tgg tac ctg cag aag 192

Leu

Leu

Tyr Be Asn Gly Tyr Asn Tyr  Leu Asp Trp Tyr  Leu Gln Lys

fifty

55 60

cca

ggg cag tct  cca cag gtc ctg atc tct ttg ggt tct aat cgg gcc 240

Pro

Gly Gln Be  Pro Gln Val Leu Ile Be Leu Gly Be Asn Arg To

65

70 75 80

tcc

ggg gtc cct gac agg ttc agt ggc agt gga tca ggc here gat ttt 288

Be

Gly Val Pro Asp Arg Phe  Be Gly  Be Gly  Be Gly Thr Asp Phe

85

90  95

here

ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336

Thr

Leu Lys Ile Be Arg Val Glu To Glu Asp Val Gly Val Tyr Tyr

100

# 5 110

tgc

atg falls gct cga falls act cca ttc act ttc ggc cct ggg acc aaa 384

Cys

Met Gln To Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys

115

120 125

gtg

gat atc aga cga act  gtg gct gca cca tct gtc ttc atc ttc ccg 432

Val

Asp Ile Arg Arg Thr  Val To To Pro Be  Val Phe Ile Phe Pro

130

135 140

cca

tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480

Pro

Be Asp Glu Gln Leu Lys Be Gly Thr To Be Val Val Cys Leu

145

150 155 160

ctg

aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528

Leu

Asn Asn Phe Tyr Pro Arg Glu To Lys val Gln Trp Lys Val Asp

165

170  175

aac

gcc ctc falls tcg ggt  aac tcc cag gag agt gtc here gag cag gac 576

Asn

To Leu Gln Be Gly  Asn Be Gln Glu Be Val Thr Glu Gln Asp

180

185 190

agc

aag gac  agc acc tac  agc ctc  agc  agc acc ctg acg ctg  agc aaa 624

Be

Lys Asp  Be Thr Tyr  Be Leu  Be  Be Thr Leu Thr Leu  Be Lys

195

200 205

gca

gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672

To

Asp Tyr Glu Lys His Lys Val Tyr  To Cys Glu Val Thr His Gln

210

215 220

ggc

ctg agc tcg ccc gtc here aag agc ttc aac agg gga gag tgt tag 720

Gly

Leu Be Be Pro Val Thr Lys Be Phe Asn Arg  Gly Glu Cys *

225

230 235

5

<210> 2 <211> 239 <212> PRT <213> Artificial sequence <220> <223> Human anti-CD40 antibody light chain CHIR-12.12 <400> 2 Met Leu wing Pro Gln Leu Leu Gly Leu Leu Met Leu Trp Val Be

one

5 10 fifteen

Gly

Be Be  Gly Asp Ile Val Met Thr Gln Be Pro Leu Be Leu Thr

twenty

25 30

Val

Thr Pro Gly Glu Pro To Be Ile Be Cys Arg Be Be Gln Be

35

40 Four. Five

Leu

Leu

Tyr Be Asn Gly Tyr Asn Tyr  Leu Asp Trp Tyr  Leu Gln Lys

fifty

55 60

Pro

Gly Gln Be  Pro Gln Val Leu Ile Be Leu Gly be Asn Arg To

65

70 75 80

Be

Gly Val Pro Asp Arg Phe  Be Gly  Be Gly  Be Gly Thr Asp Phe

85

90 95

Thr

Leu Lys Ile Be Arg Val Glu To Glu Asp Val Gly Val Tyr Tyr

100

105 110

Cys

Met Gln To Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys

115

120 125

Val

Asp Ile Arg Arg Thr  Val To To Pro Be  Val Phe Ile Phe Pro

130

135 140

Pro

Be Asp Glu Gln Leu Lys Be Gly Thr To Be Val Val Cys Leu

145

150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Be Lys Asp Be Thr Tyr Be Leu Be Be Thr Leu Thr Leu Be Lys 195 200 205 Wing Asp Tyr Glu Lys His Lys Val Tyr Wing Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Be Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

<210> 3

<211> 2016

<212> DNA

<213> Artificial sequence

<220>

<223> Coding sequence for the heavy chain of the human anti-CD40 antibody CHIR-12.12 (with introns)

<400> 3

atggagtttg ggctgagctg ggttttcctt gttgctattt taagaggtgt ccagtgtcag 60 gtgcagttgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcct ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagttata tcatatgagg aaagtaatag ataccatgca 240 gactccgtga agggccgatt caccatctcc agagacaatt ccaagatcac gctgtatctg 300 caaatgaaca gcctcagaac tgaggacacg gctgtgtatt actgtgcgag agatgggggt 360 atagcagcac ctgggcctga ctactggggc cagggaaccc tggtcaccgt ctcctcagca 420 agtaccaagg gcccatccgt cttccccctg gcgcccgcta gcaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt tggtgagagg 720 ccagcacagg gagggagggt gtctgctgga agccaggctc agcgctcctg cctggacgca 780 tcccggctat gcagtcccag tccagggcag caaggcaggc cccgtctgcc tcttcacccg 840 gaggcctctg cccgccccac tcatgctcag ggagagggtc ttctggcttt ttccccaggc 900 tctgggcagg cacaggctag gtgcccctaa cccaggccct gcacacaaag gggcaggtgc 960 tgggctcaga cctgccaaga gccatatccg ggaggaccct gcccctgacc taagcccacc 1020 ccaaaggcca aactctccac tccctcagct cggacacctt ctctcctccc agattccagt 1080 aactcccaat cttctctctg cagagcccaa atcttgtgac aaaactcaca catgcccacc 1140 gtgcccaggt aagccagccc aggcctcgcc ctccagctca aggcgggaca ggtgccctag 1200 agtagcctgc atccagggac aggccccagc cgggtgctga cacgtccacc tccatctctt 1260 cctcagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca aaacccaagg 1320

acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac gtgagccacg 1380 aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga 1440 caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc 1500 tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc 1560 cagcccccat cgagaaaacc atctccaaag ccaaaggtgg gacccgtggg gtgcgagggc 1620 cacatggaca gaggccggct cggcccaccc tctgccctga gagtgaccgc tgtaccaacc 1680 tctgtcccta cagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1740 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1800 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1860 gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1920 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1980 acgcagaaga gcctctccct gtctccgggt aaatga 2016

<210> 4

<211> 469

<212> PRT 5 <213> Artificial sequence

<220>

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

<400> 4

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Wing Ile Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Be Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Wing Arg Asp Gly Gly Ile Wing Wing Pro Gly Pro Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Be Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ala Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Wing Wing Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170

175

Thr

Val Be Trp Asn Be Gly To Leu  Thr Be Gly Val His  Thr Phe

180

185 190

Pro

To Val Leu Gln Be Be Gly Leu Tyr Be Leu Be Be Val Val

195

200 205

Thr

Val Pro Be Be Be Leu Gly  Thr Gln  Thr Tyr Ile Cys Asn Val

210

215 220

Asn

His Lys Pro Be  Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

225

230 235 240

Be

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro To Pro Glu Leu

245

250 255

Leu

Gly Gly Pro Be Val Phe  Leu Phe Pro Pro Lys Pro Lys Asp Thr

260

265 270

Leu

Met Ile Be Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275

280 285

Be

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290

295 300

Glu

Val His Asn To Lys Thr Lys Pro Arg  Glu  Glu Gln Tyr Asn Be

305

310 315 320

Thr

Tyr Arg Val Val Be Val Leu  Thr Val Leu His Gln Asp Trp Leu

325

330 335

Asn

Gly Lys Glu Tyr Lys Cys Lys Val Be  Asn Lys To Leu Pro To

340

3. 4. 5 350

Pro

Ile Glu Lys Thr Ile Be Lys To Lys Gly Gln  Pro Arg Glu

Pro

355

360 365

Gln

Val Tyr Thr Leu Pro Pro Be Arg Glu Glu Met Thr Lys Asn

Gln

370

375 380

Val

Be Leu Thr Cys Leu  Val Lys Gly Phe Tyr Pro Be Asp Ile To

385

390 395 400

Val

Glu Trp Glu Be Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405

410 415

Pro

Pro

Val Leu Asp Be Asp Gly Be Phe Phe Leu Tyr Be Lys Leu

420

425 430

Thr

Val Asp Lys Be Arg Trp Gln Gln Gly Asn Val Phe Be Cys Be

435

440 445

Val

Met His Glu To Leu His Asn His Tyr Thr Gln Lys Be Leu Be

450 455 460

Leu

Be Pro Gly Lys

465

5

<210> 5 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of variant of the human CD40 antibody CHIR-12.12 <400> 5 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val To Ile Leu Arg Gly

one

5 10 fifteen

Val

Gln Cys Gln  Val Gln Leu  Val Glu Be Gly Gly Gly  Val  Val Gln

twenty

25 30

Pro

Gly Arg Be Leu Arg Leu Be Cys To To Be Gly Phe Thr Phe

35

40 Four. Five

Be

Be

Tyr Gly Met His Trp Val Arg Gln To Pro Gly Lys Gly Leu

fifty

55 60

Glu

Trp Val To Val Ile Be Tyr  Glu  Glu Be Asn Arg Tyr His To

65

70 75 80

Asp

Be val Lys Gly Arg Phe Thr Ile Be Arg  Asp Asn Be Lys Ile

85

90 95

Thr

Leu Tyr Leu Gln Met Asn Be Leu Arg  Thr Glu Asp  Thr To Val

100

105 110

Tyr

Tyr

Cys To Arg Asp Gly Gly Ile To To Pro Gly Pro Asp

Tyr

115

120 125

Trp

Gly Gln Gly Thr Leu Val Thr Val Be Be To Be Thr Lys Gly

130

135 140

Pro

Be Val Phe  Pro Leu To  Pro Be Be Lys Be Thr Be Gly Gly

145

150 155 160

Thr

To To Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165

170 175

Thr

Val Be Trp Asn Be Gly To Leu  Thr Be Gly Val His  Thr Phe

180

185 190

Pro

To Val Leu Gln Be Be Gly Leu Tyr Be Leu Be Be Val Val

195

200 205

Thr

Val Pro Be Be Be Leu Gly  Thr Gln  Thr Tyr Ile Cys Asn Val

210

215 220

Asn

His Lys Pro Be  Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

225

230 235 240

Be

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro To Pro Glu Leu

245

250 255

Leu

Gly Gly Pro Be Val Phe  Leu Phe Pro Pro Lys Pro Lys Asp Thr

260

265 270

Leu

Met Ile Be Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275

280 285

Be

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290

295 300

Glu

Val His Asn To Lys Thr Lys Pro Arg  Glu  Glu Gln Tyr Asn Be

305

310 315 320

Thr

Tyr Arg Val Val Be Val Leu  Thr Val Leu His Gln Asp Trp Leu

325

330 335

Asn

Gly Lys Glu Tyr Lys Cys Lys Val Be  Asn Lys To Leu Pro To

340

3. 4. 5 350

Pro

Ile Glu Lys Thr Ile Be Lys To Lys Gly Gln  Pro Arg Glu

Pro

355

360 365

Gln

Val Tyr Thr Leu Pro Pro Be Arg Glu Glu Met Thr Lys Asn

Gln

370

375 380

Val

Be Leu Thr Cys Leu  Val Lys Gly Phe Tyr Pro Be Asp Ile To

385

390 395 400

Val

Glu Trp Glu Be Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405

410 415

Pro

Pro

Val Leu Asp Be Asp Gly Be Phe Phe Leu Tyr Be Lys Leu

420

425 430

Thr

Val Asp Lys Be Arg Trp Gln Gln Gly Asn Val Phe Be Cys Be

435

440 445

Val

Met His Glu To Leu His Asn His Tyr Thr Gln Lys Be Leu Be

450

455 460

Leu

Be Pro Gly Lys

465

<210> 6

<211> 239

<212> PRT

<213> Artificial sequence

5

<220>

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

<400> 6

Met

To Leu Leu To Gln Leu Leu Gly Leu Leu  Met Leu Trp Val Pro

1 5 10 15

Gly

Be Be  Gly To Ile Val Met Thr Gln Pro Pro Leu Be Be Pro

twenty

25 30

Val

Thr Leu Gly Gln Pro To Be Ile Be Cys Arg Be Be Gln Be

35

40 Four. Five

Leu

Val His Be Asp Gly Asn Thr Tyr  Leu Asn Trp  Leu Gln Gln Arg

fifty

55 60

Pro

Gly Gln  Pro  Pro Arg Leu Leu Ile Tyr Lys Phe Phe Arg Arg Leu

65

70 75 80

Be

Gly Val Pro Asp Arg Phe  Be Gly  Be Gly To Gly Thr Asp Phe

85

90 95

Thr

Leu Lys Ile Be Arg val Glu To Glu Asp Val Gly Val Tyr Tyr

100

105 110

Cys

Met Gln Val Thr Gln Phe Pro His Thr Phe Gly Gln Gly Thr Arg

115

120 125

Leu

Glu Ile Lys Arg Thr Val To To Pro Be Val Phe Ile Phe Pro

130

135 140

Pro

Be Asp Glu Gln Leu Lys Be Gly Thr To Be Val Val Cys Leu

145

150 155 160

Leu

Asn Asn Phe Tyr Pro Arg Glu To Lys Val Gln Trp Lys Val Asp

165

170 175

Asn

To Leu Gln Be Gly  Asn Be Gln Glu Be Val Thr Glu Gln Asp

180

185 190

Be

Lys Asp  Be Thr Tyr  Be Leu  Be  Be Thr Leu Thr Leu  Be Lys

195

200 205

To

Asp Tyr Glu Lys His Lys Val Tyr  To Cys Glu Val Thr His Gln

210

215 220

Gly

Leu Be Be Pro Val Thr Lys Be Phe Asn Arg  Gly Glu Cys

225

230 235

5

<210> 7 <211> 474 <212> PRT <213> Artificial sequence <220> <223> Human anti-CD40 antibody heavy chain CHIR-5.9 <400> 7 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu To Val Leu Gln Gly

l

5 10 fifteen

Val

Cys To Glu  Val Gln Leu  Val Gln Be Gly To Glu  Val Lys Lys

20 25

30

Pro

Gly Glu Be Leu Lys Ile Be Cys Lys Gly Be Gly Tyr Be Phe

35

40 Four. Five

Thr

be Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

fifty

55 60

Glu

Trp Met Gly Ile Ile Tyr Pro Gly Asp Be Asp Thr Arg Tyr Be

65

70 75 80

Pro

Be Phe Gln Gly Gln Val Thr Ile Be To Asp Lys Be Ile Be

85

90 95

Thr

To Tyr Leu Gln Trp Be Be Leu Lys To Be Asp  Thr To Met

100

105 110

Tyr

Tyr

Cys To Arg Gly Thr To To Gly Arg Asp

Tyr

Tyr

Tyr

Tyr

115

120 125

Tyr

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Be Be

130

135 140

To

Be Thr Lys Gly Pro be Val Phe Pro Leu  To Pro  To Be Lys

145

150 155 160

Be

Thr  Be Gly Gly Thr To To Leu Gly Cys Leu Val Lys Asp Tyr

165

170 175

Phe

Pro Glu Pro Val Thr Val Be Trp Asn Be Gly To Leu Thr Be

180

185 190

Gly

Val His Thr Phe Pro To Val Leu Gln Be Be  Gly Leu Tyr Be

195

200 205

Leu

Be Be Val Val Thr Val Pro Be Be Be  Leu Gly Thr Gln Thr

210

215 220

Tyr

Ile Cys Asn Val Asn His Lys Pro Be Asn Thr Lys Val Asp Lys

225

230 235 240

Arg

Val Glu Pro Lys Be Cys Asp Lys Thr His Thr Cys Pro Pro Cys

245

250 255

Pro

To  Pro Glu Leu Leu Gly Gly  Pro Be Val Phe Leu Phe

Pro

Pro

260

265 270

Lys

Pro  Lys Asp Thr Leu Met Ile Be Arg Thr Pro Glu Val Thr Cys

275

280 285

Val

Val

Val

Asp  Val Be His Glu Asp Pro Glu  Val Lys Phe Asn Trp

290

295 300

Tyr

Val Asp Gly Val Glu Val His Asn To Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu

Gln Tyr Asn Be Thr Tyr Arg Val Val Be Val Leu Thr Val Leu

325

330 335

His

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Be Asn

340

3. 4. 5 350

Lys

To Leu Pro To Pro Ile Glu  Lys Thr Ile be  Lys To  Lys Gly

355

360 365

Gln

Pro Arg Glu Pro  Gln Val Tyr Thr Leu Pro Pro Be Arg Glu Glu

370

375 380

Met

Thr Lys Asn Gln Val Be Leu Thr Cys Leu Val Lys Gly Phe Tyr

385

390 395 400

Pro

Be Asp Ile To Val Glu Trp Glu Be Asn Gly Gln  Pro Glu Asn

405

410 415

Asn

Tyr Lys Thr Thr Pro Pro Val Leu Asp Be Asp Gly Be Phe Phe

420

425 430

Leu

Tyr Be Lys  Leu Thr Val Asp Lys Be Arg Trp Gln Gln Gly Asn

435

440 445

Val

Phe Be Cys Be  Val Met His Glu To Leu His Asn His Tyr Thr

450

455 460

Gln

Lys Be Leu Be Leu Be Pro Gly Lys

465

470

5

<210> 8 <211> 474 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of variant of the human CD40 antibody CHIR-5.9 <400> 8 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu To Val Leu Gln Gly

one

5 10 fifteen

Val

Cys To Glu  Val Gln Leu  Val Gln Be Gly To Glu  Val Lys Lys

twenty

25 30

Pro

Gly Glu Be Leu Lys Ile Be Cys Lys Gly Be Gly Tyr Be Phe

35

40 Four. Five

Thr

Be Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

fifty

55 60

Glu

Trp Met Gly Ile Ile Tyr Pro Gly Asp Be Asp Thr Arg Tyr Be

65

70 75 80

Pro

Be Phe Gln Gly Gln Val Thr Ile Be To Asp Lys Be Ile Be

85 90

95

Thr

To Tyr Leu Gln Trp Be Be Leu Lys To Be Asp  Thr To Met

100

105 110

Tyr

Tyr

Cys To Arg Gly Thr To To Gly Arg Asp

Tyr

Tyr

Tyr

Tyr

115

120 125

Tyr

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Be Be

130

135 140

To

Be Thr Lys Gly Pro Be Val Phe Pro Leu  To Pro Be Be Lys

145

150 155 160

Be

Thr  Be Gly Gly Thr To To Leu Gly Cys Leu Val Lys Asp Tyr

165

170 175

Phe

Pro Glu Pro Val Thr Val Be Trp Asn Be Gly To Leu Thr Be

180

185 190

Gly

Val His Thr Phe Pro To Val Leu Gln Be Be  Gly Leu Tyr Be

195

200 205

Leu

Be Be Val Val Thr Val Pro Be Be Be  Leu Gly Thr Gln Thr

210

215 220

Tyr

Ile Cys Asn Val Asn His Lys Pro Be Asn Thr Lys Val Asp Lys

225

230 235 240

Arg

Val Glu Pro Lys Be Cys Asp Lys Thr His Thr Cys Pro Pro Cys

245

250 255

Pro

To  Pro Glu Leu Leu Gly Gly  Pro Be Val Phe Leu Phe

Pro

Pro

260

265 270

Lys

Pro  Lys Asp Thr Leu Met Ile Be Arg Thr Pro Glu Val Thr Cys

275

280 285

Val

Val

Val

Asp  Val Be His Glu Asp Pro Glu  Val Lys Phe Asn Trp

290

295 300

Tyr

Val Asp Gly Val Glu Val His Asn To Lys Thr Lys Pro Arg Glu

305

310 315 320

Glu

Gln Tyr Asn Be Thr Tyr Arg Val Val Be Val Leu Thr Val Leu

325

330 335

His

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Be Asn

340

3. 4. 5 350

Lys

To Leu Pro To Pro Ile Glu  Lys Thr Ile Be  Lys To  Lys Gly

355

360 365

Gln

Pro Arg Glu Pro  Gln Val Tyr Thr Leu Pro Pro Be Arg Glu Glu

370 375 380

Met

Thr Lys Asn Gln Val Be Leu Thr Cys Leu Val Lys Gly Phe Tyr

385

390 395 400

Pro

Be Asp Ile To Val Glu Trp Glu Be Asn Gly Gln  Pro Glu Asn

405

410 415

Asn

Tyr Lys Thr Thr Pro Pro Val Leu Asp Be Asp Gly Be Phe Phe

420

425 430

Leu

Tyr Be Lys  Leu Thr Val Asp Lys Be Arg Trp Gln Gln Gly Asn

435

440 445

Val

Phe Be Cys Be  Val Met His Glu To Leu His Asn His Tyr Thr

450

455 460

Gln

Lys Be Leu Be Leu Be Pro Gly Lys

465

470

5 10

<210> 9 <211> 612 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) ... (612) <221>misc feature <222> (0) ... (0) <223 > Coding sequence for the short isoform of human CD40 <400> 9 atg gtt cgt ctg cct ctg cag tgc gtc ctc tgg ggc tgc ttg ctg acc 48

Met

Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

one

5 10 fifteen

gct

gtc cat cca gaa cca ccc act gca tgc aga gaa aaa cag tac cta 96

To

Val His Pro Glu Pro Pro Thr  To Cys Arg Glu Lys Gln Tyr Leu

twenty

25 30

tie

aac agt cag tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg 144

Ile

Asn Be Gln Cys Cys Be Leu Cys Gln Pro Gly Gln Lys Leu Val

35

40 Four. Five

agt

gac tgc here gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa 192

Be

Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

fifty

55 60

agc

gaa ttc cta gac acc tgg aac aga gag here cac tgc cac cag cac 240

Be

Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65

70 75 80

aaa

tac tgc gac ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc 288

Lys

Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln  Lys Gly Thr

85 90

95

tca

gaa here gac acc atc tgc acc tgt gaa gaa ggc tgg cac tgt acg 336

Be

Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100

105 110

agt

gag gcc tgt gag agc tgt gtc ctg cac cgc tca tgc tcg ccc ggc 384

Be

Glu To Cys Glu  Be Cys Val Leu His Arg  Be Cys  Be Pro Gly

115

120 125

ttt

ggg gtc aag cag att gct here ggg gtt tct gat acc atc tgc gag 432

Phe

Gly Val Lys Gln Ile To Thr Gly Val Be Asp Thr Ile Cys Glu

130

135 140

ccc

tgc cca gtc ggc ttc ttc tcc aat gtg tca tct gct ttc gaa aaa 480

Pro

Cys  Pro Val Gly Phe Phe Be Asn Val Be Be To Phe Glu Lys

145

150 155 160

tgt

cac cct tgg here agg tcc cca gga tcg gct gag agc cct ggt ggt 528

Cys

His Pro Trp Thr Arg Be Pro Gly Be To Glu Be Pro Gly Gly

165

170  175

gat

ccc cat cat ctt cgg  gat cct gtt tgc cat cct ctt ggt gct ggt 576

Asp

Pro His His Leu Arg  Asp Pro Val Cys His Pro Leu Gly To Gly

180

185 190

ctt

tat falls aaa ggt ggc falls gaa gcc aac falls sooo 612

Leu

Tyr Gln Lys Gly Gly Gln Glu To Asn Gln *

195

200

<210> 10

<211> 203

<212> PRT

<213> Homo sapiens

5

<400> 10

Met

Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

one

5 10 fifteen

To

Val His Pro Glu Pro Pro Thr  To Cys Arg Glu Lys Gln Tyr Leu

twenty

25 30

Ile

Asn Be Gln Cys Cys Be Leu Cys Gln Pro Gly Gln Lys Leu Val

35

40 Four. Five

Be

Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

fifty

55 60

Be

Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65

70 75 80

Lys

Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln  Lys Gly Thr

85

90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100

105 110

Be

Glu To Cys Glu  Be Cys Val Leu His Arg  Be Cys  Be Pro Gly

115

120 125

Phe

Gly Val Lys Gln Ile To Thr Gly Val Be Asp Thr Ile Cys Glu

130

135 140

Pro

Cys  Pro Val Gly Phe Phe Be Asn Val Be Be To Phe Glu Lys

145

150 155 160

Cys

His Pro Trp Thr Arg Be Pro Gly Be To Glu Be Pro Gly Gly

165

170 175

Asp

Pro His His Leu Arg  Asp Pro Val Cys His Pro Leu Gly To Gly

180

185 190

Leu

Tyr Gln Lys Gly Gly Gln Glu To Asn Gln

195

200

5 10

<210> 11 <211> 834 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)… (834) <221> misc characteristic <222> (0)… (0) < 223> Coding sequence for the long isoform of human CD40 <400> 11 atg gtt cgt ctg cct ctg cag tgc gtc ctc tgg ggc tgc ttg ctg acc 48

Met

Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

one

5 10 fifteen

gct

gtc cat cca gaa cca ccc act gca tgc aga gaa aaa cag tac cta 96

To

Val His Pro Glu Pro Pro Thr  To Cys Arg Glu Lys Gln Tyr Leu

twenty

25 30

tie

aac agt cag tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg 144

Ile

Asn Be Gln Cys Cys Be Leu Cys Gln Pro Gly Gln Lys Leu Val

35

40 Four. Five

agt

gac tgc here gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa 192

Be

Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

fifty

55 60

agc

gaa ttc cta gac acc tgg aac aga gag here cac tgc cac cag cac 240

Be

Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65

70 75 80

aaa

tac tgc gac ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc 288

Lys

Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln  Lys Gly Thr

85

90  95

tca

gaa here gac acc atc tgc acc tgt gaa gaa ggc tgg cac tgt acg 336

Be

Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100

105 1l0

agt

gag gcc tgt gag agc tgt gtc ctg cac cgc tca tgc tcg ccc ggc 384

Be

Glu To Cys Glu  Be Cys Val Leu His Arg  Be Cys  Be Pro Gly

115

120 125

ttt

ggg gtc aag cag att gct here ggg gtt tct gat acc atc tgc gag 432

Phe

Gly Val Lys Gln Ile To Thr Gly Val Be Asp Thr Ile Cys Glu

130

135 140

ccc

tgc cca gtc ggc ttc ttc tcc aat gtg tca tct gct ttc gaa aaa 480

Pro

Cys  Pro Val Gly Phe Phe Be Asn Val Be Be To Phe Glu Lys

145

150 155 160

tgt

cac cct tgg here agc  tgt gag acc aaa gac ctg gtt gtg falls cag 528

Cys

His Pro Trp Thr Be  Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165

170  175

gca

ggc here aac aag act gat gtt gtc tgt ggt ccc cag gat cgg ctg 576

To

Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180

185 190

aga

gcc ctg gtg gtg atc ccc atc atc ttc ggg atc ctg ttt gcc atc 624

Arg

To Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe To Ile

195

200 205

ctc

ttg gtg ctg gtc ttt atc aaa aag gtg gcc aag aag cca acc aat 672

Leu

Leu

Val  Leu Val Phe Ile Lys Lys Val To Lys Lys Pro Thr Asn

210

215 220

aag

gcc ccc cac ccc  aag cag gaa ccc cag gag atc aat ttt ccc gac 720

Lys

To Pro His Pro  Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225

230 235 240

gat

ctt cct ggc tcc aac act gct gct cca gtg cag gag act tta cat 768

Asp

Leu Pro Gly Be Asn Thr To To Pro Val Gln Glu Thr Leu His

245

250  255

gga

tgc falls ccg gtc acc cag gag gat ggc aaa gag agt cgc atc tca 816

Gly

Cys Gln Pro Val Thr Gln Glu Asp  Gly Lys Glu Be Arg Ile Be

260

265 270

gtg

cag gag aga cag tga 834

Val

Gln Glu Arg Gln *

275

<210> 12

<211> 277

<212> PRT

<213> Homo sapiens

<400> 12

Met

Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

one

5 10 fifteen

To

Val His Pro Glu Pro Pro Thr  To Cys Arg Glu Lys Gln Tyr Leu

twenty

25 30

Ile

Asn Be Gln Cys Cys Be Leu Cys Gln Pro Gly Gln Lys Leu Val

35

40 Four. Five

Be

Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

fifty

55 60

Be

Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65

70 75 80

Lys

Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln  Lys Gly Thr

85

90 95

Be

Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100

105 110

Be

Glu To Cys Glu  Be Cys Val Leu His Arg  Be Cys  Be Pro Gly

115

120 125

Phe

Gly Val Lys Gln Ile To Thr Gly Val Be Asp Thr Ile Cys Glu

130

135 140

Pro

Cys  Pro Val Gly Phe Phe Be Asn Val Be Be To Phe Glu Lys

145

150 155 160

Cys

His Pro Trp Thr Be  Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165

170 175

To

Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180

185 190

Arg

To Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe To Ile

195

200 205

Leu

Leu

Val  Leu Val Phe Ile Lys Lys Val To Lys Lys Pro Thr Asn

210

215 220

Lys

To Pro His Pro  Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225

230 235 240

Asp

Leu Pro Gly Be Asn Thr To To Pro Val Gln Glu Thr Leu His

245

250 255

Gly

Cys Gln Pro Val Thr Gln Glu Asp  Gly Lys Glu Be Arg Ile Be

260

265 270

Val

Gln Glu Arg Gln

275

Claims (21)

one.

An anti-CD40 antagonist antibody or an antigen-binding fragment thereof for use in a method of treating a human subject of a cancer characterized by a neoplastic B-cell growth by combining antibody therapy with an anti-CD20 antibody or a fragment of

5

antigen-binding same, wherein said anti-CD40 antagonistic antibody or said fragment thereof of

antigen binding are selected from the group consisting of:

(a) an antibody or an antigen binding fragment thereof that binds with an epitope capable of binding

with the monoclonal antibody CHIR-5.9, which can be obtained from the deposited hybridoma cell line

in the ATCC as Patent Deposit No. PTA-5542, or with the monoclonal antibody CHIR-12.12, which

can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No.

PTA-5543;

(b) an antibody or an antigen binding fragment thereof that binds to an epitope comprising

residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12;

(c) an antibody or an antigen binding fragment thereof that binds to an epitope comprising

fifteen

residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12; Y

(d) an antibody or an antigen binding fragment thereof that competes with the monoclonal antibody

CHIR-5.9, which can be obtained from the hybridoma cell line deposited with the ATCC as a Deposit of

Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12, which can be obtained from the cell line of

hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a binding assay

competitive

2.

The use of an effective amount of an anti-CD40 antagonist antibody or a fragment thereof

antigen in the manufacture of a medicine to be used to treat a human subject of cancer

characterized by a neoplastic growth of B cells by combination antibody therapy with an anti CD20 antibody or a fragment thereof antigen binding. wherein said antibody

25

anti CD40 antagonist or said antigen binding fragment thereof are selected from the group

Constituted by:

(a) an antibody or an antigen binding fragment thereof that binds with an epitope capable of binding

with the monoclonal antibody CHIR-5.9, which can be obtained from the deposited hybridoma cell line

in the ATCC as Patent Deposit No. PTA-5542 or with the monoclonal antibody CHIR-12.12, which

can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No.

PTA-5543;

(b) an antibody or an antigen binding fragment thereof that binds to an epitope comprising

residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12;

(c) an antibody or an antigen binding fragment thereof that binds to an epitope comprising

35

residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12; Y

(d) an antibody or an antigen binding fragment thereof that competes with the monoclonal antibody

CHIR-5.9, which can be obtained from the hybridoma cell line deposited with the ATCC as a Deposit of

Patent No. PTA-5542 or the monoclonal antibody CHIR-12.12, which can be obtained from the cell line of

hybridoma deposited in the ATCC as Patent Deposit No. PTA-5543 in a binding assay

competitive

3.

An anti CD40 antagonist antibody or an antigen binding fragment thereof for use in a

procedure to inhibit the growth of a tumor comprising neoplastic B cells by therapy

of combining antibodies with an anti CD20 antibody or an antigen binding fragment thereof,

wherein said anti CD40 antagonist antibody or said antigen binding fragment thereof are

Four. Five

selected from the group consisting of:

(a) an antibody or an antigen binding fragment thereof that binds with an epitope capable of binding

with the monoclonal antibody CHIR-5.9, which can be obtained from the deposited hybridoma cell line

in the ATCC as Patent Deposit No. PTA-5542 or with the monoclonal antibody CHIR-12.12, which

can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No.

PTA-5543;

(b) an antibody or an antigen binding fragment thereof that binds to an epitope comprising

residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12;

(c) an antibody or an antigen binding fragment thereof that binds to an epitope comprising

residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12; Y

(d) an antibody or an antigen-binding fragment thereof that competes with the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the CHIR monoclonal antibody -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. 4. The use of an effective amount of an anti-CD40 antagonist antibody or an antigen binding fragment thereof in the manufacture of a medicament to be used to inhibit the growth of a tumor comprising neoplastic B cells by antibody combination therapy. with an anti-CD20 antibody or a fragment thereof to the antigen in which said anti-CD40 antagonistic antibody or said fragment thereof to the antigen are selected from the group consisting of:

(to)

an antibody or an antigen-binding fragment thereof that binds with an epitope capable of binding with the monoclonal antibody CHIR-5.9, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or with the monoclonal antibody CHIR-12.12, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543;

(b)

an antibody or an antigen binding fragment thereof that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12;

(C)

an antibody or an antigen binding fragment thereof that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12; Y

(d)

an antibody or an antigen-binding fragment thereof competing with the monoclonal antibody CHIR-5.9, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay.

5. An in vitro method for inhibiting the growth of a tumor comprising neoplastic B cells, comprising contacting said cells with an effective amount of an anti-CD40 antagonistic antibody or an antigen binding fragment thereof in combination with an anti CD20 antibody or an antigen binding fragment thereof, wherein said antibody or said antigen binding fragment thereof is selected from the group consisting of:

(to)

an antibody or an antigen-binding fragment thereof that binds with an epitope capable of binding with the monoclonal antibody CHIR-5.9, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or with the monoclonal antibody CHIR-12.12, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543;

(b)

an antibody or an antigen binding fragment thereof that binds to an epitope comprising residues 82-87 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12;

(C)

an antibody or an antigen binding fragment thereof that binds to an epitope comprising residues 82-89 of the human CD40 sequence shown in SEQ ID No. 10 or in SEQ ID No. 12; Y

(d)

an antibody or an antigen-binding fragment thereof competing with the monoclonal antibody CHIR-5.9, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543 in a competitive binding assay.

6.

The antibody, the antigen-binding fragment, the use or the method of any of claims 1-5 wherein said anti-CD40 antagonistic antibody is a human antibody.

7.

The antibody, the antigen binding fragment, the use or the method of any of claims 1-6 wherein said antibody or said antigen binding fragment thereof is selected from the group consisting of:

(i)

an antibody or an antigen-binding fragment thereof comprising a light chain variable domain containing residues of the complementarity determining region (CDR) of SEQ ID No. 2 or SEQ ID No. 6;

(ii)

an antibody or antigen binding fragment thereof comprising a heavy chain variable domain containing residues of the complementarity determining region (CDR) of SEQ ID No. 4 or SEQ ID No. 7;

(iii) an antibody or an antigen binding fragment thereof comprising a light chain variable domain containing the hypervariable loop residues of SEQ ID No. 2 or SEQ ID No. 6; Y (iv) an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain containing the hypervariable loop residues of SEQ ID No. 4 or SEQ ID No. 7.

8.

The antibody, antigen binding fragment, use or method of claim 7 wherein said antibody or said antigen binding fragment thereof 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)

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

(xii) SEQ ID No. 2 and SEQ ID No. 4; Y (xiii) SEQ ID No. 2 and SEQ ID No. 5.

9.

The antibody, antigen binding fragment, use or method of claim 7 wherein said antibody or said antigen binding fragment thereof 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)

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

(x)

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

(xi)

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

(xii) SEQ ID No. 6 and SEQ ID No. 7; Y (xiii) SEQ ID No. 6 and SEQ ID No. 8.

10.

The antibody, the antigen binding fragment, the use or the method of any preceding claim wherein said antibody is the CHIR-5.9 monoclonal antibody, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5542 or the monoclonal antibody CHIR-12.12, which can be obtained from the hybridoma cell line deposited in the ATCC as Patent Deposit No. PTA-5543.

eleven.

The antibody, the antigen-binding fragment, the method or the use of any preceding claim wherein said antigen-binding fragment of said anti-CD40 antibody or said anti-CD20 antibody are selected from the group consisting of a Fab fragment, a fragment F (ab ') 2, an Fv fragment and a single chain Fv fragment.

12.

The antibody, antigen binding fragment, use or method of any preceding claim wherein said anti CD40 antibody or said antigen binding fragment thereof binds to human CD40 antigen with an affinity (KD) between 10- 6 M and 10-7 M.

13.

The antibody, the antigen binding fragment, the use or the method of claim 12 wherein said anti CD40 antibody or said antigen binding fragment thereof binds with the human CD40 antigen with an affinity (KD) between 10 -8 M and 10-12 M.

14.

The antibody, the antigen-binding fragment, the use or the method of any preceding claim wherein said anti-CD40 antibody or said antigen-binding fragment thereof is produced in a CHO cell line.

fifteen.

The antibody, the antigen-binding fragment, the method or use of any preceding claim wherein said antibody combination therapy provides a synergistic therapeutic effect.

or in which the development of said tumor is synergistically inhibited.

16.

The antibody, the antigen-binding fragment, the method or use of any preceding claim wherein said anti-CD20 antibody is selected from the group consisting of a human anti-CD20 antibody, a murine anti-CD20 antibody, a chimeric anti-CD20 antibody and a humanized anti CD20 antibody.

17.

The antibody, the antigen binding fragment, the method or the use of any preceding claim wherein said anti CD20 antibody is IDEC-C2B8 or an anti CD20 antibody having the binding characteristics of IDEC-C2B8.

18.

The antibody, the antigen-binding fragment, the method or the use of any preceding claim wherein the cancer is selected from the group consisting of non-Hodgkin lymphoma, chronic lymphocytic leukemia, multiple myeloma, B cell lymphoma, B cell lymphoma high-grade, intermediate-grade B-cell lymphoma, low-grade B-cell lymphoma, acute lymphoblastic B-cell leukemia, myeloblastic leukemia, Hodgkin's disease, plasmacytoma, follicular lymphoma, follicular follicular small cell lymphoma, follicular cell lymphoma large, mixed follicular small cell lymphoma, diffuse small cell diffuse lymphoma, diffuse small cell lymphocytic lymphoma, prolymphocytic leukemia, lymphoplasmacytic lymphoma, marginal area lymphoma, lymphoid tissue lymphoma associated with mucous, monocytoid B cell lymphoma, lymphoma splenic, hairy cell leukemia, diffuse lymphoma d and large cells, mediastinal large B cell lymphoma, lymphomatoid granulomatosis, intravascular lymphomatosis, diffuse mixed cell lymphoma, diffuse large cell lymphoma, immunoblastic lymphoma, Burkitt lymphoma, AIDS-related lymphoma and mantle cell lymphoma.

19.

The antibody, the antigen binding fragment, the method or use of any preceding claim wherein said cancer is refractory to treatment with said anti CD20 antibody or said antigen binding fragment thereof.

twenty.

The antibody, the antigen binding fragment, the method or use of any preceding claim wherein said anti CD20 antibody or said antigen binding fragment thereof and said anti CD40 antagonist antibody or said antigen binding fragment thereof They are administered sequentially.

twenty-one.

The antibody, the antigen-binding fragment, the method or use of any preceding claim wherein said anti-CD20 antibody or said antigen-binding fragment thereof and said anti-CD40 antagonistic antibody or said antigen-binding fragment thereof They are administered simultaneously.