HK1178176B - Anti-cd40 antibodies - Google Patents

Description

anti-CD 40 antibodies

Technical Field

In general, the invention relates to humanized anti-CD 40 antibodies for diagnostic and therapeutic applications. More specifically, the invention discloses humanized anti-CD 40 antibodies and methods for treating various diseases or disorders characterized by cells expressing CD 40. Pharmaceutical compositions and kits comprising the humanized anti-CD 40 antibodies are also disclosed.

Background

CD40 is a 48 kDaI-type membrane intrinsic glycoprotein and is a member of the Tumor Necrosis Factor (TNF) receptor superfamily. CD40 is expressed on various types of cells, including normal and neoplastic B cells, interlaced cells, carcinoma cells, epithelial cells (e.g., keratinocytes), fibroblasts (e.g., synoviocytes), and platelets. CD40 is also present on monocytes, macrophages, some endothelial cells and follicular dendritic cells. CD40 is expressed early in B cell ontogeny, after the emergence of CD10 and CD19, and before the expression of CD21, CD23, CD24 and the emergence of surface immunoglobulin m (igm) on B cell precursors (Uckun et al, 1990, Blood15: 2449). CD40 has also been detected on tonsils and bone marrow-derived plasma cells (Pellat-Decounyck et al, 1994, Blood84: 2597).

The ligand for CD40 is CD40L (also known as CD154, gp39, and TRAP), which is a member of the TNF superfamily. CD40L is mainly in activated CD4⁺Transmembrane proteins expressed on T cells and a small subset of CD8+ T cells (van kootenc. and Banchereau, 2000).

The interaction of CD40 with CD40L induces both humoral and cell-mediated immune responses. CD40 modulates this ligand-receptor pair to activate B cells and other Antigen Presenting Cells (APC), including Dendritic Cells (DC) (reviewed by Toubi and Shoenfeld, 2004; Kiener et al, 1995). The function of CD40 on B cells has been extensively studied in the industry. In the germinal center of secondary lymphoid organs, activation of B cells by CD40 induces proliferation, differentiation into antibody secreting cells and isotype switching. In vitro studies have shown that CD40 activation has a direct effect on the production of cytokines (IL-6, IL-10, TNF- α, LT α), the expression of adhesion molecules and co-stimulatory receptors (ICAM, CD23, CD80 and CD86), and the increase in B-lymphocyte expression of MHC class I, MHC class II and TAP transporters (Liu et al, 1996). In most of these processes, CD40 interacts synergistically with cytokines or other receptor-ligand interactions.

The signaling of CD40 on monocytes and DCs results in prolonged survival and secretion of cytokines (IL-1, IL-6, IL-8, IL-10, IL-12, TNF- α and MIP-1 α). The ligand binding of CD40 on the APC also leads to upregulation of co-stimulatory molecules such as ICAM-1, LFA-3, CD80 and CD 86. Activation of the CD40 receptor is a key signal that allows DCs to fully mature into potent APCs, driving T cell activation (Banchereau and Steinman, 1998) (van kootenc, and Banchereau, 2000).

Recent studies in mouse models have shown that CD40 signaling on dendritic cells also plays an important role in TH17 cell production, and TH17 cells are considered as autoimmune mediators in diseases such as arthritis and multiple sclerosis (Iezzi et al, 2009) (Perona-Wright et al, 2009).

The availability of CD40 and CD40L knockout mice, as well as agonistic and antagonistic anti-mouse antibodies, allows the study of the CD40-CD40L interaction in several disease models. Administration of blocking anti-CD 40L has been shown to be beneficial in several autoimmune models, including idiopathic disease (e.g., lupus nephritis in SNF1 mice or diabetes in NOD mice) or experimentally induced forms of disease (e.g., collagen-induced arthritis (CIA) or Experimental Autoimmune Encephalomyelitis (EAE)) (Toubi and Shoenfeld, 2004). anti-CD 40LmAb inhibits CIA in mice, which blocks the development of joint inflammation, serum antibody titers against collagen, infiltration of inflammatory cells into the infrasynovial tissue, and erosion of cartilage and bone (dure et al, 1993). For both lupus nephritis and EAE, anti-CD 40L has also been shown to alleviate progressive disease, confirming the role of CD40-CD40L in the effector phase of the disease (Kalled et al, 1998); (Howard et al, 1999).

The role of CD40-CD40L interaction in EAE development was also studied in CD 40L-deficient mice carrying transgenic T cell receptors specific for myelin basic protein. The mice did not develop EAE after antigen priming, and CD4+ T cells remained dormant and did not produce INF- γ (Grewal et al, 1996).

In addition, inhibitory antibodies against CD40 show beneficial effects in models of inflammatory diseases such as EAE. Lamann and co-workers demonstrated that antagonistic mice against human CD40mAbmu5D12 and chimeric forms of this mAb were effective in preventing clinical expression of chronic demyelinating EAE in inbred marmosets (Laman et al, 2002); (Boon et al, 2001). Follow-up studies showed that therapeutic treatment with the chimeric anti-human CD40 antibody reduced MRI detectable inflammation and delayed the expansion of pre-existing brain lesions in the marmoset EAE model (Hart et al, 2005).

anti-CD 40 antibodies with agonistic activity were tested in a mouse model of arthritis and some contradictory results were obtained. As expected for immunostimulants, agonistic anti-mouse CD40mAbFGK45 was shown to exacerbate disease in the DBA/1 mouse model of CIA (Tellander et al, 2000). However, in another chronic CIA model FGK45, as well as in another agonistic anti-mouse CD40mAb (3/23), both showed positive therapeutic effects (Mauri et al, 2000). This treatment group postulated that agonistic antibodies in this therapeutic treatment regimen produce beneficial effects by inducing an immune bias towards a Th2 response and decreasing IFN- γ levels and increasing IL-4 and IL-10 levels (Mauri et al, 2000).

It has also been recorded that graft rejection can be prevented by blocking the CD40/CD154 interaction. The use of the chimeric anti-CD 40 antagonist ch5D12 in rhesus monkey kidney xenograft studies showed that antagonism of CD40 was sufficient to achieve disease remission and extend mean survival beyond 100 days. When ch5D12 was combined with an anti-CD 86 antibody and administered only at the beginning of the allograft study, followed by extended treatment with cyclosporine, a mean survival time of more than 4 years was obtained, indicating that this combination could effectively induce tolerance (Haanstra et al, 2005).

Thus, there are numerous preclinical studies demonstrating that the CD40-CD40L diad has a crucial role in driving a potent T cell-dependent immune response. Thus, blocking CD40 signaling is recognized as a suitable and necessary therapeutic strategy to suppress pathogenic autoimmune responses in diseases such as RA, multiple sclerosis, or psoriasis. However, to date, no CD40 antibody has been approved for therapeutic intervention for such disorders, as anti-CD 40 antibodies have previously been shown in development to have significant side effects. Thus, there remains a great need for therapeutic agents that can intervene in the role of CD40-CD40L and block CD40 signaling. This need may be met by a novel humanized anti-CD 40 antibody that specifically binds CD40 and which exhibits antigen binding specificity, affinity, and pharmacokinetic and pharmacodynamic properties that make it useful for therapeutic intervention in CD 40-based disorders.

Summary of The Invention

The present invention provides a humanized monoclonal antibody, wherein the antibody specifically binds human CD40, has an antagonistic activity in B cell proliferation with an IC50 of less than 1nM and no agonism at concentrations up to 100 μ g/ml, and wherein the antibody is further characterized by an in vivo half-life of at least 10 days in a non-human primate.

The humanized monoclonal antibody is further characterized by a half-life of the antibody in cynomolgus monkeys (cynomolgus monkey) of greater than 8 days at a dose of less than 30 mg/kg.

In exemplary embodiments, the antibodies of the invention comprise a heavy chain sequence selected from any one of seq id No. 1 to seq id No. 4 and a light chain sequence selected from any one of seq id No. 5 to seq id No. 8.

In other embodiments, the antibody is a humanized antibody or an antigen-binding fragment of an antibody having a heavy chain variable region amino acid sequence of any one of: SEQ ID NO. 1 to 4, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, or SEQ ID NO. 73.

In other embodiments, the antibody is a humanized antibody or antigen-binding fragment of an antibody comprising the following light chain variable domain amino acid sequences: SEQ ID NO. 5 to SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 74, SEQ ID NO. 75, or SEQ ID NO. 76.

In particular embodiments, the monoclonal antibodies described herein are characterized in that they comprise a heavy chain and a light chain, wherein the heavy chain CDR1 sequence is selected from seq id No. 9 to seq id No. 11, the heavy chain CDR2 sequence is selected from seq id No. 12 to seq id No. 15, and the heavy chain CDR3 sequence is selected from seq id No. 16 to seq id No. 17; and wherein the light chain CDR1 sequence is selected from SEQ ID NO:18 to SEQ ID NO:21, the light chain CDR2 sequence is SEQ ID NO:22 to SEQ ID NO:23, and the light chain CDR3 sequence is selected from SEQ ID NO:24 to SEQ ID NO: 25.

In particular embodiments, the monoclonal antibody described herein is characterized in that it comprises the heavy chain CDR1 sequence seq id No. 10, the heavy chain CDR2 sequence seq id No. 13 and the heavy chain CDR3 sequence seq id No. 16, and wherein the antibody comprises the light chain CDR1 sequence seq id No. 19, the light chain CDR2 sequence seq id No. 22 and the light chain CDR3 sequence seq id No. 24.

In other embodiments, the monoclonal antibody described herein is characterized in that it comprises the heavy chain CDR1 sequence seq id No. 9, the heavy chain CDR2 sequence seq id No. 14 and the heavy chain CDR3 sequence seq id No. 16, and wherein the antibody comprises the light chain CDR1 sequence seq id No. 20, the light chain CDR2 sequence seq id No. 22 and the light chain CDR3 sequence seq id No. 24.

In another embodiment, the monoclonal antibody described herein is characterized in that it comprises the heavy chain CDR1 sequence seq id No. 9, the heavy chain CDR2 sequence seq id No. 14 and the heavy chain CDR3 sequence seq id No. 16, and wherein the antibody comprises the light chain CDR1 sequence seq id No. 20, the light chain CDR2 sequence seq id No. 22 and the light chain CDR3 sequence seq id No. 24.

In another embodiment, the monoclonal antibody described herein is characterized in that it comprises the heavy chain CDR1 sequence seq id No. 11, the heavy chain CDR2 sequence seq id No. 15 and the heavy chain CDR3 sequence seq id No. 17, and wherein the antibody comprises the light chain CDR1 sequence seq id No. 21, the light chain CDR2 sequence seq id No. 23 and the light chain CDR3 sequence seq id No. 25.

The individual sequences of the heavy chains in preferred antibodies of the invention are also described herein. For example, the invention relates to an anti-CD 40 antibody comprising a heavy chain variable domain sequence of any one of SEQ ID NOs: 1 to 4. The anti-CD 40 antibody is further characterized in that it comprises the light chain variable domain sequence of any one of SEQ ID NO. 5 to SEQ ID NO. 8.

Also included are humanized antibodies or antibody fragments having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31; SEQ ID NO. 37 and SEQ ID NO. 36; SEQ ID NO. 38 and SEQ ID NO. 36; SEQ ID NO:39 and SEQ ID NO: 36; SEQ ID NO. 40 and SEQ ID NO. 36.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 28 and SEQ ID NO. 26.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 29 and SEQ ID NO. 26.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 30 and SEQ ID NO. 26.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 32 and SEQ ID NO. 31.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 33 and SEQ ID NO. 31.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 34 and SEQ ID NO. 31.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 35 and SEQ ID NO. 31.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 37 and SEQ ID NO. 36.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 38 and SEQ ID NO. 36.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 39 and SEQ ID NO. 36.

In another embodiment, the invention relates to a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 40 and SEQ ID NO. 36.

Another embodiment relates to an isolated antibody or antigen-binding fragment that specifically binds human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence at least 90% identical to the amino acid sequence of the framework region in the human variable domain heavy chain amino acid sequence of seq id No. 27, seq id No. 28, seq id No. 29, or seq id No. 30; and comprises a light chain amino acid sequence at least 90% identical to the corresponding light chain variable domain of SEQ ID NO. 26.

Another embodiment relates to an isolated antibody or antigen-binding fragment that specifically binds human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence at least 90% identical to the amino acid sequence of the framework region in the human variable domain heavy chain amino acid sequence of seq id No. 32, seq id No. 33, seq id No. 34, or seq id No. 35; and comprises a light chain amino acid sequence at least 90% identical to the corresponding light chain variable domain of SEQ ID NO. 31.

In another aspect, the invention relates to the isolated antibody or antigen binding fragment of the embodiments of the previous paragraph, wherein the heavy chain amino acid sequence is seq id No. 32; in another embodiment the heavy chain amino acid sequence is SEQ ID NO. 33; in another embodiment the heavy chain amino acid sequence is SEQ ID NO: 34; in another embodiment the heavy chain amino acid sequence is SEQ ID NO 35.

Also included are isolated antibodies or antigen-binding fragments that specifically bind to human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence at least 90% identical to the amino acid sequence of the framework region in the human variable domain heavy chain amino acid sequence of seq id No. 37, seq id No. 38, seq id No. 39, or seq id No. 40; and comprises a light chain amino acid sequence at least 90% identical to the corresponding light chain of SEQ ID NO. 36.

In another aspect, the invention relates to the isolated antibody or antigen binding fragment of the embodiments of the previous paragraph, wherein the heavy chain amino acid sequence is seq id No. 37; in another embodiment the heavy chain amino acid sequence is SEQ ID NO 38; in another embodiment the heavy chain amino acid sequence is SEQ ID NO: 39; in another embodiment the heavy chain amino acid sequence is SEQ ID NO: 40.

The antibodies of the invention are further characterized by the inability of the antibodies to stimulate cytokine production by B cells in the absence of CD 40L.

The antibody of the present invention is also characterized in that the antibody binds to human CD40 in the presence of 50% human serum and the binding rate (ontate) is reduced by less than two-fold.

The antibody of the present invention is also characterized in that the antibody inhibits IgM and IgG production in a mammal at a concentration of 1 mg/kg.

The antibodies of the invention can be used in a variety of therapeutic, prophylactic, diagnostic and other methods. For example, the invention describes a method of blocking the function of human CD40 in a mammal comprising administering to the mammal a composition comprising an antibody of the invention in an amount sufficient to block a CD 40-mediated immune response in the mammal.

Also included herein is a method of treating or ameliorating graft-versus-host disease in a mammal comprising administering to the mammal a composition comprising an antibody of the invention in an amount sufficient to reduce one or more symptoms of graft-versus-host disease in the animal.

For example, autoimmune or inflammatory diseases may include, but are not limited to, rheumatoid arthritis, multiple sclerosis, proliferative lupus glomerulonephritis, Inflammatory Bowel Disease (IBD), psoriasis, Idiopathic Thrombocytopenic Purpura (ITP), Crohn's disease, Systemic Lupus Erythematosus (SLE), Hashimoto's thyroiditis, primary myxoedema, thyrotoxicosis/graves disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune myocarditis, Addison's disease, menopause, type I diabetes, goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, chronic hepatitis (HBsAg negative), crypto-cirrhosis, Sjogren's syndrome, Crohn's disease, idiopathic thrombocytopenia, rheumatoid arthritis, dermatomyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus, and systemic vasculitis. In an exemplary embodiment, the mammal has rheumatoid arthritis.

The methods of the invention may further comprise administering a second therapeutic agent selected from the group consisting of: TNF antagonists, disease modifying antirheumatics, CTLA4 antagonists, anti-IL-6 receptor mAb and anti-CD 20 mAb.

In particular embodiments, the inflammatory disease or autoimmune disease is an inflammatory disease or autoimmune disease associated with cells expressing both CD40 and CD 20.

In a particular embodiment, the treatment involves administration of the antibody composition by a parenteral route of administration.

In particular embodiments, the treatment involves administering the antibody composition intravenously or subcutaneously.

Other methods of the invention include inhibiting B cell production of antibodies in a human patient comprising administering to the human patient an effective amount of an anti-CD 40 antibody of the invention.

More specifically, human patients suffer from inflammatory or autoimmune diseases associated with cells expressing CD 40.

In an exemplary embodiment, the human patient has an autoimmune disease selected from the group consisting of an autoimmune or inflammatory disease selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, proliferative lupus glomerulonephritis, Inflammatory Bowel Disease (IBD), psoriasis, Idiopathic Thrombocytopenic Purpura (ITP), crohn's disease and Systemic Lupus Erythematosus (SLE), hashimoto's thyroiditis, primary mucoedema, thyrotoxicosis/graves 'disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune myocarditis, addison's disease, premature menopause, type I diabetes, goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, chronic active hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus and systemic vasculitis.

Another method of the invention relates to inhibiting the growth of a cell expressing human CD40 antigen comprising administering to the cell an antibody or antigen-binding fragment of the invention that specifically binds to human cell surface CD40 antigen, wherein binding of the antibody or antigen-binding fragment to the CD40 antigen inhibits the growth or differentiation of the cell.

Also included is a method of treating a subject having a CD 40-associated disorder, comprising administering to the subject an antibody or antigen-binding fragment of the invention that specifically binds to human CD40, wherein binding of the antibody or antigen-binding fragment to CD40 inhibits growth or differentiation of cells of a CD 40-associated disorder. The cell can be, but is not limited to, a B lymphoid stem cell (lymphoblastoid cells), a pancreatic cell, a lung cell, a breast cell, an ovarian cell, a colon cell, a prostate cell, a skin cell, a head and neck cell, a bladder cell, a bone cell, or a kidney cell.

Therapeutic methods that inhibit cell growth or differentiation may be used to treat chronic lymphocytic leukemia, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, Non-Hodgkin's lymphoma, Hodgkin's disease, Waldenstrom's macroglobulinemia, or Kaposi's sarcoma.

Also included is a method of inducing clearance of peripheral B cells comprising administering to said cells an antibody or antigen-binding fragment of the invention that specifically binds to a human cell surface CD40 antigen, wherein binding of the antibody or antigen-binding fragment to the CD40 antigen induces clearance of said cells.

In particular embodiments, the antibody or antigen-binding fragment is administered to an individual having an immune disorder. For example, the immune disorder is rheumatoid arthritis or systemic lupus erythematosus.

Also included is a method of treating rheumatoid arthritis in an individual comprising administering to the individual an antibody of the invention, wherein the antibody is an antagonist antibody that blocks CD40 function in the individual.

Preferably, the antibody is administered in an amount effective to inhibit B cell differentiation and antibody isotype switching in the individual.

In other embodiments, the antibody is administered in an amount effective to inhibit the production of cytokines and chemokines and the upregulation of adhesion molecules in the T cells and macrophages of the individual. Preferably, the individual is administered in an amount effective to inhibit dendritic cell activation in the individual.

In other embodiments, the method is further characterized by administering the antibody in an amount effective to inhibit the down-regulation of pro-inflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandin production, and adhesion molecules in non-immune cells of the individual.

In particular embodiments, the antibody is administered in combination with a regimen comprising the administration of methotrexate and/or the administration of Enbrel/Humira.

The individuals receiving this therapy were those with rheumatoid arthritis who were non-responders to methotrexate treatment alone.

In particular embodiments, the method comprises treating the subject with a regimen comprising administration of methotrexate and/or administration of Enbrel/Humira.

The methods of the invention are further characterized by the efficacy of treating the subject with the antagonistic anti-CD 40 antibody is superior to treatment with methotrexate alone, Enbrel alone, and a combination of Enbrel + methotrexate.

The methods of the invention are further characterized by efficacy of treatment of the subject with the antagonistic anti-CD 40 antibody in patients who do not respond adequately to methotrexate being superior to treatment with Enbrel + MTX.

In particular embodiments, the antibody is administered in combination with a regimen comprising an anti-TNF agent.

In particular embodiments, an individual may be described as a responder to treatment with rheumatoid arthritis and an anti-TNF drug alone. In such embodiments, the method may comprise treating the subject with a regimen comprising treatment with an anti-TNF drug and the antagonistic anti-CD 40 antibody.

In particular embodiments, the efficacy of treatment of the subject with the antagonist anti-CD 40 antibody is superior to treatment with an anti-TNF drug.

In other embodiments, the method is characterized by efficacy of treatment of the subject with the antagonist anti-CD 40 antibody superior to treatment with Orencia or Rituxan in patients who do not respond adequately to anti-TNF drugs alone.

The present invention also includes a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment as described herein; and (ii) a pharmaceutically acceptable excipient. In the compositions, the antibody or antigen-binding fragment thereof can be advantageously conjugated to a second drug (e.g., a cytotoxic agent, a PEG carrier, an enzyme, or a label).

Also included herein are isolated polynucleotides encoding a heavy chain variable region amino acid sequence of any one of: SEQ ID NO. 1 to 4, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, or SEQ ID NO. 73.

Also included herein are isolated polynucleotides encoding a light chain variable region amino acid sequence of any one of: SEQ ID NO. 5 to SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 74, SEQ ID NO. 75, or SEQ ID NO. 76.

The invention further relates to the use of an antibody as described herein for the manufacture of a medicament for blocking human CD40 function in a mammal, wherein the medicament blocks a CD 40-mediated immune response in the mammal.

In one embodiment, the invention relates to the manufacture of a medicament for treating or ameliorating graft-versus-host disease in a mammal.

In an exemplary embodiment, the medicament is prepared for treating an autoimmune or inflammatory disease selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, proliferative lupus glomerulonephritis, Inflammatory Bowel Disease (IBD), psoriasis, Idiopathic Thrombocytopenic Purpura (ITP), crohn's disease and Systemic Lupus Erythematosus (SLE), hashimoto's thyroiditis, primary mucoedema, thyrotoxicosis/graves 'disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune myocarditis, addison's disease, premature menopause, type I diabetes, goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, chronic active hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus and systemic vasculitis.

In some embodiments, the medicament may further comprise a second therapeutic agent selected from the group consisting of: TNF antagonists, disease modifying antirheumatics, CTLA4 antagonists, anti-IL-6 receptor mAb and anti-CD 20 mAb.

The medicament may be prepared for use in parenteral routes of administration. The medicament may be prepared for intravenous or subcutaneous use.

Another embodiment encompasses the use of an antibody described herein in the manufacture of a medicament for inhibiting B cell production of an antibody in a human patient.

Another embodiment encompasses the use of an antibody described herein for the preparation of a medicament for inhibiting the growth and/or differentiation of cells expressing the human CD40 antigen.

Another embodiment encompasses the use of an antibody as described herein in the manufacture of a medicament for treating an individual having a CD 40-associated disorder, wherein binding of the antibody or antigen-binding fragment to CD40 in the medicament inhibits growth or differentiation of cells of the CD 40-associated disorder.

The medicament may be prepared for use in treating a CD 40-associated disorder selected from the group consisting of: b lymphoid stem cells, pancreatic cells, lung cells, breast cells, ovarian cells, colon cells, prostate cells, skin cells, head and neck cells, bladder cells, bone cells, or kidney cells.

The medicament may be prepared for use in the treatment of chronic lymphocytic leukemia, burkitt's lymphoma, multiple myeloma, T-cell lymphoma, non-hodgkin's lymphoma, hodgkin's disease, waldenstrom's macroglobulinemia or kaposi's sarcoma.

Another embodiment includes the use of an antibody of the invention in the manufacture of a medicament for inducing clearance of peripheral B cells, wherein the antibody or antigen-binding fragment in the medicament specifically binds to a human cell surface CD40 antigen, wherein binding of the antibody or antigen-binding fragment to the CD40 antigen induces clearance of the cells.

The medicament may be prepared for use in treating an individual suffering from an immune disorder.

The medicament may be prepared for use in the treatment of rheumatoid arthritis or systemic lupus erythematosus.

Another embodiment includes the use of an antibody of the invention in the manufacture of a medicament for treating rheumatoid arthritis in an individual.

The medicament may be prepared for use in inhibiting B cell differentiation and antibody isotype switching in the subject.

The medicament may be prepared for use in inhibiting cytokine and chemokine production and the upregulation of adhesion molecules in T cells and macrophages of the individual.

The medicament may be prepared for use in inhibiting dendritic cell activation in the subject.

The medicament may be prepared for use in inhibiting the production of pro-inflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandins and down-regulation of adhesion molecules in non-immune cells of the individual.

In certain embodiments, the medicament is prepared as a combination medicament for administration in combination with a regimen comprising the administration of methotrexate and/or the administration of Enbrel/Humira.

In other embodiments, the medicament is prepared as a combination medicament and the medicament comprises an anti-TNF drug in addition to the antibody of the invention.

Drawings

FIG. 1: a: the binding curve of the humanized antibody to CD 40-transfected HEK-293 cells and the corresponding EC50 values were measured by flow cytometry. B: comparison of binding of antibody a, antibody B and antibody C to HEK cells transfected with CD40 was measured by flow cytometry. Shown is representative data for one experiment.

FIG. 2: the binding curve of the humanized antibody to RAMOS cells and the corresponding EC50 value were measured by flow cytometry. Shown is representative data for one experiment.

FIG. 3: antagonistic activity of mouse and humanized antibodies was tested in a human primary B cell proliferation assay. (A) Shown are representative antibody titration curves and resulting IC50 values for each mouse precursor antibody. Shown are representative data for one donor. (B) Shown is the superposition of inhibition curves for antagonism of various humanized anti-CD 40 antibodies compared to 4D 11.

FIG. 4: summary of test results for antagonistic (IC50) and agonistic (SI = stimulation index) activities of humanized antibodies in human primary B cell proliferation assays. Various anti-CD 40 antibodies, 4D11, G28.5, and 5D12 are shown for comparison.

FIG. 5: inhibition of CD 40-induced up-regulation of CD86 by antibody B, antibody a and antibody C was tested in a human whole blood assay. The 4D11 anti-CD 40 antibody is shown for comparison. The IgG1 isotype control showed no effect in this analysis. Shown are representative data for one donor.

FIG. 6: summary of the results of testing antibody B for inhibition of CD 40-induced up-regulation of CD86 on human purified B cells and in human whole blood. Shown are data at two points (A) IC50 and (B) IC 90. The IgG1 isotype control showed no effect in either assay. Data for multiple donors (n =4-5) are summarized in the table.

FIG. 7: inhibition of CD 40-induced up-regulation of CD86 by antibody B, antibody a and antibody C was tested in a cynomolgus whole blood assay. The IgG1 isotype control showed no effect in this analysis. Shown are representative data for one donor.

FIG. 8: plasma concentration time profiles of antibody A (left panel) and antibody B (right panel) in cynomolgus monkeys after administration of 1mg/kg and 10mg/kg of each antibody. Data are summary of 3 animals given each antibody.

FIG. 9: percentage change of CD86 positive B cells from cynomolgus monkeys before administration (antibody B) and (antibody a) and at 3 time points after treatment with each antibody. Antibody B (top panel) and antibody A were administered to 3 animals at 1mg/kg (left panel) or 10mg/kg (right panel), respectively.

FIG. 10: at injection 1.25X10⁶Content of (A) human IgG and (B) human IgM in NSG mice 2 weeks after human PBMC. Mice were treated with vehicle, isotype control and antibodies (antibody a, antibody B and antibody C) at a dose of 1mg/kg one day prior to transfer of human PBMCs.

FIG. 11: binding of various mouse anti-human CD40 antibodies to human platelets.

FIG. 12: summary of the results of the binding comparisons of antibody B and anti-CD 40mAb4D11 on human B cells and platelets in whole blood.

FIG. 13: ADCC activity of wild type and knockout IgG1 constructs.

Detailed Description

CD 40-mediated signaling has been recognized as involved in a variety of target conditions. Although a number of preclinical data are available showing that intervention in such conditions would be therapeutically beneficial, there remains a need for antagonistic anti-CD 40 antibodies that can be used to treat autoimmune diseases. A preferred embodiment of the invention relates to a humanized antibody that recognizes CD 40. In particular embodiments, the sequence of the humanized antibody has been determined based on the sequence of certain major mouse antibodies.

The terms "CD 40" and "CD 40 surface antigen" refer to the approximately 48kD glycoprotein expressed on the surface of normal and neoplastic B cells, which serves as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al, 1987, J.Immunol.138: 788-785). cDNA molecules encoding CD40 have been isolated from a library prepared from the Burkitt lymphoma cell line Raji (Stamenkovic et al, 1989, EMBO J.8: 1403).

As used herein, a cell line that endogenously expresses CD40 is characterized by any cell that surface expresses CD40, including, but not limited to, normal and neoplastic B cells, interlaced dendritic cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsils cells, and plasma cells from the bone marrow. In some embodiments, the CD40 molecule is a human CD40 molecule.

The antibodies of the invention specifically bind to human recombinant and native CD 40. A humanized monoclonal antibody (wherein the antibody specifically binds to human CD 40) having antagonist activity with an IC50 of less than 1nM in B cell proliferation and no agonism at concentrations up to 100 μ g/ml, and wherein the antibody is further characterized by an in vivo half-life of at least 10 days in a non-human primate.

Preferably, the antibody specifically binds to CD40 with an EC50 of less than 1nM in CD40-Fc conjugates and to CD40 with an EC50 of less than 2.5nM in CD40 expressing cells. The antagonistic properties of the antibody were defined as having a B cell or dendritic cell antagonistic activity IC50 of less than 1 nM. The antibodies also have superior pharmacokinetic properties with an in vivo half-life that is extended compared to other anti-CD 40 antibodies (e.g., anti-CD 40 antibody 4D 11).

As used herein, a cell line expressing CD40 is characterized by any cell that expresses CD40 on its surface, including, but not limited to, normal and neoplastic B cells, interlaced cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsillar cells, and plasma cells from the bone marrow. In some embodiments, the CD40 molecule is a human CD40 molecule.

The antibodies of the invention recognize specific "CD 40 epitope" and "CD 40 epitope". These terms as used herein refer to molecules (e.g., peptides) or molecular fragments that are immunoreactive with an anti-CD 40 antibody and include, for example, the CD40 epitope that can be recognized by any antibody having the following heavy/light chain sequence combinations: any one of light chain SEQ ID NO. 26 and heavy chain SEQ ID NO. 27, 28, 29 or 30; or any of the light chain SEQ ID NO. 31 and heavy chain SEQ ID NO. 32, 33, 34 or 35; or any of the light chain SEQ ID NO. 36 and heavy chain SEQ ID NO. 37, 38, 39 or 40. The CD40 epitope may be included in a protein, protein fragment, peptide, or the like. The most common epitopes are proteins, short oligopeptides, oligopeptides (i.e., organic compounds that mimic the antibody binding properties of the CD40 antigen), or combinations thereof.

The general structure of antibodies or immunoglobulins is well known to those skilled in the art and the molecule is an heterotetrameric glycan protein, typically about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotetrameric molecule is formed via a covalent disulfide bond between two identical heavy chains in the heterodimer. Although the light and heavy chains are linked together by a disulfide bond, the number of disulfide bonds between the two heavy chains may vary depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at the amino terminus_H) Followed by three or four constant domains (C)_H1、C_H2、C_H3And C_H4) And C_H1And C_H2The hinge region in between. Each light chain has two domains, the amino-terminal variable domain (V)_L) And a carboxyl groupBasal-terminal constant domain (C)_L)。V_LDomain and V_HDomains are non-covalently bound, and C_LThe domains are typically linked to C via disulfide bonds_H1The domains are covalently linked. It is believed that particular amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain (Chothia et al, 1985, J.mol.biol.186: 651-663).

Certain domains within the variable domain differ widely, i.e., "superdenaturalized", between different antibodies. The hypervariable domain contains residues which are directly involved in the binding and specificity of each particular antibody for its specific antigenic determinant. The hyperdegeneration in both the light chain variable domain and the heavy chain variable domain is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence alignment in the following documents: kabat et al, 1991, sequence of proteins of immunologicalcalest, 5th edition, public health service, national institutes of health, Bethesda, MD.; while HVLs are structurally defined according to the three-dimensional structure of the variable domains, as described by Chothia and Lesk, 1987, j.mol.biol.196: 901 — 917. Where the two methods allow slightly different identification of the CDRs, the structural definition is preferred. As defined by Kabat, in the light chain variable domain, CDR-L1 is located approximately at residues 24-34, CDR-L2 is located approximately at residues 50-56, and CDR-L3 is located approximately at residues 89-97; in the heavy chain variable domain, CDR-H1 is located approximately at residues 31-35, CDR-H2 is located approximately at residues 50-65, and CDR-H3 is located approximately at residues 95-102. The CDRs 1,2, CDR3 of the heavy and light chains thus define unique functional properties specific for a given antibody.

The three CDRs within each heavy and light chain are separated by Framework Regions (FRs) containing sequences that are generally less variable. From the amino-terminus to the carboxy-terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The larger beta-sheet configuration of the FRs places the CDRs within each chain in close proximity to each other and to the CDRs in the other chain. The resulting conformation contributes to the antigen binding site (see Kabat et al, 1991, NIHPubl. Nos. 91-3242, Vol.I, p.647-669), but not all CDR residues must be directly involved in antigen binding.

FR residues and Ig constant domains are not directly involved in antigen binding, but contribute to antigen binding and/or mediate antibody effector functions. It is believed that some FR residues have a significant effect on antigen binding in at least three ways: non-covalent binding directly to the epitope, interaction with one or more CDR residues, and affecting the interface between the heavy and light chains. The constant domains are not directly involved in antigen binding, but mediate various Ig effector functions, such as the involvement of antibodies in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

The light chain of vertebrate immunoglobulins can be assigned to one of two significantly different classes (kappa (κ) and lanrda (λ)) based on the amino acid sequence of the constant domain. By comparison, the heavy chains of mammalian immunoglobulins are classified into the following five main categories according to the sequence of the constant domains: IgA, IgD, IgE, IgG and IgM. IgG and IgA are further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂The constant domains of heavy chains corresponding to different immunoglobulin classes are referred to as α, γ, and μ, respectively.

The terms "antibody," "anti-CD 40 antibody," "humanized anti-CD 40 antibody," and "variant humanized anti-CD 40 antibody" are used herein in the broadest sense and specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., variable domains and other portions of antibodies) that express the desired biological activity (e.g., CD40 binding).

The term "monoclonal antibody" (mAb) refers to an antibody in a substantially homogeneous population of antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic determinant (i.e., an "epitope"). Thus, the modifier "monoclonal" indicates a population of antibodies that is substantially homogeneous and directed against the same epitope, and is not to be construed as requiring production of the antibody by any particular method. It is understood that monoclonal antibodies can be prepared by any technique or method known in the art; including, for example, the hybridoma method (Kohler et al, 1975, Nature256:495), or recombinant DNA methods known in the art (see, for example, U.S. Pat. No. 4,816,567), or methods for isolating recombinantly produced monoclonal antibodies using phage antibody libraries by techniques described in the following references: clackson et al, 1991, Nature 352: 624-; and Marks et al, 1991, j.mol.biol.222: 581-597.

Chimeric antibodies are composed of antibody heavy and light chain variable regions from one species (e.g., non-human mammal, such as a mouse) and heavy and light chain constant regions of another species (e.g., human) antibody, and can be obtained by: a DNA sequence encoding an antibody variable region from a first species (e.g., mouse) is ligated to a DNA sequence encoding an antibody constant region from a second species (e.g., human), and the host is transformed with an expression vector containing the ligated sequences, thereby making it possible to produce a chimeric antibody. Alternatively, in a chimeric antibody, one or more regions or domains in the heavy and/or light chain may also be identical, homologous or variants thereof, or from consensus or germline sequences, to the corresponding sequences in a monoclonal antibody from another immunoglobulin class or isotype. Chimeric antibodies can include fragments of such antibodies, provided that the antibody fragment exhibits the desired biological activity of its parent antibody, e.g., binds to the same epitope (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, 1984, Proc. Natl. Acad. Sci. USA 81: 6851-.

The terms "antibody fragment," "anti-CD 40 antibody fragment," "humanized anti-CD 40 antibody fragment," "variant humanized anti-CD 40 antibody fragment" refer to a portion of a full-length anti-CD 40 antibody in which the variable regions or functional capacity, e.g., specific CD40 epitope binding, is retained. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')₂Fd, Fv, scFv-Fc fragment, diabody (diabody), and linear antibodySingle-chain antibodies, minibodies (minibodies), diabodies formed from antibody fragments, and multispecific antibodies formed from antibody fragments.

Full-length antibodies can be treated with enzymes such as papain or pepsin to generate useful antibody fragments. Papain digestion is used to generate two identical antigen-binding antibody fragments, called "Fab" fragments (each with a single antigen-binding site) and a residual "Fc" fragment. Fab fragments also contain a light chain constant domain and a heavy chain C_H1A domain. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites and still being capable of cross-linking antigens.

Fab' fragments differ from Fab fragments in that they are at C_H1Additional residues are present at the C-terminus of the domain, including one or more cysteines from the antibody hinge region. F (ab')₂Antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge region. Other chemical couplings of antibody fragments are also known.

The "Fv" fragment contains the entire antigen recognition and binding site, which is composed of a dimer of one heavy chain variable domain in tight, non-covalent association with one light chain variable domain. In this configuration, the three CDRs of each variable domain interact, thereby generating a V_H-V_LThe surface of the dimer defines the antigen binding site. The six CDRs collectively confer antigen binding specificity to the antibody.

"Single chain Fv" or "scFv" antibody fragments are fragments comprising antibody V_HAnd V_LA single-chain Fv variant of a domain wherein said domain is present in a single polypeptide chain. Single-chain Fv's recognize and bind antigen. The scFv polypeptide may also optionally contain a site V_HDomain and V_LPolypeptide linkers between domains to facilitate scFv formation into the desired three-dimensional structure for antigen binding (see, e.g., Pluckthun, 1994, InThermatologyof monoclonal antibodies, Vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, p.269-315).

Other known antibody fragments include those comprising a pair of strandsFd section (V) of the string_H-C_H1-V_H-C_H1) To form an antibody fragment of a pair of antigen binding regions. The "linear antibody" may be bispecific or monospecific as described, for example, in Zapata et al, 1995, ProteinEng.8(10): 1057-1062.

A humanized antibody or a fragment thereof is a specific type of chimeric antibody that comprises an immunoglobulin amino acid sequence variant or a fragment thereof that binds to a predetermined antigen and that comprises one or more FRs having substantially a human immunoglobulin amino acid sequence and one or more CDRs having substantially a non-human immunoglobulin amino acid sequence. This non-human amino acid sequence is often referred to as an "import" sequence, and is typically taken from an "import" antibody domain, particularly a variable domain. Typically, a humanized antibody comprises at least the CDRs or HVLs of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain. The present invention describes a specific humanized anti-CD 40 antibody comprising the CDRs derived from a murine monoclonal antibody inserted between the FRs of the heavy and light chain variable domains of the human germline sequences as shown in tables 3 and 4. It will be appreciated that certain murine FR residues may be important to the function of the humanized antibody, and that certain human germline sequence heavy and light chain variable domain residues will therefore be modified to be identical to those in the corresponding murine sequences.

In another aspect, the humanized anti-CD 40 antibody comprises substantially all of at least one (typically two) variable domain (e.g., as contained in, e.g., Fab ', F (ab')2, Fabc, and Fv fragments), wherein all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin, and in particular herein all of the CDRs are murine sequences as detailed herein in tables 1-4 below, and all or substantially all of the FRs are FRs of a human immunoglobulin consensus sequence or germline sequence. In another aspect, the humanized anti-CD 40 antibody also includes at least a portion of an Fc region of an immunoglobulin, typically a human immunoglobulin. Typically, an antibody contains both a light chain and at least a heavy chain variable domain. If appropriate, the antibody may also comprise heavy chain C_H1Hinge, C_H2、C_H3And/or C_H4One or more of the zones.

Human sourceThe chemo-anti-CD 40 antibody can be selected from any immunoglobulin class (including IgM, IgG, IgD, IgA and IgE) and any isotype (including IgG)₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂). For example, the constant domain may be a complement-fixed constant domain in which the humanized antibody is expected to exhibit cytotoxic activity, and the isotype is typically an IgG₁. If such cytotoxic activity is not desired, the constant domain may be of another isotype, e.g., IgG₂. Alternative humanized anti-CD 40 antibodies may comprise sequences from more than one immunoglobulin class or isotype, and those skilled in the art may be skilled in selecting particular constant domains to optimize the desired effector function. In particular embodiments, the antibodies provided herein are IgG1 antibodies, and more specifically IgG1 antibodies that knock out effector function.

The FR and CDR or HVL of the humanized anti-CD 40 antibody need not correspond exactly to the parent sequence. For example, one or more residues in the imported CDR or HVL or consensus FR or germline FR sequences can be altered (e.g., mutagenized) by substitution, insertion or deletion such that the resulting amino acid residues are no longer identical to the original residues at the corresponding positions in either parent sequence, but the antibody still retains the function of binding to CD 40. The changes may generally not be widely varied and may be conservative changes. Typically, at least 75% of the humanized antibody residues will correspond to those in the parent consensus FR or germline FR and input CDR sequences, more typically at least 90%, and most typically more than 95% or more than 98% or more than 99% of the residues.

Influencing the interface between the heavy and light chain variable regions ('V')_L-V_HInterface ") are those that affect the proximity or orientation of the two chains relative to each other. Some residues that may be involved in interchain interactions include V_LResidues 34, 36, 38, 44, 46, 87, 89, 91, 96 and 98 and V_HResidues 35, 37, 39, 45, 47, 91, 93, 95, 100 and 103 (using the numbering system described in Kabat et al, sequence of proteins of immunologicals Interest (national institutes of health, Bethesda, Md., 1987))). U.S. Pat. No. 6,407,213 also discusses, for example, V_LResidues 43 and 85 and V_HResidues 43 and 60 may also participate in this interaction. Although the residues are described only for human IgG, they are applicable to other species as well. Important antibody residues that are reasonably expected to participate in the inter-chain interactions are selected for substitution in the consensus sequence.

The terms "consensus sequence" and "consensus antibody" refer to an amino acid sequence comprising the most commonly occurring amino acid residue at each position in all immunoglobulins (e.g., human immunoglobulin variable domains) of any particular class, isotype, or subunit structure. The consensus sequence may be based on immunoglobulins of a particular species or multiple species. A "consensus" sequence, structure, or antibody is understood to encompass a consensus human sequence as described in certain embodiments, and refers to an amino acid sequence comprising the most commonly occurring amino acid residue at each position in all human immunoglobulins of any particular class, isotype, or subunit structure. Thus, a consensus sequence contains an amino acid sequence that has at each position an amino acid present in one or more known immunoglobulins, but which may not exactly replicate the entire amino acid sequence of any single immunoglobulin. The variable region consensus sequence is not available from any naturally occurring antibody or immunoglobulin and variants thereof (Kabat et al, 1991, sequences of proteins of immunologica interest, 5th edition, public health service, national institutes of health, Bethesda, Md.). The FRs of the heavy and light chain consensus sequences and variants thereof provide useful sequences for the preparation of humanized anti-CD 40 antibodies. See, for example, U.S. patent nos. 6,037,454 and 6,054,297.

Human germline sequences occur naturally in the human population. The combination of germline genes results in antibody diversity. The germline antibody sequences of the antibody light chains are from conserved human germline kappa or lambda v-genes and j-genes. Similarly, the heavy chain sequences are from germline v-, d-and j-genes (LeFranc, M-P and LeFranc, G, "the immunologlobulinFactsBook" academic Press, 2001).

As used herein, "variant," "anti-CD 40 variant," "humanized anti-CD 40 variant," or "variant humanized anti-CD 40" each refer to a humanized anti-CD 40 antibody having at least a heavy chain variable murine CDR derived from any one of SEQ ID NOs 1 through 4, or a light chain murine CDR sequence derived from a murine monoclonal antibody represented by any one of SEQ ID NOs 5 through 8, and an FR sequence derived from a human consensus sequence. Variants include those having one or more amino acid changes in one or both of the light or heavy chain variable domains, provided that the amino acid changes do not substantially impair binding of the antibody to CD 40. Exemplary humanized antibodies generated herein include those referred to as antibody A, antibody B and antibody C, and the respective heavy and light chain sequences thereof are shown in SEQ ID NO. 26 through SEQ ID NO. 40.

An "isolated" antibody is one that has been identified and has been isolated and/or recovered from a component of its natural environment. Contaminant components in the natural environment of an antibody are those that can interfere with diagnostic or therapeutic applications of the antibody, and can be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect, the antibody can be purified to a degree of separation of at least greater than 95% by weight of the antibody.

Since at least one component of the natural environment of the antibody is not present, an isolated antibody includes an antibody in situ within the recombinant cell from which it was produced. However, isolated antibodies can generally be prepared by at least one purification step in which recombinant cellular material is removed.

The term "antibody performance" refers to factors contributing to the recognition of an antigen by an antibody or the efficacy of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect antibody properties such as folding, and can affect physical factors such as: initial rate of antibody binding to antigen (k)_a) Dissociation constant (k) of antibody and antigen_d) The affinity constant (Kd) of the antibody to the antigen, the conformation of the antibody, the protein stability and the half-life of the antibody.

The term "epitope-tagged" as used herein refers to the fusion of an anti-CD 40 antibody with an "epitope tag". An "epitope tag" is a tag having sufficient amino acids to provide a table for antibody productionA polypeptide at position, and which is designed such that it does not interfere with the desired activity of the humanized anti-CD 40 antibody. The epitope tag is typically sufficiently unique such that antibodies raised against the epitope tag do not substantially cross-react with other epitopes. Suitable tag polypeptides generally contain at least 6 amino acid residues and usually contain about 8 to 50 amino acid residues or about 9 to 30 residues. Examples of epitope tags and antibodies that bind epitopes include influenza HA tag polypeptide and its antibody 12CA5(Field et al, 1988mol. cell. biol. 8: 2159-; the C-myc tag and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (Evan et al, 1985, mol. cell. biol.5 (12): 3610-3616); and the herpes simplex virus glycoprotein D (gD) tag and its antibodies (Paborsky et al, 1990, protein engineering3 (6): 547-553). In certain embodiments, the epitope tag is a "salvage receptor binding epitope". The term "salvage receptor binding epitope" as used herein refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) An epitope in the Fc region that is responsible for extending the in vivo serum half-life of an IgG molecule.

In some embodiments, the antibodies of the invention may be conjugated to a cytotoxic agent. A cytotoxic agent is any substance that inhibits or prevents cell function and/or causes cell destruction. The term is intended to include radioisotopes (e.g., I)¹³¹、I¹²⁵、Y⁹⁰And Re¹⁸⁶) Chemotherapeutic drugs and toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin), and fragments thereof. The cytotoxic agent can be conjugated to the humanized antibody of the invention by standard procedures, and it can be used, for example, to treat patients for whom antibody therapy is indicated.

A "chemotherapeutic drug" is a chemical compound that can be used to treat cancer. There are a variety of examples of chemotherapeutic drugs that can be conjugated to the therapeutic antibodies of the invention. Examples of the chemotherapeutic drugs include alkylating agents such as thiotepa (thiotepa) and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, e.g. benzodipa, carbineQuinone (carboquone), meturedopa and uredopa; ethyleneimine and methylmelamine including hexamethylmelamine (altretamine), triethylenemelamine, triethylenephosphoramide sulfide, and trimethylolmelamine; annonaceous acetogenin (especially bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan); bryostatin (bryostatin); callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin1 and cryptophycin 8); dolastatin (dolastatin); auristatin (including the analogs monomethyl-auristatin e and monomethyl-auristatin f); duocarmycin (duocarmycin) (including the synthetic analogs KW-2189 and CBI-TMI); punicin (eleutherobin); pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chloramphazine), chlorophosphamide (chlorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenyleneterester), prednimustine (prednimustine), trofosfamide (trofosfamide) and uramustine (uracil); nitroureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranirnustine); antibiotics, e.g. enediyne (enediyne) antibiotics (e.g. calicheamicin), especially calicheamicin gamma 1I and calicheamicinSee, e.g., AngewChem.Intl.Ed.Engl. (1994)33: 183-); dynemicin, including dynemicin a; bisphosphonates, such as clodronate (clodronate); esperamicin (esperamicin); novel oncostatin chromophores (neocarzinostatin chromophores) and related chromoprotein enediyne antibiotics chromophores (relatedchromoprotenenediynediotic chromophores), aclaciNomysin, actinomycin, authamycin, azaserine, bleomycin, actinomycin C, carbamycin, carminomycin, carcinomycin, chromomycin, dactinomycin, daunorubicin, ditorubicin, 6-diazo-5-oxo-L-norleucine (6-diazo-5-oxo-L-norleucin), doxorubicin (Adriamycin)^TM) (including morpholino-doxorubicin (morpholino-doxorubicin), cyanomorpholino-doxorubicin (cyanomorpholo-doxorubicin), 2-pyrrolino-doxorubicin (2-pyrrolino-doxorubicin), deoxydoxorubicin (deoxydoxorubicin)), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcelomycin), mitomycins (mitomycin) such as mitomycin C (mitomycin C), mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pellomycin (polyplomycin), podomycin (puromycin), adriamycin (doxorubicin), doxorubicin (pyridomycin), doxorubicin (streptozocin), streptomycin (streptozocin, mitomycin, mito; antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-fluorouracil) (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin and trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (6-mercaptopurine), thiamiprine (thiamiprine), and thioguanine (thioguanine); pyrimidine analogs such as, for example, ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), carmofur (carmofur), cytarabine (cytabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), and floxuridine (floxuridine); androgens such as carposterone (calusterone), methyl androsterone propionate (dromostanolone propionate), epitioandrostanol (epitiostanol), mepiquane (mepiquitane), and testolactone (testolactone); anti-adrenergic agents (anti-acquired), examplesSuch as aminoglutethimide (aminoglutethimide), mitotane (mitotane) and trilostane (trilostane); folic acid supplements, such as folinic acid (frolicicacid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; idazot (edatraxate); desphosphamide (defofamine); colchicine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etiolate (ellitiniumacetate); epothilone (epothilone); etoglut (etoglucid); gallium nitrate (gallimnitrate); hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinol (maytansinoid) classes such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanmol; rhizobia (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllinic acid); 2-ethylhydrazine (2-ethylhydrazine); procarbazine (procarbazine);razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2', 2 "-trichlorotriethylamine (2, 2', 2" -trichlorotriethylamine); trichothecenes (trichothecenes) (especially T-2 toxin, veracurin A, bacillosporin A (roridinin A) and anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); bromopropylpiperazine (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide; thiotepa; taxanes (taxoids), e.g. paclitaxel (paclitaxel) ((R))Bristol-Myers SquibbOncology, Princeton, N.J.) and doxPaclitaxel (doxetaxel) ((R))Rhone-PoulencRorer, Antony, France); chlorambucil; gemcitabine (Gemcitabine) (Gemzar)^TM) (ii) a 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); an ifosfamide; mitoxantrone; vincristine (vincristine); vinorelbine (vinorelbine) (Navelbine)^TM) (ii) a Nuantro (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine (capecitabine); and a pharmaceutically acceptable salt, acid or derivative of any of the above. Also included in this definition are anti-hormonal agents used to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (tamoxifen) (including Nolvadex)^TM) Raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxyttamoxifen, troloxifene (trioxifene), keoxifene, LY117018, onapristone (onapristone), and toremifene (Fareston)^TM) (ii) a Aromatase inhibitors which inhibit aromatase and which regulate the production of estrogen in the adrenal gland, e.g. 4(5) -imidazole, aminoglutethimide, megestrol acetate (Megace)^TM) Exemestane (exemestane), formestane (formestane), fadrozole (fadrozole), vorozole (rivosor)^TM) Letrozole (Femara)^TM) And anastrozole (Arimidex)^TM) (ii) a And antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and a pharmaceutically acceptable salt, acid or derivative of any of the above. Any one or more of the drugs may be administered to the person of the inventionThe humanized antibodies are conjugated to provide therapeutic agents useful in the treatment of various disorders.

Antibodies may also be conjugated to prodrugs. A "prodrug" is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted to a more active form. See, for example, Wilman, 1986, "Prodrangun cancer Chemotherapy", Biochemical society transactions, 14, pp.375-382, 615th MeetingBellfast; and Stella et al, 1985, "produgs: AchemiachichottargetDrugDelivery ", DirectDrugDelivery, Borchardt et al (ed.), pp.247-. Prodrugs that may be used include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs and optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs and other 5-fluorouridine prodrugs, which are convertible to non-cytotoxic drugs having higher activity. Examples of cytotoxic drugs that can be derivatized into prodrug forms include, but are not limited to, those chemotherapeutic drugs described above.

The antibodies of the invention may also be conjugated with a label, either alone or with a further second drug (prodrug, chemotherapeutic drug, etc.), for diagnostic as well as therapeutic monitoring purposes. The label is distinct from the other second drug, which refers to a detectable compound or composition drug, and which may be conjugated directly or indirectly to the humanized antibody of the invention. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels), or in the case of an enzymatic label, the label may catalyze a detectable chemical change in the substrate compound or composition. Labeled humanized anti-CD 40 antibodies can be prepared and used in a variety of applications, including in vitro and in vivo diagnostics.

The antibodies of the invention may be formulated as part of a liposome formulation to achieve in vivo delivery thereof. "liposomes" are vesicles composed of various types of lipids, phospholipids and/or surfactants. Liposomes can be used to deliver compounds or formulations to a mammal, such as the humanized anti-CD 40 antibodies disclosed herein, optionally conjugated or combined with one or more pharmaceutically active drugs and/or labels. The components of liposomes are typically arranged in a bilayer structure, similar to the lipid arrangement of biological membranes.

Certain aspects of the invention relate to isolated nucleic acids encoding one or more domains of the humanized antibodies of the invention. An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of antibody nucleic acid. An isolated nucleic acid molecule is distinct from nucleic acid molecules that are present in natural cells.

In various aspects of the invention, one or more domains of the humanized antibody are recombinantly expressed. Such recombinant expression may employ one or more control sequences, i.e., polynucleotide sequences required for expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for use in prokaryotic cells include, for example, promoter, operator, and ribosome binding site sequences. Eukaryotic control sequences include, but are not limited to, promoters, polyadenylation signals, and enhancers. The control sequences are useful for expression and production of humanized anti-CD 40 antibodies in prokaryotic and eukaryotic host cells.

A nucleic acid sequence is "operably linked" when it is placed in functional association with another nucleic acid sequence. For example, if a presequence or secretory leader is expressed as a preprotein that is involved in the secretion of a polypeptide, then the presequence or secretory leader is operably linked to a nucleic acid encoding the polypeptide; a promoter or enhancer is operably linked to a sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous sequences, and in the case of a secretory leader, contiguous sequences and in reading frame. However, enhancers are optionally contiguous. Ligation may be accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers may be used.

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, "transformants" and "transformed cells" include primary individual cells and cultures derived therefrom without regard to the number of transfers.

The term "mammal" for therapeutic purposes means any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, cats, cattle, and the like. Preferably, the mammal is a human.

As used herein, a "disorder" is any condition that may benefit from treatment with the humanized anti-CD 40 antibodies described herein. The term includes chronic as well as acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder. Non-limiting examples or conditions to be treated herein include cancer, hematologic malignancies, benign and malignant tumors, leukemias and lymphoid malignancies, and inflammatory, angiogenic, autoimmune and immunological conditions.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by dysregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

The term "CD 40-associated disorder" or "CD 40-associated disease" as used herein refers to a condition in which it is desirable to modify or eliminate cells that express CD 40. The conditions include cells expressing CD40 that exhibit abnormal proliferation or cells expressing CD40 that are associated with cancerous or malignant growth. More specifically, examples of cancers showing abnormal expression of CD40 antigen include B lymphoid stem cells, burkitt's lymphoma, multiple myeloma, T cell lymphoma, kaposi's sarcoma, osteosarcoma, epidermal and endothelial tumors, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer (melanoma), bladder cancer and renal cancer. Such conditions include, but are not limited to, leukemia, lymphoma (including B-cell lymphoma and non-hodgkin's lymphoma), multiple myeloma, waldenstrom's macroglobulinemia; solid tumors, including sarcomas (e.g., osteosarcoma, Ewing's sarcoma), malignant melanoma, adenocarcinomas including ovarian adenocarcinoma, kaposi's sarcoma/kaposi's tumor, and squamous cell carcinoma.

CD 40-related disorders also include immune system diseases and disorders, such as autoimmune disorders and inflammatory disorders. Such conditions include, but are not limited to, Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), scleroderma, Sjogren's syndrome, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., ulcerative colitis and crohn's disease), pulmonary inflammation, asthma, and Idiopathic Thrombocytopenic Purpura (ITP).

The phrase "preventing … growth" or "growth inhibitory" as used herein refers to inhibiting the growth or proliferation of cells, particularly neoplastic cell types expressing the CD40 antigen. Thus, growth inhibition (for example) significantly reduces the percentage of S-phase neoplastic cells.

The term "intravenous infusion" refers to the introduction of a drug into the vein of an animal or human patient over a period of time greater than about 15 minutes, typically between about 30 to 90 minutes.

The term "intravenous bolus" or "intravenous bolus" refers to the administration of a drug into the veins of an animal or human such that the body receives the drug in about 15 minutes or less, typically 5 minutes or less.

The term "subcutaneous administration" refers to the introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug reservoir. Pinching or pulling the skin up and away from the underlying tissue may create a pocket.

The term "subcutaneous infusion" refers to the introduction of a drug by relatively slow, sustained delivery from a drug reservoir under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, over a period of time, including (but not limited to) 30 minutes or less, or 90 minutes or less. Optionally, the infusion may be performed by subcutaneously implanting a drug delivery pump (implanted under the skin of an animal or human patient), wherein the pump delivers a predetermined amount of drug over a predetermined period of time (e.g., 30 minutes, 90 minutes, or a period of time spanning an entire treatment regimen).

The term "subcutaneous bolus" refers to administration of a drug under the skin of an animal or human patient, wherein the bolus drug is delivered for less than about 15 minutes; in another aspect, less than 5 minutes; and in another aspect, less than 60 seconds. In another aspect, administration is within a pouch between the skin and the base tissue, wherein the pouch may be created by pinching or pulling the skin up away from the base tissue.

The term "therapeutically effective amount" is used to refer to an amount of active agent that alleviates or ameliorates one or more symptoms of the disorder being treated. In this case, it is an amount that has a beneficial patient outcome (e.g., growth arresting effect) or causes cell loss. In one aspect, the therapeutically effective amount has apoptotic activity, or is capable of inducing cell death. In another aspect, a therapeutically effective amount refers to a target serum concentration that has been shown to be effective, for example, in slowing disease progression. Efficacy may be measured in a conventional manner depending on the condition to be treated. For example, in neoplastic diseases or disorders characterized by cells expressing CD40, efficacy can be measured by assessing time to disease progression or determining response rate.

As used herein, "treatment" and "therapy" and similar terms are intended to include therapeutic as well as prophylactic or inhibitory measures against a disease or disorder that produces any clinically desirable or beneficial effect, including, but not limited to, alleviation or relief of one or more symptoms of the disease or disorder, regression of the disease or disorder, slowing or stopping the progression of the disease or disorder. Thus, for example, the term treating includes administering a drug before or after the onset of symptoms of a disease or disorder, thereby preventing or eliminating one or more signs of the disease or disorder. As another example, the term includes administering a drug after clinical expression of a disease, thereby counteracting the symptoms of the disease. In addition, administration of a drug after onset and after clinical symptoms have occurred encompasses "treatment" or "therapy" as used herein, wherein administration affects a clinical parameter of the disease or disorder, such as the degree of tissue damage or the amount or extent of metastasis, whether or not the treatment results in amelioration of the disease. In addition, the results should be considered effective in treating the underlying disorder, provided that the composition of the invention, alone or in combination with another therapeutic agent, reduces or ameliorates at least one symptom of the disorder being treated, whether or not all symptoms of the disorder are reduced, as compared to when the humanized CD40 antibody composition is not used.

The term "package insert" refers to instructions typically included in commercial packaging for therapeutic products that contain information regarding the indications, usage, administration, contraindications, and/or warnings for using the therapeutic products.

Antibodies

Humanized anti-CD 40 antibodies and compositions and articles of manufacture comprising one or more humanized anti-CD 40 antibodies of the invention are described and disclosed herein. Also described herein are binding agents that include antigen-binding fragments of humanized anti-CD 40 antibodies. The humanized anti-CD 40 antibodies and binding agents can prevent cell growth, cause depletion of cells expressing CD40, or induce or cause cytotoxic or cytostatic effects against target cells. The humanized anti-CD 40 antibodies and binding agents are useful for treating a variety of diseases or disorders characterized by the proliferation of cells expressing the CD40 surface antigen. The humanized anti-CD 40 antibody and the CD40 binding agent each include at least one portion (i.e., an antigen-binding fragment) that specifically recognizes an epitope of CD 40.

In preliminary characterization, murine antibodies were selected based on CD40 binding characterization.

From the initial study, murine antibodies were selected having the heavy chain variable regions shown in table 1 below and the light chain variable regions shown in table 2 below:

table 1: CD40 murine leader-VH sequence

Table 2: CD40 murine leader-VK sequence

The human framework sequence for each mouse leader sequence was selected based on framework homology, CDR structures, conserved standard residues (conserved canonical residues), conserved interface packaging residues (conserved interface packaging residues), and other parameters.

The murine heavy and light chain CDRs for various murine selected antibodies are shown in tables 3 and 4, respectively:

table 3: heavy chain CDR sequence

The H-CDR1 listed above uses the sequence numbered by the Chothia numbering system (Al-Lazikani et Al, (1997) JMB273, 927-948). The Kabats numbers for the sequences are indicated in bold italics for residues in CDR1 and CDR2 above and IMGT numbers are shown underlined. The H-CDR3 sequence of each of 2H11, 10F2, and 19B10 isTTSYYVGTYGY(SEQ ID NO:77) and the H-CDR3 sequence of 20E2ARQDGYRYAMDY(SEQIDNO:78)。

Table 4: light chain CDR sequences

Similarly, the Chothia numbering system is used in table 4, and Kabats numbers for sequences are shown in bold italics and IMGT numbers are shown in underlined text.

Fab showing better or equal binding compared to the chimeric parent Fab is selected and converted to IgG. Clones from the 20E2 series were converted into two different IgG patterns: a) IgG4DM (double mutant: (double mutant) (B))doublemutant)) has two mutations in the Fc/hinge region, Ser228Pro reduces half of the molecule formation and Leu235Glu further reduces Fc γ R binding; b) IgG1KO (knock-out: (knock-out) effector function) has two mutations Leu234Ala and Leu235Ala in the Fc region, which reduce effector functions such as Fc γ R and complement binding. Both IgG formats are described in the literature. Example 1 illustrates humanization of three candidates in more detail. The result of this humanization is the generation of humanized antibody sequences having the heavy and light chain sequences shown below:

in some embodiments, the antigen-binding fragment may block proliferation or prevent cell growth or cause clearance, death, or loss thereof of a cell, for example, via binding to a CD40 surface antigen, for example. For example, in T and B cell malignancies, anti-tumor effects (e.g., growth arrest with or without cell loss or apoptosis) are often elicited when malignant cells are exposed to stimuli that cause activation of normal lymphocytes. It has been observed that this activation-induced growth prevents signals with receptors via antigen or co-stimulation (see, e.g., Ashwell et al, 1987, Science 237: 61; Bridges et al, 1987, J.Immunol.139: 4242; Page and Defranco, 1988, J.Immunol.140: 3717; and Beckwith et al, 1990, J.Natl.cancer Inst.82: 501). CD40 stimulation inhibits B cell lymphoma growth due to specific binding of antibodies or soluble ligands (see, e.g., Funakoshi et al, 1994, Blood 83: 2787-. Inhibiting malignant cell growth in this manner and drugs directed against the CD40 surface antigen are examples of suitable drugs.

CD 40-specific drugs include antigen-binding fragments of humanized anti-CD 40 antibodies that bind CD40 (e.g., human CD40 or variants thereof). The CD 40-specific drugs and antibodies may optionally be conjugated or fused to cytotoxic or chemotherapeutic drugs. In aspects where the humanized antibody binds to CD40 surface antigen and results in clearance of a cell type expressing CD40, binding is generally characterized by homing to CD40 surface antigen cells in vivo. Suitable binding agents bind to the CD40 antigen with sufficiently high affinity and/or avidity (avidity) to allow CD40 specific drugs to be used as therapeutic agents by specifically targeting antigen-expressing cells.

In some aspects, the humanized antibody reduces binding of CD40 ligand to CD40 by at least 45%, at least 50%, at least 60%, or at least 75% or at least 80%, or at least 90%, or at least 95%.

In some embodiments, a humanized anti-CD 40 antibody (including antigen binding fragments thereof, such as heavy and light chain variable domains) comprises an amino acid sequence derived from residues of CDR antibody a (heavy chain sequence = seq id no: 27; seq id no: 28; seq id no:29 or seq id no: 30; light chain sequence = seq id no:26), antibody B (heavy chain sequence = seq id no: 32; seq id no: 33; seq id no: 34; or seq id no: 35; light chain sequence = seq id no:31) and antibody C (heavy chain sequence = seq id no: 37; seq id no: 38; seq id no:39 or seq id no: 40; light chain sequence = seq id no:36) described above and amino acid residues derived from a human immunoglobulin framework region. The humanized anti-CD 40 antibody optionally includes specific amino acid substitutions in the consensus or germline framework regions.

Specific amino acid residue substitutions at the framework positions can improve various aspects of antibody performance (including binding affinity and/or stability) relative to those shown in humanized antibodies formed by "direct exchange" of CDRs or HVLs into human germline framework regions, as shown in the examples below.

In some embodiments, the invention features other monoclonal antibodies having the heavy chain (VH) sequences of SEQ ID NO:1 through SEQ ID NO:4 and the light chain (VL) sequences of SEQ ID NO:5 through SEQ ID NO:8 (see tables 1 and 2 above). The CDR sequences of the murine antibodies are shown in tables 3 and 4, placing the CDRs in the FRs of human consensus heavy and light chain variable domains can produce humanized antibodies useful in the invention.

In certain embodiments, the humanized anti-CD 40 antibodies disclosed herein comprise at least a heavy chain or light chain variable domain having the CDRs or HVLs of the murine monoclonal antibody shown in tables 1-4 above and the FRs of the human germline heavy and light chain variable domains. In exemplary embodiments, the humanized antibodies produced herein are: antibody A, antibody B and antibody C, and the respective heavy and light chain sequences thereof are shown in SEQ ID NO. 26 to SEQ ID NO. 40.

In particular embodiments, antibodies having the following are contemplated: 27, 28, 29 or 30, and 26. Alternative antibodies include those having the following: the heavy chain sequence is SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34 or SEQ ID NO. 35, and the light chain sequence is SEQ ID NO. 31. In other embodiments, humanized antibodies are provided having: a heavy chain sequence of SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 or SEQ ID NO. 40 and a light chain sequence of SEQ ID NO. 36.

The CDRs of the sequences are shown in tables 3 and 4. In particular embodiments, chimeric antibodies having a switch CDR region between the exemplary immunoglobulins (i.e., one or both CDRs of antibody a are switched, e.g., with a similar CDR from antibody C) are expected to produce useful antibodies.

In certain embodiments, the humanized anti-CD 40 antibody is an antibody fragment. Various antibody fragments have been outlined above and various techniques have been developed to produce them. Fragments can be obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, 1992, journal of Biochemical and physical methods 24: 107-117; and Brennan et al, 1985, Science 229: 81). Alternatively, the fragments may be produced directly in recombinant host cells. For example, Fab '-SH fragments can be recovered directly from E.coli (E.coli) and chemically coupled to form F (ab')₂Fragments (see, e.g., Carter et al, 1992, Bio/Technology10: 163-. By another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Other techniques for generating antibody fragments will be apparent to those skilled in the art.

Certain embodiments include F (ab') of a humanized anti-CD 40 antibody₂A fragment comprising the heavy chain sequence of any one of seq id No. 27, seq id No. 28, seq id No. 29 or seq id No. 30 and the light chain sequence of seq id No. 26. Alternative antibodies include those having the following: the heavy chain sequence is SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34 or SEQ ID NO. 35, and the light chain sequence is SEQ ID NO. 31. In other embodiments, humanized antibodies are provided having: a heavy chain sequence of SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 or SEQ ID NO. 40 and a light chain sequence of SEQ ID NO. 36. Such embodiments can include those comprising the F (ab')₂The whole antibody of (1).

In some embodiments, the antibody or antibody fragment comprises a constant region that mediates effector function. The constant region may provide an antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) response against a target cell expressing CD 40. The effector domain may be, for example, the Fc region of an Ig molecule. Typically, CD40 binding agents recruit and/or activate cytotoxic white blood cells (e.g., Natural Killer (NK) cells, phagocytic cells (e.g., macrophages), and/or serum complement components).

The effector domain of the antibody may be from any suitable vertebraAnimal species and isotypes. The ability of isoforms from different animal species to mediate effector functions varies. For example, the ability of a human immunoglobulin to mediate CDC and ADCC/ADCP, respectively, generally has the following order: IgM ≈ IgG₁≈IgG₃>IgG₂>IgG₄And IgG₁≈IgG₃>IgG₂/IgM/IgG₄. Murine immunoglobulins generally mediate CDC and ADCC/ADCP, respectively, in the following order: murine IgM ≈ IgG₃>>IgG_2b>IgG_2a>>IgG₁And IgG_2b>IgG_2a>IgG₁>>IgG₃. In another embodiment, murine IgG_2aMediate ADCC while murine IgG_2aAnd IgM mediates CDC.

Antibody modification

Humanized anti-CD 40 antibodies and medicaments may include modifications to a humanized anti-CD 40 antibody or antigen binding fragment thereof. For example, it may be desirable to modify an antibody with respect to effector function to enhance the effectiveness of the antibody in treating cancer. One such modification is the introduction of cysteine residues into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus generated may have improved internalization capacity and/or enhanced complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC). See, for example, Caron et al, 1992, j.expmed.176: 1191-1195; and scopes, 1992, j.immunol.148: 2918-2922. Hetero-bifunctional cross-linking agents can also be used to prepare homodimeric antibodies with enhanced anti-tumor activity, as described by Wolff et al, 1993, cancer research 53: 2560, 2565. Alternatively, the antibody can be engineered to contain dual Fc regions, thereby enhancing the complement lysis and ADCC capabilities of the antibody. See Stevenson et al, 1989, Anti-cancer great design 3: 219-230.

Antibodies with improved ability to support ADCC have been generated by modifying the glycosylation pattern of the Fc region of the antibody. This is possible because the antibody is at C_H2Glycosylation at asparagine residue N297 in the domain is involved in the interaction between IgG and Fc γ receptors necessary for ADCC. Host cell lines have been engineered to express antibodies with altered glycosylation, such as increased bisecting (bisecting) N-acetylglucosamine or decreased fucose. Decreasing fucose enhances ADCC activity to a greater extent than increasing the presence of bisecting N-acetylglucosamine. In addition, ADCC enhancement of low fucose antibodies was not associated with the Fc γ RIIIaV/F polymorphism.

Modifying the amino acid sequence in the Fc region of an antibody is an alternative to glycosylation engineering to enhance ADCC. The Fc gamma receptor has been determined in human IgG by extensive mutational analysis₁A binding site of (a). This resulted in the generation of humanized IgG with Fc mutations₁An antibody, said mutation increasing binding affinity to Fc γ RIIIa and enhancing ADCC in vitro. In addition, Fc variants have been obtained with a number of different alterations in binding properties, such as enhanced binding to specific Fc γ R receptors and unchanged or reduced binding to other Fc γ R receptors.

Another aspect includes an immunoconjugate comprising a humanized antibody or fragment thereof conjugated to a cytotoxic agent, such as a chemotherapeutic drug, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that may be used to form useful immunoconjugates include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Colletoxin A chain, α -sarcina, Aleurites fordii (Aleurites fordii) protein, Carcinia americana (Phytolacca) protein (PAPI, PAPII, and PAP-S), Momordica charantia (Momordicacharantia) inhibitors, curculin (curcin), crotin, Saponaria officinalis (Saonafia) inhibitors, gelonin, mitogellin, restrictocin, phenomycin (enomycin), trichothecene, etc. a variety of radionuclides useful for the production of such immunoconjugates include human anti-CD 84 antibodies²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶Re。

conjugates of humanized anti-CD 40 antibodies with cytotoxic or chemotherapeutic agents can be made by known methods using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3- (2-pyridyldimercapto) propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidates (e.g. dimethyl adipate HCL), active esters (e.g. disuccinimidyl suberate), aldehydes (e.g. glutaraldehyde), bis-azido compounds (e.g. bis (p-azidobenzoyl) hexanediamine), bis-azido derivatives (e.g. bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g. toluene 2, 6-diisocyanate) and bis-active fluorine compounds (e.g. 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be identified as vietta et al, 1987, Science 238: 1098, as described in the preceding paragraph. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugation of radionucleotides to antibodies. Conjugates can also be formed using a cleavable linker.

In another embodiment, the antibody may be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretargeting. In this procedure, an antibody-receptor conjugate is administered to a patient, followed by self-circulation removal of unbound conjugate using a clearing agent, and subsequent administration of a "ligand" (e.g., avidin) that selectively binds to the receptor, which is conjugated to a cytotoxic agent (e.g., a radionuclide).

The humanized anti-CD 40 antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in the following references: epstein et al, 1985, proc.natl.acad.sci.usa82: 3688; hwang et al, 1980, Proc.Natl.Acad.Sci.USA77: 4030; and U.S. patent nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed, for example, in U.S. patent No. 5,013,556.

Particularly useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter membrane with a specified pore size to obtain liposomes with the desired diameter. Fab' fragments of the antibodies disclosed herein can be combined with liposomes via disulfide interchange reactions, as described in Martin et al, 1982, j.biol.chem.257: 286-. Chemotherapeutic drugs (e.g., doxorubicin) are optionally contained within the liposomes. See, for example, Gabizon et al, 1989, J.NationalCancerInst.81(19): 1484.

The antibodies described and disclosed herein may also be used in ADEPT (antibody-directed enzyme prodrug therapy) procedures by conjugating the antibody with an enzyme that activates the prodrug, which converts the prodrug (e.g., a peptidyl chemotherapeutic drug) to an active anticancer drug. See, for example, WO81/01145, WO88/07378, and U.S. Pat. No. 4,975,278. The enzyme component of the immunoconjugates useful for ADEPT is an enzyme that acts on the prodrug to convert it to a more active, cytotoxic form. Specific enzymes useful for ADEPT include (but are not limited to): alkaline phosphatase for converting a phosphate-containing prodrug into a free drug; arylsulfatase for converting the sulfate-containing prodrug into a free drug; a cytosine deaminase for converting non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as Serratia (Serratia) protease, thermolysin, subtilisin, carboxypeptidase, and cathepsin (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs; a D-alanylcarboxypeptidase for converting a prodrug containing a D-amino acid substituent; carbohydrate-cleaving enzymes, such as β -galactosidase and neuraminidase, which are used to convert glycosylated prodrugs into free drugs; a beta-lactamase for converting a beta-lactam derived drug into a free drug; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, for converting drugs derivatized at the amine nitrogen with a phenoxyacetyl group or a phenylacetyl group, respectively, into free drugs. Alternatively, an antibody with enzymatic activity ("abzyme") can be used to convert the prodrug into a free active drug (see, e.g., Massey, 1987, Nature 328: 457-458). Antibody-abzyme conjugates can be prepared by known methods for delivering abzymes to a tumor cell population, for example, by covalently binding the enzyme to the humanized anti-CD 40 antibody/heterobifunctional cross-linking reagent described above. Alternatively, fusion proteins can be constructed using recombinant DNA techniques, which comprise at least the antigen binding region of an antibody disclosed herein and at least a functionally active portion of the above-described enzymes linked thereto (see, e.g., Neuberger et al, 1984, Nature 312: 604-608).

In certain embodiments, it may be desirable to use humanized anti-CD 40 antibody fragments rather than whole antibodies to, for example, promote tumor penetration. It may be desirable to modify antibody fragments to extend their serum half-life. This can be achieved, for example, by incorporating salvage receptor binding epitopes into the antibody fragment. In one approach, appropriate regions in the antibody fragment may be altered (e.g., mutated) or the epitope may be incorporated into a peptide tag, e.g., by DNA or peptide synthesis, which is then fused terminally or medially to the antibody fragment. See, for example, WO 96/32478.

In other embodiments, covalent modifications of the humanized anti-CD 40 antibody are also included. Covalent modifications include modifications to the following groups: cysteinyl, histidinyl, lysyl and amino-terminal residues, arginyl, tyrosyl, carboxyl-side groups (aspartyl or glutamyl), glutamyl and aspartyl residues or seryl or threonyl residues. Another type of covalent modification involves chemical or enzymatic coupling of the glycoside to an antibody. If applicable, the modification can be prepared by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modification of antibodies can be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or amino-or carboxy-terminal residues.

Removal of any carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically. Chemical deglycosylation is described in the following documents: hakimuddin et al, 1987, arch, biochem, biophysis, 259: 52 and Edge et al, 1981, anal. biochem., 118: 131. enzymatic cleavage of the carbohydrate moiety on antibodies can be achieved by using a variety of endoglycosidases and exoglycosidases, such as Thotakura et al, 1987, meth. enzymol 138: 350.

Another type of covalent modification that may be used comprises linking the antibody to one of a variety of non-proteinaceous polymers (e.g., polyethylene glycol, polypropylene glycol, or polyalkylene oxide) in a manner described in one or more of the following: U.S. Pat. No. 4,640,835, U.S. Pat. No. 4,496,689, U.S. Pat. No. 4,301,144, U.S. Pat. No. 4,670,417, U.S. Pat. No. 4,791,192, and U.S. Pat. No. 4,179,337.

Humanized and amino acid sequence variants

Amino acid sequence variants of the anti-CD 40 antibody can be prepared by introducing appropriate nucleotide changes into the anti-CD 40 antibody DNA or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the anti-CD 40 antibodies of the examples herein. Any combination of deletions, insertions, and substitutions are performed to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processes of the humanized or variant anti-CD 40 antibody, such as changing the number or position of glycosylation sites.

A method that can be used to identify certain residues or regions of preferred mutagenic positions in anti-CD 40 antibodies is known as "alanine scanning mutagenesis" as described in Cunningham and Wells (Science, 244: 1081-1085 (1989)). Here, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with a neutral or negatively charged amino acid (typically alanine) to affect the interaction of the amino acid with the CD40 antigen. Those amino acid positions that show functional sensitivity to substitution are then modified by introducing additional or other variants at or against the substitution site. Thus, where the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning mutagenesis or random mutagenesis is performed at the target codon or region and the expressed anti-CD 40 antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions (ranging in length from one residue to polypeptides containing hundreds or more residues) as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-CD 40 antibody fused to an epitope tag. Other insertional variants of the anti-CD 40 antibody molecule include fusions of enzymes or polypeptides that increase the serum half-life of the antibody to the N-or C-terminus of the anti-CD 40 antibody.

Another type of variant is an amino acid substitution variant. The variants have at least one amino acid residue removed from the anti-CD 40 antibody molecule and a different residue inserted at that position. The most interesting sites for substitution mutagenesis include the hypervariable regions, but also FR alterations are encompassed. Conservative substitutions are shown in table 5 under the heading "preferred substitutions". If the substitution results in an alteration in biological activity, more important alterations (designated "exemplary substitutions," or as further described below with reference to amino acid species) can be introduced and the product screened.

TABLE 5

In protein chemistry, it is generally accepted that the biological properties of an antibody can be achieved by selecting substitutions that differ significantly in their effect of maintaining the following properties: (a) the structure of the polypeptide backbone in the substitution region, e.g., in a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Natural residues are classified into the following classes according to common side chain properties:

(1) hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidity: asp, glu;

(4) alkalinity: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one of the classes for another.

Any cysteine residues not involved in maintaining the proper conformation of the humanized or variant anti-CD 40 antibody may also be substituted (typically with serine) to improve the oxidative stability of the molecule, prevent aberrant cross-linking, or provide a recognized point of incorporation with cytotoxic or cytostatic compounds. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

One type of substitutional variant involves substituting one or more hypervariable region residues in a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further development may have improved biological properties relative to the parent antibody from which they were generated. A convenient method of generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6 to 7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the encapsulated M13 gene III product within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which significantly facilitate antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to determine the contact point between the antibody and human CD 40. The contact residues and nearby residues are candidate substitution residues for the techniques described herein. Immediately after the variants are generated, the set of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another class of amino acid variants of antibodies alters the original glycosylation pattern of the antibody. By "altered" is meant the deletion of one or more carbohydrate moieties present in the antibody and/or the addition of one or more glycosylation sites not present in the antibody.

In some embodiments, it may be desirable to modify the antibodies of the invention to add glycosylation sites. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of an aspartic acid residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the side chain of aspartic acid. Thus, the presence of any of the tripeptide sequences in a polypeptide may result in a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Thus, to glycosylate a particular protein (e.g., an antibody), the amino acid sequence of the protein is engineered to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). It may also be altered (for O-linked glycosylation sites) by the addition or substitution of one or more serine or threonine residues in the original antibody sequence.

Nucleic acid molecules encoding amino acid sequence variants of anti-CD 40 antibodies are prepared by a variety of methods known in the art. The methods include, but are not limited to, isolation from a natural source (in the case of natural amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis, either by pre-prepared variant or non-variant forms of the anti-CD 40 antibody.

Polynucleotides, vectors, host cells and recombinant methods

Other embodiments contemplate that the antibody comprises a sequence encoding a humanized anti-CD 40 antibodyThe isolated polynucleotides listed, vectors and host cells comprising the polynucleotides, and recombinant techniques for producing humanized antibodies. The isolated polynucleotide may encode any desired form of an anti-CD 40 antibody, including, for example, full-length monoclonal antibodies, Fab ', F (ab')₂And Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

Some embodiments include an isolated polynucleotide comprising a sequence encoding an antibody or antibody fragment having the heavy chain variable region amino acid sequence of any one of: 1 to 4 of SEQ ID NO, 27 of SEQ ID NO, 28 of SEQ ID NO, 29 of SEQ ID NO, 30 of SEQ ID NO, 32 of SEQ ID NO, 33 of SEQ ID NO, 34 of SEQ ID NO, 35 of SEQ ID NO, 37 of SEQ ID NO, 38 of SEQ ID NO, 39 of SEQ ID NO or 40 of SEQ ID NO. Some embodiments include isolated polynucleotides comprising sequences encoding an antibody or antibody fragment having the light chain variable domain amino acid sequence of SEQ ID NO. 26, SEQ ID NO. 31, or SEQ ID NO. 36.

In one aspect, the isolated polynucleotide sequence encodes an antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31; SEQ ID NO. 37 and SEQ ID NO. 36; SEQ ID NO. 38 and SEQ ID NO. 36; SEQ ID NO:39 and SEQ ID NO: 36; SEQ ID NO. 40 and SEQ ID NO. 36.

A polynucleotide comprising a sequence encoding a humanized anti-CD 40 antibody or fragment or chain thereof may be fused to one or more regulatory or control sequences known in the art and may be contained in a suitable expression vector or host cell known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain may be independently fused to a polynucleotide sequence encoding a constant domain (e.g., a human constant domain) to allow for the production of a complete antibody. Alternatively, the polynucleotides or portions thereof may be fused together to provide a template for the production of single chain antibodies.

For recombinant production, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or expression. A variety of vectors suitable for expression of recombinant antibodies can be used. Carrier components generally include (but are not limited to) one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer component, a promoter, and a transcription termination sequence.

Humanized anti-CD 40 antibodies may also be produced as fusion polypeptides in which the antibody is fused to a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence of choice is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that are unable to recognize and process the signal sequence of the humanized anti-CD 40 antibody, the signal sequence may be replaced by a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leader sequence, and the like. For yeast secretion, the native signal sequence can be replaced by, for example, the following signals: the leaders from the yeast invertase alpha-factor (including Saccharomyces (Saccharomyces) and Kluyveromyces (Kluyveromyces) alpha-factor leaders), acid phosphatase, Candida albicans (C.albicans) glucoamylase or the signals described in WO 90/13646. In mammalian cells, mammalian signal sequences can be used as well as viral secretory leaders (e.g., the herpes simplex virus gD signal). The DNA of the precursor region is joined in reading frame to DNA encoding the humanized anti-CD 40 antibody.

Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomously replicating sequence. Such sequences for a variety of bacteria, yeasts and viruses are well known in the art. The origin of replication from the plasmid pBR322 is suitable for most Gram (Gram) negative bacteria, the 2- υ plasmid origin is suitable for yeast, and a variety of viral origins (SV40, polyoma, adenovirus, VSV, and BPV) can be used to clone vectors in mammalian cells. Typically, mammalian expression vectors do not require an origin of replication component (typically only the SV40 origin may be used because it contains an early promoter).

Expression and cloning vectors may contain genes encoding selectable markers to facilitate identification of expression. Typical selectable marker genes encode the following proteins: conferring resistance to antibiotics or other toxins (e.g., ampicillin (ampicillin), neomycin, methotrexate, or tetracycline); alternatively, a complement that is auxotrophic; or in other alternatives to supply specific nutrients not present in the complex medium, such as a gene encoding the D-alanine racemase for Bacilli (Bacillus).

One example of a selection scheme utilizes a drug to prevent growth of the host cell. Those cells successfully transformed with the heterologous gene produce a protein conferring drug resistance and can therefore survive this selection. The dominant selection example uses the drugs neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those that allow identification of cells that can take up the nucleic acid encoding the humanized anti-CD 40 antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and-II (e.g., primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like. Cells transformed with the DHFR selection gene were first identified by culturing all transformants in medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (e.g., DG 44).

Alternatively, host cells transformed or co-transformed with DNA sequences encoding an anti-CD 40 antibody, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3' -phosphotransferase (APH), particularly wild-type hosts containing endogenous DHFR, may be selected by growing the cells in media containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin (kanamycin), neomycin, or G418. See, for example, U.S. patent No. 4,965,199.

If recombinant production is carried out using yeast cells as host cells, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb et al, 1979, Nature 282: 39) can be used as a selectable marker. The TRP1 gene provides a selection marker for yeast mutant strains that lack the ability to grow in tryptophan (e.g., ATCC No. 44076 or PEP4-1) (Jones, 1977, Genetics85: 12). The presence of a trp1 lesion in the yeast host cell genome provides an effective environment to detect transformation by growth in the absence of tryptophan. Similarly, Leu2p deficient yeast strains (e.g., ATCC20,622 or 38,626) were complemented by known plasmids having the Leu2 gene.

In addition, Kluyveromyces may be transformed with a vector derived from the 1.6 μm circular plasmid pKD 1. Alternatively, expression systems for large scale production of recombinant bovine chymosin have been reported to be useful in Kluyveromyces lactis (K.lactis.) (VandenBerg, 1990, Bio/Technology8: 135). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of the genus Kluyveromyces have also been disclosed (Fleer et al, 1991, Bio/Technology9: 968-.

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid molecule encoding an anti-CD 40 antibody or polypeptide chain thereof. Promoters suitable for use in prokaryotic hosts include the phoA promoter, the beta-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters (e.g., the tac promoter). Other known bacterial promoters are also suitable. Promoters for use in bacterial systems may also contain Shine-Dalgamo (s.d.) sequences operably linked to DNA encoding the humanized anti-CD 40 antibody.

Various eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream from the transcription start site. Another sequence found 70 to 80 bases upstream from the transcription start point of many genes is a CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence, possibly adding to the 3' end of the coding sequence the signal of the poly a tail. All of the sequences are inserted into eukaryotic expression vectors in a suitable manner.

Examples of suitable promoter sequences for yeast hosts include the promoters for the following enzymes: 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Another advantage of inducible promoters is that transcription is controlled by growth conditions. The promoter includes the yeast promoter regions of the following enzymes: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, nitrogen metabolism-related derivative enzymes, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP73,657. Yeast enhancers may also be used advantageously with yeast promoters.

Transcription of humanized anti-CD 40 antibodies from vectors in mammalian host cells is controlled, for example, by promoters obtained from: viral genomes, such as polyoma, fowlpox, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian Virus 40(SV 40); a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter; a heat shock promoter, provided that the promoter is compatible with a host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments, which also contain the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIIIE restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. patent No. 4,419,446. A modification of this system is described in U.S. patent No. 4,601,978. See also Reyes et al, 1982, Nature297: 598-. Alternatively, the rous sarcoma virus (Roussarcomavir) long terminal repeat can be used as a promoter.

Another useful component that can be used in recombinant expression vectors is an enhancer sequence, which is used to increase transcription of DNA encoding the humanized anti-CD 40 antibody in higher eukaryotes. Various enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer (bp100-270) located late in the replication origin (lateside), the cytomegalovirus early promoter enhancer, the polyoma enhancer located late in the replication origin, and the adenovirus enhancer. A description of the enhancer modules for the activation of eukaryotic promoters can also be found in Yaniv, 1982, Nature297: 17-18. The enhancer may be spliced into the vector at a position 5' or 3' to the sequence encoding the humanized anti-CD 40 antibody, but is preferably located at a site 5' to the promoter.

Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human cells or nucleated cells from other multicellular organisms) may also contain sequences necessary for termination of transcription and for stabilizing the mRNA. The sequences are generally obtained from the 5 'and occasionally the 3' untranslated regions of eukaryotic or viral DNA or cDNA. The region contains a nucleotide segment that is transcribed as a polyadenylated fragment in the untranslated portion of the mRNA encoding the anti-CD 40 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and expression vectors disclosed therein. In some embodiments, the humanized anti-CD 40 antibody can be expressed using the CHEF system. (see, for example, U.S. patent No. 5,888,809, the disclosure of which is incorporated herein by reference.)

Suitable host cells for cloning or expressing the DNA in the vectors herein are the above mentioned prokaryotic cells, yeast cells or higher eukaryotic cells. Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example: enterobacteriaceae (Enterobacteriaceae), for example Escherichia (Escherichia) (e.g.Escherichia coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (e.g.Salmonella typhimurium), Serratia (e.g.Serratia marcescens) and Shigella (Shigella), and Bacillus (e.g.Bacillus subtilis) and Bacillus licheniformis (e.g.Bacillus subtilis 41P disclosed in lichen 266,710 published 4.12.1989), Pseudomonas (e.g.Pseudomonas) and Streptomyces (Streptomyces). A preferred E.coli cloning host is E.coli 294(ATCC31,446), but other strains are also suitable, such as E.coli B, E.coli X1776(ATCC31,537) and E.coli W3110(ATCC27,325). The described embodiments are illustrative and not restrictive.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding humanized anti-CD 40 antibodies. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. However, a variety of other genera, species and strains can generally be used and are useful herein, such as Schizosaccharomyces (Schizosaccharomyces pombe); kluyveromyces hosts such as kluyveromyces lactis, kluyveromyces fragilis (k.fragilis) (ATCC12,424), kluyveromyces bulgaricus (k.bulgaricus) (ATCC16,045), kluyveromyces wilkerii (k.wickramii) (ATCC24,178), kluyveromyces farinosus (k.walltii) (ATCC56,500), kluyveromyces drosophilus (k.drosophilarium) (ATCC36,906), kluyveromyces thermotolerans (k.thermotolerans), and kluyveromyces marxianus (k.marxianus); yarrowia (EP402,226); pichia pastoris (Pichia pastoris) (EP183,070); candida (Candida); trichoderma reesei (Trichoderma reesei) (EP244,234); neurospora crassa (Neurosporacrassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis (Schwanniomyces occidentalis); and filamentous fungi such as Neurospora (Neurospora), Penicillium (Penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) hosts (e.g., Aspergillus nidulans (a. nidulans) and Aspergillus niger (a. niger)).

Suitable host cells expressing the glycosylated humanized anti-CD 40 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells, including, for example, various baculovirus strains and variants and corresponding permissive insect host cells from, for example, the following hosts: spodoptera frugiperda (spodoptera rugerida) (caterpillars), aedes aegypti (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (drosophila melanogaster), and bombyx mori (silkworms). Various strains of transfection virus are available from published sources, such as the L-1 variant of Autographa californica (NPV) and the Bm-5 strain of Bombyx mori NPV, and are particularly useful for transfecting Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

In another aspect, expression of humanized anti-CD 40 is performed in a vertebrate cell. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure and technology is widely available. Examples of mammalian host cell lines which may be used are monkey kidney CV1 transformed by SV40 (COS-7, ATCCRL 1651), human embryonic kidney lines (293 lines or 293 cells subcloned for growth in suspension culture, Graham et al, 1977, J.GenVirol.36: 59), baby hamster kidney cells (BHK, ATCCCL 10), Chinese hamster ovary cells/-DHFR 1(CHO, Urlaub et al, 1980, Proc.Natl.Acad.Sci.USA77: 4216; e.g.DG 44), mouse testis support cells (TM4, Mather, 1980, biol.Reprod.23: 243) 251), MDCC 1 CCCL70, African green monkey kidney cells (ATCO-76, CCRL-1587), human cervical cancer cells (HELA, CCCL2), canine kidney cells (CCCK, CCCC) 539), bovine mouse kidney cells (MRC 1 CCCL 3575), human mammary mouse lung cells (ATCCTr) 7372, mCRT 35383, mCCTR 2, ATCC No. 35, ATCC No. 25, ATCC No. 2, ATCC No. 25, ATCC No. 2, No. 2, FS4 cells and a human hepatoma line (HepG 2).

Host cells are transformed with the above-described expression or cloning vectors for the production of humanized anti-CD 40 antibodies and cultured in common nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

The host cells used to produce the humanized anti-CD 40 antibodies described herein can be cultured in a variety of media. Commercially available media are suitable for culturing host cells, such as Ham's F10(Sigma-Aldrich, St. Louis, Mo.), minimal essential medium ((MEM), Sigma-Aldrich), RPMI-1640 (Sigma-Aldrich), and Dulbecco's modified eagle's Medium (((DMEM), Sigma-Aldrich). In addition, any of the media described in one or more of the following references may be used as the host cell culture medium: ham et al, 1979, meth.enz.58: 44; barnes et al, 1980, anal. biochem.102: 255; U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, U.S. Pat. No. 5,122,469, WO90/103430 and WO 87/00195. Any of the media may be supplemented as necessary with hormones and/or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium salts, magnesium salts, and phosphate salts), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc., are those previously used for the selected host cell for expression and will be apparent to those skilled in the art.

When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced within the cell, the cell may be disrupted as a first step to release the protein. Particulate debris, i.e., host cells or lysed fragments, can be removed by centrifugation or ultrafiltration. Carter et al, 1992, Bio/Technology10:163-167 describe the procedure for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (ph3.5), EDTA, and phenylmethanesulfonyl fluoride (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation. In case the antibody is secreted into the culture medium, the supernatant from the expression system is typically first concentrated using a commercially available protein concentration filter (e.g., Amicon or millipore pellicon ultrafiltration unit). A protease inhibitor such as PMSF may be included in any of the preceding steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of adventitious contaminants. Antibodies can be isolated from host cells using a variety of methods.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a common purification technique. The suitability of protein a as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (see, e.g., Lindmark et al, 1983J. Immunol. meth.62: 1-13). Protein G is recommended for all mouse isoforms and human gamma 3 (see, e.g., Guss et al, 1986 EMBO.5: 1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices may be used. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) benzene, allow faster flow rates and shorter processing times than can be achieved using agarose. Provided that the antibody comprises C_H3Domain, then BakerbondABX^TMResins (j.t.baker, phillips burg, n.j.) can be used for purification. Depending on the antibody to be recovered, other protein purification techniques may also be used, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin Sepharose^TMChromatography, anion or cation exchange resins (e.g. polyaspartic acid)Column) chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preliminary purification step, the mixture comprising the target antibody and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 and 4.5, which is typically performed at low salt concentrations (e.g., about 0-0.25M salt).

Also included are nucleic acids that hybridize under low, medium, and high stringency conditions as defined herein to all or a portion of a nucleotide sequence (e.g., a portion encoding a variable region) represented as an isolated polynucleotide sequence encoding an antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain comprising the following amino acid sequences, respectively: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31; SEQ ID NO. 37 and SEQ ID NO. 36; SEQ ID NO. 38 and SEQ ID NO. 36; SEQ ID NO:39 and SEQ ID NO: 36; SEQ ID NO. 40 and SEQ ID NO. 36. The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80% (e.g., at least 90%, at least 95%, or at least 98%) identical to the sequence of a portion or all of the nucleic acid encoding the anti-CD 40 polypeptide (e.g., heavy or light chain variable region) or its complement. Hybridizing nucleic acids of the type described herein may be used, for example, as cloning probes, primers (e.g., PCR primers), or diagnostic probes.

Some embodiments include an isolated polynucleotide comprising a sequence encoding an antibody or antibody fragment having a heavy chain variable region amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of: 1 to 4 of SEQ ID NO, 27 of SEQ ID NO, 28 of SEQ ID NO, 29 of SEQ ID NO, 30 of SEQ ID NO, 32 of SEQ ID NO, 33 of SEQ ID NO, 34 of SEQ ID NO, 35 of SEQ ID NO, 37 of SEQ ID NO, 38 of SEQ ID NO, 39 of SEQ ID NO or 40 of SEQ ID NO. Some embodiments include an isolated polynucleotide comprising a sequence encoding an antibody or antibody fragment having a light chain variable domain amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of: SEQ ID NO. 5 to 8, SEQ ID NO. 26, SEQ ID NO. 31 or SEQ ID NO. 36.

In one aspect, the isolated polynucleotide sequence encodes an antibody or antibody fragment having a heavy chain variable domain and a light chain variable region each comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of an antibody or antibody fragment having a heavy chain variable domain and a light chain variable region, respectively, comprising the amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31; SEQ ID NO. 37 and SEQ ID NO. 36; SEQ ID NO. 38 and SEQ ID NO. 36; SEQ ID NO:39 and SEQ ID NO: 36; SEQ ID NO. 40 and SEQ ID NO. 36.

In another aspect, the invention relates to a polynucleotide as described in the embodiments of the previous paragraph, wherein the encoded antibody or antibody fragment comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of an antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain, respectively, comprising the amino acid sequences: in one embodiment the sequences are SEQ ID NO 27 and SEQ ID NO 26, respectively; in another embodiment, SEQ ID NO 28 and SEQ ID NO 26, respectively; in another embodiment, SEQ ID NO. 29 and SEQ ID NO. 26; in another embodiment, SEQ ID NO 30 and SEQ ID NO 26, respectively; in another embodiment, SEQ ID NO:32 and SEQ ID NO:31, respectively; in another embodiment, SEQ ID NO 33 and SEQ ID NO 31; in another embodiment, SEQ ID NO:34 and SEQ ID NO:31, respectively; in another embodiment, SEQ ID NO 35 and SEQ ID NO 31; in another embodiment, SEQ ID NO 37 and SEQ ID NO 36, respectively; in another embodiment, SEQ ID NO 38 and SEQ ID NO 36, respectively; in another embodiment, SEQ ID NO:39 and SEQ ID NO:36, respectively; and in another embodiment SEQ ID NO 40 and SEQ ID NO 36, respectively.

The term "identical" or "percent identity" as used herein in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned (for maximum correspondence). To determine percent identity, the sequences are aligned for optimal alignment purposes (e.g., gaps can be introduced in a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then aligned. A position in the first sequence is identical to a corresponding position in the second sequence if the position is occupied by the same amino acid residue or nucleotide at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity% = number of identical positions/total number of positions (e.g., number of overlapping positions) × 100). In some embodiments, the two sequences being compared are the same length after gaps have been introduced in the sequences, as appropriate (e.g., excluding additional sequences that extend beyond the sequences being compared). For example, in comparing variable region sequences, leader sequences and/or constant domain sequences are not considered. In a sequence alignment between two sequences, a "corresponding" CDR refers to a CDR at the same position in both sequences (e.g., CDR-H1 for each sequence).

Determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. Preferred non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87: 2264. 2268), which are based on the algorithms of Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873 and 5877. This algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al (1990, J.mol.biol.215: 403-. BLAST nucleotide search can be performed using NBLAST program (score =100, word length =12) to obtain nucleotide sequences homologous to nucleic acids encoding proteins of interest. BLAST protein searches can be performed using the XBLAST program (score =50, word length =3) to obtain amino acid sequences homologous to the protein of interest. For comparison purposes, to obtain gap-added alignment, the alignment can be determined, for example, by Altschul et al, 1997, nucleic acids as cidsRes.25: 3389 GappedBLAST was used as described in 3402. Alternatively, PSI-Blast can be used to perform an iterative search that detects distant contacts between molecules (same article). When utilizing BLAST, gappedBLAST, and PSI-BLAST programs, the preset parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred non-limiting example for alignment sequences is the algorithm of Myers and Miller (CABIOS (1989)). This algorithm has been incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using an alignment program to compare amino acid sequences, a PAM120 weighted residue table, a gap length penalty equal to 12, and a gap penalty equal to 4 can be used. Other algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, such as Torellis and Robotti, 1994, comput.appl.biosci.10: 3-5; and FASTA, such as Pearson and Lipman, 1988, proc.natl.acad.sci.usa 85: 2444-8. Within FASTA, ktup is a control option to set sensitivity and search speed. If ktup =2, finding similar regions in the two aligned sequences by examining the alignment residue pairs; if ktup =1, a single aligned amino acid is checked. Ktup may be set to 2 or 1 for protein sequences, or 1 to 6 for DNA sequences. If ktup is not specified, the default is 2 for protein and 6 for DNA. Alternatively, protein sequence alignment can be performed using the CLUSTALW algorithm, as described by Higgins et al, 1996, methods enzymol.266: 383-402.

Non-therapeutic applications

The antibodies described herein can be used as affinity purifiers. In this method, the antibody is immobilized on a solid phase such as a protein A resin using methods well known in the art. The immobilized antibody is contacted with a sample containing the CD40 protein (or fragment thereof) to be purified, and the carrier is subsequently washed with a suitable solvent, whereby substantially all material in the sample other than the CD40 protein bound to the immobilized antibody can be removed. Finally, the carrier is washed with another suitable solvent, thereby releasing the CD40 protein from the antibody.

Humanized anti-CD 40 antibodies may also be used in diagnostic assays to detect and/or quantify CD40 protein, e.g., to detect CD40 expression in specific cells, tissues or serum.

In some embodiments, for example for diagnostic purposes, it may be advantageous to label the antibody with a detectable moiety. A variety of detectable labels may be used, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The label can be indirectly conjugated to the antibody using a variety of known techniques. For example, an antibody may be conjugated to biotin and any of the three broad classes of labels described above may be conjugated to avidin, or vice versa. Biotin binds selectively to avidin and the label can thus be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody may be conjugated to a smaller hapten (e.g., digoxin) and one different type of the label described above may be conjugated to an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label to the antibody can be achieved.

Exemplary radioisotope labels include³⁵S、¹⁴C、¹²⁵I、³H and¹³¹I. the antibodies can be labeled with radioisotopes using, for example, techniques described in the following documents: currentprotocol immunology, volumes 1 and 2, 1991, edited by Coligen et al, Wiley-Interscience, New York, N.Y., Pubs. Radioactivity can be measured, for example, by scintillation counting.

Exemplary fluorescent labels include those from: rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine (rhodamine) and its derivatives, dansyl (dansyl), Lissamine (Lissamine), phycoerythrin, and texas red (TexasRed). The fluorescent label can be conjugated to the antibody via known techniques, such as those disclosed in currentprotocol immunology (supra). A fluorometer can be used to quantify fluorescence.

A variety of well-characterized enzyme-substrate labels are known in the art (for example, for review see U.S. patent No. 4,275,149). Enzymes generally catalyze the development of a chromogenic substrate with a chemical change that can be measured using a variety of techniques. For example, the change may be a change in color of the substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. A chemiluminescent substrate can be electronically excited by a chemical reaction and can subsequently emit light that can be measured using, for example, a chemiluminescence meter or provide energy to a fluorescent acceptor.

Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferases, U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. The technology of conjugating enzymes with antibodies is described, for example, in the following documents: o' Sullivan et al, 1981, methods for the preparation of enzyme-antibody conjugate for use in enzyme elmunoassay, methods in enzyme (edited by j.langone and h.vanvunaks), Academicpress, n.y., 73: 147-166.

Examples of enzyme-substrate combinations include (for example): horseradish peroxidase (HRPO) with catalase as a substrate, wherein the catalase oxidizes a dye precursor (e.g., o-phenylenediamine (OPD) or 3,3',5,5' -tetramethylbenzidine hydrochloride (TMB)); alkaline Phosphatase (AP) with p-nitrophenyl phosphate as chromogenic substrate; and β -D-galactosidase (. beta. -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-. beta. -D-galactosidase or fluorogenic substrate 4-methylumbelliferyl-. beta. -D-galactosidase).

One skilled in the art will appreciate a variety of other enzyme-substrate combinations. A general review of such combinations is found in U.S. patent No. 4,275,149 and U.S. patent No. 4,318,980.

In another embodiment, an unlabeled humanized anti-CD 40 antibody is used and detected with a labeled antibody that binds the humanized anti-CD 40 antibody.

The antibodies described herein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich (sandwich) assays, and immunoprecipitation assays. See, for example, Zola, Monoclonal antibodies: amanaloftechnologies, p.147-.

Diagnostic kit

The humanized anti-CD 40 antibody can be used in a diagnostic kit, i.e., a packaged combination of a predetermined amount of reagents and instructions for performing a diagnostic assay. If the antibody is labeled with an enzyme, the kit may include the substrate and cofactor required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like. The relative amounts of the various reagents can vary widely so that the concentration of the reagents in solution can significantly optimize the sensitivity of the assay. The reagents may be provided in dry powder form (typically by lyophilization) which includes excipients which, upon dissolution, provide a solution of the reagents with a suitable concentration.

Therapeutic uses

In another embodiment, the humanized anti-CD 40 antibodies disclosed herein can be used to treat various disorders associated with the expression of CD40 as described herein.

The humanized anti-CD 40 antibody or drug is administered by any suitable means, including parenterally, subcutaneously, intraperitoneally, intrapulmonary, and intranasally, and if local immunosuppressive therapy is desired, intralesional administration (including infusion or otherwise contacting the graft with the antibody prior to transplantation) is performed. The humanized anti-CD 40 antibody or drug may be administered, for example, in infusion or bolus form. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the humanized anti-CD 40 antibody is suitably administered by pulse infusion, particularly at decreasing doses of the antibody. In one aspect, administration is effected by injection, most preferably intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term.

In preventing or treating a disease, the appropriate dosage of the antibody may depend on a variety of factors, such as the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, as defined above, and the discretion of the attending physician. The antibody is administered to the patient at one time or in an appropriate manner over a series of treatments.

Depending on the type and severity of the disease, the initial candidate dose of antibody administered to the patient is about 1 μ g/kg to 20mg/kg (e.g., 0.1-15mg/kg), whether administered, for example, by one or more divided administrations or by continuous infusion. Depending on the factors mentioned above, typical daily doses may range from about 1 μ g/kg to 100mg/kg or higher. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may be used. The progress of this therapy can be readily monitored by conventional techniques and assays. Exemplary dosage regimens are disclosed in WO 94/04188.

The term "inhibit" as used herein in the same context as "ameliorate" and "reduce" means reduce one or more characteristics of a disease.

The antibody compositions can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause, the site of delivery of the drug, the method of administration, the timing of administration, and other factors known to the practitioner. The "therapeutically effective amount" to be administered of the antibody may depend on the factors and is the minimum amount required to prevent, ameliorate or treat a condition associated with CD40 expression.

The antibody need not be, but is optionally, formulated with one or more drugs currently used to prevent or treat the disorder. The effective amount of the additional agent will depend on the amount of humanized anti-CD 40 antibody present in the formulation, the type of disorder or treatment, and other factors described above. Such other drugs are typically used at the same doses and routes of administration as used above or at about 1% to 99% of the doses used above.

CD40 related disorders

anti-CD 40 antibodies or drugs can be used to treat or prevent CD 40-expressing cancers or immune disorders characterized by expression of CD40, for example, due to inappropriate activation of immune cells (e.g., lymphocytes or dendritic cells). The expression of CD40 may be due, for example, to an increased level of CD40 protein on the cell surface and/or to altered antigenicity of the expressed CD 40. Treatment or prevention of an immune disorder according to the methods described herein is achieved by administering to an individual in need of such treatment or prevention an effective amount of an anti-CD 40 antibody or medicament, whereby the antibody (i) binds to an activated immune cell expressing CD40 and associated with the disease state, and (ii) exerts a cytotoxic, cytostatic, or immunosuppressive effect on the activated immune cell.

Immune diseases characterized by inappropriate activation of immune cells and which can be treated or prevented by the methods described herein can be classified according to, for example, the type of underlying hypersensitivity reaction of the disorder. The reactions can be generally classified into four categories: allergic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cell-mediated immune (CMI) reactions (also known as delayed-type hypersensitivity (DTH) reactions). (see, e.g., fundamentals immunology (edited by William E. Paul, ravenPress, N.Y., 3 rd edition, 1993))

Specific examples of the immune diseases include the following diseases: rheumatoid arthritis, autoimmune demyelinating diseases (e.g., multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveal retinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hematologic diseases, inflammatory bowel diseases (e.g., crohn's disease or ulcerative colitis), anaphylaxis, Sjogren's syndrome, type I diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine deficiency, Schmidt's syndrome, autoimmune uveitis, addison's disease, epinephrine, thyroiditis, hashimoto's thyroiditis, autoimmune thyroiditis, pernicious anemia, gastric atrophy, and the like, Chronic hepatitis, lupus-like hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, deslerian syndrome (Dressler's disease), autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dyskinesia, fingertip sclerosis, and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, dermatitis, atopic rhinitis, Goodpasture's syndrome (Goodpasture's disease), Chagas 'disease, sarcoidosis, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's disease, Rheumatic fever, asthma, recurrent abortion, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, aviary lung, toxic epidermal necrolysis, Alport's syndrome (Alport' ssyndrome), alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu 'sarteris, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, triple aspirin (Samper' ssdrome), eczema, lymphomatoid granulomatosis, Behcet's disease, Kaplan's syndrome (Caplan 'ssdrome), Kawasaki's disease, dengue fever, encephalomyelitis, and encephalomyelitis, Endocardial myocardial fibrosis, endophthalmitis, persistent elevated erythema, psoriasis, fetal erythroblastosis, eosinophilic fasciitis, post-transfusion purpura syndrome (Shulman's sydrome), Felty's syndrome (Felty's sydrome), filariasis, cyclitis, chronic cyclitis, heteroclitis, focuss's cyclitis (Fuch's), IgA nephropathy, henoschonlein purpura (Henoch-schoneinpurpa), graft-versus-host disease, transplant rejection, cardiomyopathy, myasthenia syndrome (Eaton-Lambertsyndrome), recurrent polychondritis, cryoglobulinemia, Waldenstrom's macroglobulinemia, Evan's syndrome (Evan' ssynme), acute respiratory distress syndrome, pulmonary inflammation, osteoporosis, delayed hypersensitivity, and gonadal autoimmunity.

Thus, the methods described herein encompass the treatment of disorders of the following cells: b lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th₁Lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, hashimoto's thyroiditis, Graves 'disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease) or Th₂Lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft-versus-host disease). Generally, disorders involving dendritic cells involve Th₁Lymphocytes or Th₂Disorders of lymphocytes.

Rheumatoid Arthritis (RA) is one of the most common inflammatory autoimmune diseases, affecting about 1% of the population. Despite the availability of effective treatments (e.g., MTX and anti-TNF drugs), there remains a substantial unmet medical need, particularly for those patients who do not respond adequately to anti-TNF therapy (about 30% of patients). In addition, up to 50% of patients discontinue TNF antagonist therapy within 5 years, primarily due to adverse events, but also due to the increasing number of patients who have been shown to lose therapeutic benefit. Therefore, it is important to establish effective therapies that target inflammation and joint damage in RA, and do not rely solely on direct inhibition of TNF. A very attractive approach is to target the costimulatory cellular pathway. One of the key receptor-ligand pairs in co-stimulation is CD40/CD 40L. This system allows interactions between immune cells and non-immune cells, which are important in the pathogenesis of RA. Blocking CD40 with the antagonist antibodies of the invention may have one or more of the following effects in RA:

1) inhibition of B cell differentiation and antibody isotype switching;

2) inhibition of cytokine and chemokine production and adhesion molecule upregulation in T cells and macrophages;

3) inhibiting dendritic cell activation; and

4) inhibiting the production of pro-inflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandins and down-regulating adhesion molecules in non-immune cells such as epithelial, endothelial and stromal cells.

Methods of achieving one or more of the above effects are expressly contemplated herein. In addition to RA, the compositions of the present invention are particularly useful in methods of treating multiple sclerosis, psoriasis (including psoriatic arthritis), juvenile rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and solid organ transplantation.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease with a prevalence of about 1% in adults. The disease continues to cause significant morbidity and early mortality (death is primarily due to accelerated cardiovascular disease). It has been determined that joint damage occurs very early in the course of the disease, with radiographic evidence showing bone erosion in up to 30% of patients at diagnosis, and increasing to 60% after 1 year. Current guidelines recommend initiation of treatment with traditional disease modifying antirheumatic drugs (DMARDs) within 3 months after a definitive diagnosis has been confirmed. DMARDs may reduce or prevent joint damage and maintain joint function. Currently, the rheumatologist chooses Methotrexate (MTX) as the initial DMARD therapy for most patients.

TNF antagonist etanercept (etanercept)Infliximab (infliximab)Adalimumab (adalimumab)CTLA4 antagonist abatacept (abatacept)anti-IL-6 receptor mAb tuzumab (tocilizumab) and anti-CD 20mAb rituximab (rituximab)Can be used for treating RA. Current guidelines generally recommend the use of a biological DMARD to treat active RA after finding an inadequate response to conventional DMARDs.

Recent studies in patients with early invasive RA who have not previously received MTX treatment have shown that the combination of MTX and TNF antagonists is superior to monotherapy using either. The most striking result is that combination therapy has significant radiological benefits. Therefore, a combination of MTX and TNF inhibitors should be used in patients with invasive disease and the greatest risk of invasive phenotype (e.g., high activity score, impaired function, seropositive for Rheumatoid Factor (RF) or anti-cyclic citrullinated peptide antibody (CCP), elevated CRP, radiographic erosion). However, it is expected that in clinical practice TNF antagonists will rarely be used as first-line therapies. An investigation conducted by american rheumatologists at month 4 of 2005 showed that the factors that most affect the decision to use TNF antagonists were: failure of MTX or multiple DMARDs, overall physician evaluation, impaired function, and radiographic deterioration or erosion. Currently, it is estimated that 20% of RA patients in the united states receive TNF inhibitor therapy.

Current treatments, including biological therapies, are not sufficiently helpful for most RA patients due to drug intolerance and drug toxicity or lack of response. Up to 50% of patients discontinue TNF antagonist therapy within 5 years, primarily due to adverse events, but also due to the increasing number of patients who have demonstrated a loss of response.

In some embodiments, the immune disorder is a T cell-mediated immune disorder, e.g., a T cell disorder, wherein activated T cells associated with the disorder express CD 40. An anti-CD 40 antibody or drug may be administered to exclude the activated T cells expressing CD 40. In a specific embodiment, administration of the anti-CD 40 antibody or drug excludes activated T cells expressing CD40, while the anti-CD 40 or drug excludes substantially resting T cells. In this context, "substantially nonexclusive" means that less than about 60% or less than about 70% or less than about 80% of the resting T cells are nonexclusive.

The anti-CD 40 antibodies and medicaments described herein may also be used to treat or prevent CD40 expressing cancers. Treating or preventing a CD 40-expressing cancer according to the methods described herein is achieved by administering to an individual in need of such treatment or prevention an effective amount of an anti-CD 40 antibody or drug, whereby (i) the antibody or drug binds to a CD 40-expressing cancer cell, and (ii) a cytotoxic or cytostatic effect is produced to exclude or inhibit proliferation of a CD 40-expressing cancer cell.

CD 40-expressing cancers that can be treated or prevented by the methods described herein include, for example, leukemias, such as acute leukemia, acute lymphocytic leukemia, acute myelogenous leukemia (e.g., myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, or erythroleukemia), chronic leukemia, chronic myelogenous (myelogenous) leukemia, or chronic lymphocytic leukemia; polycythemia vera; lymphoma (e.g., hodgkin's disease or non-hodgkin's disease); multiple myeloma, waldenstrom's macroglobulinemia; heavy chain disease; solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor (Ewing ' stumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, renal cell cancer, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms ' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, cervical cancer, Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma (menengioma), melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma or esophageal carcinoma).

Pharmaceutical compositions and administration thereof

A composition comprising a CD40 binding agent (e.g., an anti-CD 40 antibody) can be administered to an individual having an immune disorder or a CD 40-expressing cancer or at risk of developing the disease. The invention further provides the use of a CD40 binding agent (e.g., an anti-CD 40 antibody) in the manufacture of a medicament for the prevention or treatment of a CD40 expressing cancer or an immune disorder. The term "individual" as used herein means any mammalian patient to which a CD40 binding agent may be administered, including, for example, humans and non-human mammals, such as primates, rodents, and dogs. Individuals specifically intended to be treated using the methods described herein include humans. In the prevention or treatment of an immune disorder or a CD40 expressing cancer, the antibody or drug may be administered alone or in combination with other compositions.

Preferred antibodies for use in the pharmaceutical composition are those humanized antibodies or antibody fragments comprising a heavy chain variable region amino acid sequence having any one of: 1 to 4 of SEQ ID NO, 27 of SEQ ID NO, 28 of SEQ ID NO, 29 of SEQ ID NO, 30 of SEQ ID NO, 32 of SEQ ID NO, 33 of SEQ ID NO, 34 of SEQ ID NO, 35 of SEQ ID NO, 37 of SEQ ID NO, 38 of SEQ ID NO, 39 of SEQ ID NO or 40 of SEQ ID NO.

Some embodiments include isolated polynucleotides comprising sequences encoding an antibody or antibody fragment having the light chain variable domain amino acid sequence of SEQ ID NO. 26, SEQ ID NO. 31, or SEQ ID NO. 36. Particularly preferred humanized antibody compositions comprise an antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the following amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31; SEQ ID NO. 37 and SEQ ID NO. 36; SEQ ID NO. 38 and SEQ ID NO. 36; SEQ ID NO:39 and SEQ ID NO: 36; SEQ ID NO. 40 and SEQ ID NO. 36. The invention encompasses isolated polynucleotides encoding any one of the following heavy chain sequences: SEQ ID NO. 1 to 4, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, or SEQ ID NO. 73. Other embodiments relate to isolated nucleic acids encoding a light chain sequence of any one of seq id nos: SEQ ID NO. 5 to SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 74, SEQ ID NO. 75, or SEQ ID NO. 76.

In certain embodiments, the compositions of the invention can be used in methods to reduce signs and symptoms, induce a major clinical response and reduce the progression of structural damage in patients with moderate to severe active RA and who do not respond adequately to MTX alone, provided that RA is intended to be treated. Examples of current such therapies are: Enbrel/Humira (data using Humira and Enbrel in two cases)Obtained from the same patient population). The compositions of the invention may be used in place of or in combination with Enbrel/Humira therapy for individuals who are non-responsive to MTX alone. Preferably, in such embodiments, the efficacy of the compositions of the invention will be superior to Enbrel + MTX in patients that do not respond adequately to methotrexate, as determined, for example, by: at 6 months, compound plus ACR20 from MTX>85% (GS: 71% for Enbrel + MTX and 27% for placebo + MTX, 59% for Humira + MTX and 24% for placebo + MTX at 12 months)^*. Other criteria for superior efficacy of the compositions of the present invention may include: similar to Enbrel, the progression of structural damage was inhibited over a one year period (Humira + MTX of 0.1 and placebo + MTX of 2.7 for mean modified Sharp score after 52 weeks)^*. In other embodiments, the composition produces a "major clinical response" superior to Enbrel in patients who have not adequately responded to methotrexate, as measured by: ACR70 (20% for Humira + MTX, 4% for placebo + MTX)^*。

In other embodiments, the compositions of the present invention may be needed to reduce signs and symptoms, induce a major clinical response and reduce the progression of structural damage in patients with moderate to severe active RA who have not adequately responded to anti-TNF drugs. Current gold standard (Goldstandard): non-anti-TNF biologic therapies. Preferably, in said individual, the composition of the invention has a potency not lower than that of a non-anti-TNF biologic therapy (e.g. Orencia, Rituxan) by historical comparison in patients who have not adequately responded to anti-TNF drugs: at 6 months, compounds plus DMARD had >50% ACR20 (GS: 50% Orencia + DMARD and 20% placebo + DMARD). In other embodiments, similar to Rituxan, the compositions of the invention inhibit the progression of structural damage over a period of one year as assessed by accepted X-ray scoring for joint erosion and articular luminal narrowing (Rituxan + MTX is 1.0 and placebo + MTX is 2.31 for the mean modified Sharp score after 52 weeks).

A variety of delivery systems are known and can be used to administer CD40 binding agents. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. CD40 binding agents can be administered, for example, by infusion, bolus injection, or injection, and can be administered with other biologically active agents, such as chemotherapeutic drugs. Administration may be systemic or local. In a preferred embodiment, administration is by subcutaneous injection. The formulation for such injection may be prepared, for example, in a pre-filled syringe, which may be administered once every other week.

The safety profile of the antibodies of the invention may be determined and preferably include one or more of the following features: no clinically significant adverse interactions with other drugs commonly used in the treatment of rheumatoid arthritis (e.g., DMARDs, steroids, NSAIDs); outage rates due to security or tolerance issues are no higher than Enbrel; the serious infection rate is not higher than that of an anti-TNF medicament or other common biological medicaments; the frequency and/or severity of injection site reactions or infusion reactions was similar to Enbrel; no or minimal (less than 5%) resistance developed after multiple treatment cycles; non-or minimally neutralizing antibodies; there is no evidence to suggest that platelet aggregation/activation that may cause thromboembolic events in vivo or platelet/endothelial dysfunction that may cause bleeding may be enhanced.

In particular embodiments, the CD40 binder composition is administered by injection, by catheter, by suppository, or by implant which is a porous, non-porous, or gelatinous material comprising membranes or fibers such as silicone rubber membranes. Typically, an anti-CD 40 antibody or a material to which the drug is not adsorbed is used when the composition is administered.

In other embodiments, the anti-CD 40 antibody or drug is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249: 1527-. In another embodiment, polymeric materials may be used. (see, for example, medical application of controlled Release, Inc. and Wise eds., CRCPRress, BocaRaton, Fla., 1974); controlledDrugBioavailability, drug product design Performance (Smolen and Ball eds., Wiley, NewYork, 1984); Range and Peppas, 1983, Macromol. Sci. Rev. Macromol. chem.23: 61. also Levy et al, 1985, Science 228: 190; During et al, 1989, an Ann. Neurol.25: 351; Howard et al, 1989, J. Neurosurg.71: 105.) other controlled release systems are discussed, for example, in Langer (supra).

A CD40 binding agent (e.g., an anti-CD 40 antibody) can be administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of the binding agent and one or more pharmaceutically compatible ingredients.

In typical embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition suitable for intravenous or subcutaneous administration to a human. Typically, the composition for injectable administration is a solution in sterile isotonic aqueous buffer. If desired, the drug may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to reduce pain at the injection site. Typically, the ingredients are supplied individually or mixed together in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent. If the drug is to be administered by infusion, it may be dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. In the event that the medicament is to be administered by injection, an ampoule containing sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.

In addition, the pharmaceutical composition may be provided in the form of a pharmaceutical kit comprising (a) a container containing the CD40 binding agent (e.g., anti-CD 40 antibody) in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent may be used to reconstitute or dilute the lyophilized anti-CD 40 antibody or drug. Optionally, the container may be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice indicates approval by the agency of manufacture, use or sale for human administration.

An effective amount of a CD40 binding agent (e.g., an anti-CD 40 antibody) in treating or preventing an immune disorder or a CD40 expressing cancer can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help determine optimal dosage ranges. The exact dosage used in the formulation may also depend on the route of administration and the stage of the immune disorder or CD40 expressing cancer and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

For example, toxicity and therapeutic efficacy of anti-CD 40 antibodies or drugs can be measured by determining ED in cell cultures or experimental animals₅₀(dose therapeutically effective in 50% of the population) by standard pharmaceutical procedures. CD40 binding agents (e.g., anti-CD 40 antibodies) that exhibit a greater therapeutic index are preferred. Given that CD40 binding agents exhibit toxic side effects, a delivery system that targets CD40 binding agents to the site of affected tissue can be used to minimize potential damage to cells that do not express CD40 and thereby reduce side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in humans. The dose of CD40 binding agent is generally at circulating concentrations with very low or no toxicity (including ED)₅₀) Within the range. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any CD 40-binding agent used in the method, the therapeutically effective dose can first be estimated from cell culture analysis. The dosage can be formulated in animal models to achieve a circulating plasma concentration range as determined in cell culture, which includes the IC₅₀(i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms). This information can be used to more accurately determine the dosage available to a person. The plasma content can be measured, for example, by high performance liquid chromatography, ELISA, or the like.

Typically, the dose of anti-CD 40 antibody or CD40 binding agent administered to a patient with an immune disorder or a CD40 expressing cancer is typically from about 0.1mg/kg to about 100mg/kg of the individual's body weight. The dose administered to the subject is from about 0.1mg/kg to about 50mg/kg of the subject's body weight, from about 1mg/kg to about 30mg/kg, from about 1mg/kg to about 20mg/kg, from about 1mg/kg to about 15mg/kg, or from about 1mg/kg to about 10 mg/kg.

Exemplary doses include, but are not limited to, 1ng/kg to 100 mg/kg. In some embodiments, the dose is about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, or about 16 mg/kg. The dose may be administered, for example, at the following frequency: daily, once per week (once a week), twice per week, three times per week, four times per week, five times per week, six times per week, once every two weeks or once a month, once every two months or once every three months. In particular embodiments, the dose is about 0.5 mg/kg/week, about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5 mg/kg/week, about 6 mg/kg/week, about 7 mg/kg/week, about 8 mg/kg/week, about 9 mg/kg/week, about 10 mg/kg/week, about 11 mg/kg/week, about 12 mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15 mg/kg/week, or about 16 mg/kg/week. In some embodiments, the dose is in the range of about 1 mg/kg/week to about 15 mg/kg/week.

In some embodiments, a pharmaceutical composition comprising a CD40 binding agent may further comprise a therapeutic drug conjugated or unconjugated to the binding agent. The anti-CD 40 antibody or CD40 binding agent may be co-administered in combination with one or more therapeutic agents for the treatment or prevention of an immune disorder or a CD40 expressing cancer. For example, the combination therapy may include cytostatics, cytotoxic agents or immunosuppressive agents. Combination therapy may also include, for example, administration of an agent that targets a receptor or receptor complex other than CD40 on the surface of an activated lymphocyte, dendritic cell, or CD 40-expressing cancer cell. Examples of such drugs include a second non-CD 40 antibody that binds to a molecule on the surface of an activated lymphocyte, dendritic cell, or CD 40-expressing cancer cell. Another example includes ligands that target such a receptor or receptor complex. Typically, the antibody or ligand binds to a cell surface receptor on an activated lymphocyte, dendritic cell, or CD 40-expressing cancer cell, and enhances the cytotoxic or cytostatic effect of the anti-CD 40 antibody by delivering a cytostatic or cytotoxic signal into the activated lymphocyte, dendritic cell, or CD 40-expressing cancer cell.

Such combination therapy administration may produce additive or synergistic effects on disease parameters (e.g., symptom severity, number of symptoms, or frequency of relapse).

For a combination treatment regimen, in a specific embodiment, the anti-CD 40 antibody or CD40 binding agent is administered concurrently with the therapeutic agent. In another specific embodiment, the therapeutic agent is administered before or after the administration of the anti-CD 40 antibody or CD40 binding agent, i.e., at least one hour and up to several months, e.g., at least one hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, or 3 months, before or after the administration of the anti-CD 40 antibody or CD40 binding agent.

Useful classes of cytotoxic or immunosuppressive agents include, for example, tubulin inhibitors, auristatins (e.g., MMAE or MMAF), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono (platinum), di (platinum), and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lecxons, nitrosoureas, cisplatin (platinols), preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids (vinca alkaloids), and the like.

Individual cytotoxic or immunosuppressive agents include, for example, androgens, Anthranomycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, thionine sulfate, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine (colchicine), cyclophosphamide, cytarabine, cytochalasin B (cytochalasin B), dacarbazine, dactinomycin (actinomycin), daunorubicin, dacarbazine (decarbazine), docetaxel (docetaxel), doxorubicin, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gramicidin D (micidagind), hydroxyurea, idarubicin, ifosfamide, irinotecan (irinotecan), lostemustine (CCC), nucloethamine, mechlorethamine, 6-mercaptopurine, 6-mercaptourea, and mecaptan, Methotrexate, mithramycin (mithramycin), mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, mithramycin (plicamycin), procarbazine (procarbazine), streptozotocin (streptozotocin), teniposide (teniposide), 6-thioguanine, thiotepa, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

In some exemplary embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB or AEVB), DNA minor groove binders (e.g., enediyne and lecxon), dacarbazine, taxanes (e.g., paclitaxel and docetaxel), puromycin, vinca alkaloids, CC-1065, SN-38, topotecan, morpholinyl-doxorubicin, rhizoxin, cyanomorpholinyl-doxorubicin, echinomycin (echinomycin), combretastatin (combretastatin), fusin (netropsin), epothilones a and B, estramustine, cryptophysins (cryptophysins), cimadrol (cemadatin), maytansinoids (maytansinoids), discodermolide (codermolide), artemisinin or mitoxantrone.

In some embodiments, the cytotoxic agent is a common chemotherapeutic agent, such as doxorubicin, paclitaxel, melphalan, vinca alkaloid, methotrexate, mitomycin C, or etoposide. In addition, effective drugs such as CC-1065 analogs, calicheamicin, maytansine, dolastatin 10 analogs, rhizoxin, and actinomycin (palytoxin) can be linked to the anti-CD 40 antibody or drug thereof.

In particular embodiments, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin 10) or a derivative thereof. Typically, an auristatin E derivative is, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF and MMAE. The synthesis and structure of auristatin E and its derivatives are described, for example, in the following documents: U.S. patent application publication Nos. 2004-0157782A1 and 2005-0238649; international patent application nos. PCT/US03/24209, PCT/US02/13435 and U.S. Pat. nos. 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, the disclosures of which are incorporated herein by reference.

In a specific embodiment, the cytotoxic agent is a DNA minor groove binding agent. (see, for example, U.S. patent No. 6,130,237). For example, in some embodiments, the minor groove binder is a CBI compound. In other embodiments, the minor groove binder is an enediyne (e.g., calicheamicin).

Examples of tubulin inhibitors include, but are not limited to, taxanes (e.g.,(paclitaxel),(docetaxel)), T67(Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other tubulin inhibitors include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), thiabendazole (nocodazole), colchicine and colchicine (colcimid), estramustine, nostoc, cerivalMaduotin, maytansinoids, combretastatin, discodermolide and punicin.

In some embodiments, the cytotoxic agent is a maytansinoid, another class of tubulin inhibitors. For example, in particular embodiments, the maytansinoid is maytansine or DM-1(ImmunoGen corporation; see also Chari et al, 1992, cancer Res.52: 127-.

In some embodiments, the therapeutic agent is not a radioisotope.

In some embodiments, the cytotoxic or immunosuppressive agent is an antimetabolite. The antimetabolite can be, for example, a purine antagonist (e.g., azathioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir (acyclovir), gancyclovir (gancyclovir), zidovudine (zidovudine), vidarabine (vidarabine), ribavirin (ribavarin), azidothymidine, cytarabine, amantadine, dideoxyuridine, iododeoxyuridine, foscarnet (posamet), or trifluridine (trifluridine).

In other embodiments, the cytotoxic or immunosuppressive agent is tacrolimus (tacrolimus), cyclosporine, or rapamycin (rapamycin). In other embodiments, the cytotoxic agent is aldesleukin (aldesleukin), alemtuzumab (alemtuzumab), alitretinoin (alitretinin), allopurinol (allopurinol), hexamethylmelamine, amifostine (amifostine), anastrozole, arsenic trioxide, bexarotene (bexarotee), carprogesterone, capecitabine, celecoxib (celecoxib), cladribine (cladribine), dalteparin alpha (darbepoteinalfa), dinil interleukin 2 (denileindifutox), dexrazoxane (dexrazoxane), drovatazone propionate, epirubicin, alfa etilinagliptin (Epoetinalfa), estramustine, exemestane (Filgrastim), fludarabine (fludarabine), valacitinib (fludarabine), valtretin (valtretin), valtretin (alfa), valtretin (alfuzumab), valtretin (doxylamine), valtretin (valtretin), valtretin (doxylamine, valtretin), valtretin (doxylamine), valnemine, valnemoramide (valtretin), valtretin (doxylamine, valtretin (e), valnemulin (doxylamine, valtretin), valbutin (e), valtrexatilin, valtretin (e), valtrexatilin, valtamicine, valtamarine), valtamarine, levamisole (levamisole), mechlorethamine (meclorethamine) or mechlorethamine (nitrogenmustard), megestrol, mesna (mesna), methotrexate, methoxsalen (methoxsalen), mitomycin C, mitotane, nandrolone phenylpropionate (nandrolone epenproxeronoate), opril interleukin (opreverlvekin), oxaliplatin (oxaliplatin), disodium pamidronate (pamidronate), pegase (pegademase), pemetrexen (pegfilgrastim), pentastatin, bronopolazine, mithramycin, porphin sodium (porfimersoium), procarbazine, quinacrine (quinacrine), labirinase (brazidine), ranitidine (rivalimide), sargasamide (sargentamicin), vincristine (vincristine), vincristolomide (vincristolomide), vincristoloside, vincristine (vincristoloside), vincristoloside (vincristoloside, vincristoloside (vincristoloside), medetoricitabine, vinpocetine, rituximab (vincristoloside), medroxithromovamycin), vincristine (vincristine), medetoricitabine, vinpocetine), medroxypromide, foscamole (vincristoloside, fosami10, vincristoloside, foscamole, rituximab (vincristolol, Vinorelbine and zoledronate (zoledronate).

In other embodiments, the medicament is a humanized anti-HER 2 monoclonal antibody; RITUXAN (rituximab; Genentech, S.S. S.F.); a chimeric anti-CD 20 monoclonal antibody; OVAREX (AltaRex, Inc., MA); PANOREX (GlaxoWellcome, NC; murine IgG2a antibody); CetuximabErbitux (Imlonesystems, NY; anti-EGFRIGG chimeric antibody); vitaxin (MedImmune, MD); campath I/H (Leukosite, MA; humanized IgG1 antibody); SmartMI95(ProteinDesignLabs, Calif.; humanized anti-CD 33IgG antibody); LymphoCide (Immunodics, NJ; humanized anti-CD 22IgG antibody); SmartID10(ProteinDesignLabs, Calif.; humanized anti-HLA-DR antibody); oncolym (Techniclone, CA; radiolabeled murine anti-HLA-Dr 10 antibody); allomone (BioTransplant, CA; humanized anti-CD 2 mAb); avastin (Genentech, CA; anti-VEGF humanized antibody); epratuzumab (Epratuzamab) (Immunomedics, NJ and Amgen, CA; anti-CD 22 antibody); and CEAcide (Immunomedics, NJ; humanized anti-CEA antibody).

Other suitable antibodies include, but are not limited to, antibodies to the following antigens: CA125, CA15-3, CA19-9, L6, Lewis Y, Lewis X, alpha-fetoprotein, CA242, placental alkaline phosphatase, prostate specific antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor antibody, P97, MUC1-KLH, CEA, gp100, MART1, prostate specific antigen, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, mucin, P21, MPG, and Neu oncogene products.

In some embodiments, the therapeutic agent is an immunosuppressive agent. The immunosuppressive agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, or methotrexate. Alternatively, the immunosuppressive agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analog (e.g., prednisone or dexamethasone).

Suitable cyclooxygenase inhibitors include meclofenamic acid (meclofenamic acid), mefenamic acid (mefenamic acid), carprofen (carprofen), diclofenac (diclofenac), diflunisal (diflunisal), fenbufen (fenbufen), fenoprofen (fenoprofen), ibuprofen (ibuprofen), indomethacin (indomethacin), ketoprofen (ketoprofen), nabumetone (nabumetone), naproxen (naproxen), sulindac (sulindac), tenoxicam (tenoxicam), tolmetin (tolmetin), and acetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol (masoprocol), phenidone (phenidone), lonapalene (lanopalen), indazolone, naphazitrom, benzofuranol, alkyl hydroxylamines) and non-redox inhibitors (e.g., hydroxythiazole, methoxyalkylthiazole, benzopyran and derivatives thereof, methoxytetrahydropyran, boswellic acid and acetylated derivatives of boswellic acid and cycloalkyl-substituted quinolinylmethoxyphenylacetic acid) and precursors of redox inhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g., phenols, propyl gallate, flavonoids and/or natural substrates containing flavonoids, hydroxylated derivatives of flavonoids, flavonols, dihydroquercetin, luteolin, galangin, olobopol (orobol), chalcone (chalcone) derivatives, 4,2',4' -trihydroxychalcone, anthranenol, N-hydroxyurea, benzofuranol, ebselen (ebselen) and reduced selenase activity enhancing substances), iron chelators (e.g., hydroxamic acid and its derivatives, N-hydroxyurea, 2-benzyl-1-naphthol, catechol, hydroxylamine, carnosol troloxC, catechol, naphthol, sulfasalazine, zileuton (zyreuton), 5-hydroxyaminobenzoic acid and 4- (omega-arylalkyl) phenyl alkanoic acids), Imidazole-containing compounds (e.g., ketoconazole (ketoconazole) and itraconazole (itraconazole)), phenothiazine, and benzopyran derivatives.

Other suitable lipoxygenase inhibitors include eicosanoid inhibitors (including, for example, stearidonic acid, eicosatetraenoic acid, docosapentaenoic acid, docosahexaenoic acid and esters thereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), veraprostol (viprostol), 15-monohydroxyeicosatetraenoic acid, 15-monohydroxy-eicosatrienoic acid and 15-monohydroxyeicosapentaenoic acid and leukotriene B5, C5 and D5), compounds that interfere with calcium flux, phenothiazine, diphenylbutylamine, verapamil (verapamil), fucoside (fuscoside), curcumin, chlorogenic acid (chlororogenic acid), caffeic acid, 5,8,11, 14-eicosatetraenoic acid (ETYA), hydroxyphenylvinylformamide, chloronaparin, heptaphylline (esculin), oxazine, xenolone, baicalein (baicalein), Propylnaproxen pyridine (proxicromil), thioether, diallyl sulfide, and bis- (1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol (calceinol), ondansilast (ontazolast), RhoerBay-x-1005, Ciba-GeigyCGS-25019C, ebselen, LeoDenmark ETH-615, LillyLY-293111, OnoONO-4057, TerumoTMK-688, Boehringer Ingleheim BI-RM-270, LillyLY213024, LillyLY 4086, LillyLY292728, OnoOLB 457, Pfizer1056, PerduederrickF 42, Rhone-PoulencRorerRP66153, SmithKlineBeechAB B-201146, SmithKlinemSB-201993, SmithKlineBeechAB-569, WarderSC-5639, WarderSC-WolK-569, Rorch SC-5669rmySC-56699, Rorch-5669rmySC-569, Rorch-56699, Rorche-Rockrex-LeurC-WO 989, Rhorex-LeurIII, RhorIII-LeurIII-LeurC-LeurIII-LR-H-125, LipyLY-5635, LiporK-LR-3, RhorC-35, RhorC-LeurC-2, LeurC-9, LeurIII-2, LeurIII-2, LeurIII-F-LeurIII-III-F-III, and its-2-F-III-2-F-III-2-III, and their derivatives, LeurIII-III, SmithKlineBeechAMSK and F-104493, LeoDenmarkSR-2566, tanabeT-757 and TeijinetEI-1338.

Article of manufacture

In another aspect, articles of manufacture comprising materials useful for treating the above-described conditions are included. The article includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition effective to treat a condition and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a humanized anti-CD 40 antibody. Indicia on or associated with the container indicates that the composition is for use in treating a selected condition. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with written instructions for use.

The invention is further illustrated in the following examples, which are not intended to limit the scope of the invention.

Examples

Example 1: generation of humanized anti-CD 40 antibodies

Murine antibodies 20E2 and 2H11 are shown in tables 1 and 2 above. Humanization of 20E2 and 2H11 clones was completed. Libraries were prepared to alter human and murine residues so that either human or murine residues may be present at any given position. This library was prepared for those amino acids that differ between human germline and murine antibodies. Only clones that retain the function of the parent murine antibody were selected.

In this way, antibodies a, B and C are humanized antibodies derived from the mouse antibody 20E2 (antibody a and B) or 2H11 (antibody C) cloned into human IgG1-KO (KO = knockout)/kappa backbone. IgG1-KO has two mutations Leu234Ala and Leu235Ala in the Fc region, thereby reducing Fc γ R and complement binding.

The results of the humanization yielded different humanized heavy and light chain variable sequences, as shown below:

SEQ ID NO:41 (variable light chain sequence):

DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO:42 (variable heavy chain sequence):

EVQLVKSGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKN

SLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO:43 (variable light chain sequence):

DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO:44 (variable heavy chain sequence):

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKN

SLYLQMNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS

SEQ ID NO:45 (variable light chain sequence):

DIVMTQSPDSLAVSLGEKVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDVAVYYCQNDYTYPLTFGAGTKVEIK

SEQ ID NO:46 (variable heavy chain sequence):

EVQLVESGGGLVKPGGSRRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKN

SLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS.

SEQ ID NO:47 (variable light chain sequence):

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO:48 (variable heavy chain sequence):

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKN

SLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO:49 (variable light chain sequence):

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDVAVYYCQNDYTYPLTFGAGTKVEIK

SEQ ID NO:50 (variable light chain sequence):

EVQLVESGGGLVKPGGSRRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO:51 (variable light chain sequence):

DIVMTQSPDSLAVSLGEKVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDLAVYYCQNDYTYPLTFGAGTKVEIK.

SEQ ID NO:52 (variable light chain sequence):

DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDF

TLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO:53 (variable heavy chain sequence):

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO:54 (variable light chain sequence):

QIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDFTLTISSL

QPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:55 (variable light chain sequence):

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLLIYSTSNLASGVPARFSGSGSGTDFTLTISSL

QPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:56 (variable light chain sequence):

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSL

QPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:57 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS.

SEQ ID NO:58 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:59 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDSKYAPKFQGKVTMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS.

SEQ ID NO:60 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWIGRIDPEDGDSKYAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:61 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQAPGQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:62 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDTKFAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:63 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDTKFAPKFQGKVTMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:64 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWIGRIDPEDGDTKFAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:65 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWMGRIDPEDGDTKFAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:66 (variable heavy chain sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQAPGQGLEWMGRIDPEDGDTKFAPKFQGKATMTADTSTS

TVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:67 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGRVTMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:68 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGRVTMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:69 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGKVTMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:70 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQAPGKGLEWMGRIDPEDGDTKYDPKFQGKATMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:71 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGKATMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:72 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQAPGKGLEWIGRIDPEDGDTKYDPKFQGKATMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:73 (variable heavy chain sequence):

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVQQAPGKGLEWMGRIDPEDGDTKYDPKFQGRVTMTADTSTD

TAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:74 (variable heavy chain sequence) 1, from antibody 10F2 Hum:

DIQMTQSPSSLSASVGDRVTITCSATSSVSYILWFQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSL

QPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:75 (variable heavy chain sequence) 2, from antibody 10F2 Hum:

DIQMTQSPSSLSASVGDRVTITCSATSSVSYILWFQQKPGKAPKLLIYSTSNLASGVPARFSGSGSGTDFTLTISSL

QPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:76 (variable light chain sequence):

QIQMTQSPSSLSASVGDRVTITCSATSSVSYILWFQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDFTLTISSL

QPEDFATYYCQQRTFYPYTFGGGTKVEIK

exemplary humanized antibodies of the invention are those having the heavy and light chain sequences set forth in the table below. The bold underlined sequences in the table below are variable domains, while the normal non-underlined sequences are constant domains:

the variable regions were subcloned into one or two different suitable IgG expression vectors:

A) human IgG1-KO (knock out)/kappa model with Leu234Ala and Leu235Ala double mutations in the Fc region to reduce effector functions such as Fc γ R and complement binding

B) Human IgG4-DM (double mutant)/kappa mode with Ser228Pro mutation in the hinge region to reduce the incidence of IgG4 half-molecule and Leu235Glu mutation to further reduce Fc γ R binding

Two candidates (antibody a and antibody B) were purified and evaluated by the following criteria:

CCF appearance (turbidity)

Filter characteristics of CCF

Yield of gamma protein A

Turbidity after elution and neutralization

Soluble aggregates (SEC)

Purity/concentration mode (SDS)

-charge mode (IEF)

Example 2: in vitro data

Antibody A, antibody B and antibody C were characterized with antibodies 4D11(Kirin/Astellas) and PG-102(Pangenetics) prepared based on the disclosed sequences. Data for antibody a, antibody B, antibody C, and 4D11 are shown below. PG-102 showed agonist activity and only incompletely inhibited B cell proliferation (not shown). Table 2.2 summary of the data obtained. The data are set forth in more detail after table 2.2.

TABLE 2.2 summary of in vitro data for antibody A, antibody B and antibody C and the 4D11 anti-CD 40 antibody to Kirin

^*SI, stimulation index;^**ratio of>1 means an increase in binding to dog relative to human.

A. Binding of humanized antibodies to cellular CD40 and recombinant CD40 proteins

The specific binding of humanized antibodies to cellular CD40 was analyzed by flow cytometry using HEK293 cells transfected with human CD 40. Concentration-dependent binding was observed for antibody a, antibody B and antibody C. The antibody shows a similar binding curve as shown in fig. 1B. The EC50 values of the antibodies of the invention and Kirin antibody 4D11 were both in the same range of about 1nM, which is most likely the sensitivity limit of the assay due to the large amount of CD40 contained in the transfected cells. The specific binding of the humanized antibody to cellular CD40 on human Ramos cells also showed concentration-dependent binding. The antibodies showed slightly different binding curves (shown in figure 2) and EC50 values between 0.21nM to 1.22 nM. No binding was detected on CD40 negative cells, e.g., untransfected HEK293 cells or T cell line HSB-2, thereby confirming selective binding to CD40 (data not shown).

The affinity of antibody a, antibody B and antibody C for binding to human CD40-Fc protein was measured via ForteBioOctet and the dissociation constant (K) was found_D)<100 pM. Kd below 100pM cannot be accurately determined due to the bivalent avidity effects of the antibody and CD 40-Fc. In addition, binding to CD40-Fc was analyzed in the absence and presence of 50% human serum, and no significant effect of serum on binding was observed (data not shown).

B. Activity of humanized antibodies in B cell activation/proliferation assays

The activity of humanized antibodies was tested in a B cell proliferation assay in which human B cells from peripheral blood were stimulated with recombinant CD40L in the presence of IL-2 and IL-4. Antibody a, antibody B and antibody C were shown to be effective in inhibiting B cell proliferation (shown in fig. 3A and 3B). Comparison with the inhibition curves and IC50 values of BI antibody and Kirin antibody 4D11 shows that the 4D11 antibody has higher potency when tested between multiple donors (fig. 3B and 4). When tested for agonistic activity in the absence of CD40L, antibodies (antibody B, antibody a, and antibody C) did not induce any B cell proliferation above background levels at concentrations up to 10 μ g/ml (67nM) similar to the 4D11 antibody (shown in figure 4).

The competing antibody 4D11 appeared to be slightly more effective with an average IC50 of about 0.02nM and no agonistic effects. Data for three BI antibodies and 4D11 are summarized in fig. 4 and table 2.2 above. Another competing antibody, PG-102 (from clone 5D12), was also tested in this assay, which showed a significant agonist effect in the absence of CD40L to stimulate B cell proliferation (fig. 4). Thus, our lead candidates are clearly distinguishable from PG-102 due to lack of agonist activity.

In a second assay, the inhibition of CD86 upregulation by antibodies was evaluated in human B cells. In this case, the assay can be performed on human whole blood or in purified B cells, both in the presence of exogenous CD 40L. Consistent with the B cell proliferation data, antibodies B, a and C tested in human whole blood were shown to be effective in inhibiting CD 40-mediated up-regulation of CD86 (shown in fig. 5), as measured by flow cytometry. Antibody C showed similar potency to 4D11 in this assay, whereas antibody B and antibody a were slightly less potent. Comparison of antibodies B and 4D11 on purified B cells or in whole blood showed that the potency of antibody B (IC50 and IC90 values) was relatively unchanged in purified B cells compared to B cells in the presence of other CD 40-containing cells or serum, while the potency of 4D11 was dramatically changed in whole blood conditions (shown in figure 6).

Similar data were obtained when whole blood samples were used to evaluate the inhibition of CD86 upregulation by antibody B, antibody a, and antibody C in cynomolgus monkey B cells (shown in figure 7). Antibody B, antibody a and antibody C tested in cynomolgus whole blood were shown to be effective in inhibiting CD 40-mediated up-regulation of CD86 as measured by flow cytometry. Thus the antibodies all showed functional cross-reactivity to cynomolgus CD40 and had similar potency to human CD 40.

Activities of antibody BIgG1KOb and antibody BIgG1WT for their ability to mediate antibody-dependent cellular cytotoxicity were evaluated (fig. 13). In this assay, RAMOS cells were incubated with human PBMC at an effector to target cell ratio of 50: 1. The amounts of antibody BIgG1KOb and antibody BIgG1WT were increased stepwise from 20. mu.g/ml and the extent of cell death was monitored by the release of LDH. Data shown are from a representative experiment. The data show that antibody AIgG1Wt20E2-12-RIgG1WT is a potent mediator of ADCC and that antibody BIgG1KOb, which contains a mutation that abrogates effector function, has no ADCC activity.

Example 3: pharmacokinetic/pharmacodynamic study

A. Intravenous administration of antibody A and antibody B in a cynomolgus monkey in a single dose of 1mg/kg or 10mg/kg

Each of antibody a and antibody B was administered intravenously at 1mg/kg and 10mg/kg to male cynomolgus monkeys (N = 3/dose). Blood samples were collected from 0-504 hours (3 weeks), serum was recovered, and samples were stored at-20 ℃ until analysis. Samples were analyzed by the sandwich ELISA described above. The serum concentration-time curves and pharmacokinetic parameters of both antibodies in monkeys after two intravenous administrations are summarized in figure 8 and table 2.7.1 (antibody a) and table 2.7.2 (antibody B) shown below. Both antibodies showed dose-dependent pharmacokinetics, indicating that at low doses, clearance is primarily due to target-mediated disposition, while at higher doses, the antibodies are cleared primarily by catabolism. Other mabs targeting membrane-bound targets (e.g., CD19, CD20, EGFR, CD146, and HER2) have been observed to have similar dose-dependent pharmacokinetic profiles. At 1mg/kg and 10mg/kg, the clearance of antibody A was 0.8mL/h/kg and 0.1mL/h/kg, respectively. At 1mg/kg and 10mg/kg, the clearance of antibody B was 0.7mL/h/kg and 0.1mL/h/kg, respectively. Similarly, at 1mg/kg and 10mg/kg doses, the half-lives of antibody a were 1 day and 13 days, respectively, and antibody B at the same dose had half-lives of 2 days and 13 days, respectively. Although antibody B had a slightly longer half-life at lower doses than antibody a at the same dose, it is not expected that this difference would be understood as longer lasting exposure after long term administration. The AUC for both compounds was super-proportional and the volume of distribution (Vss) for both compounds was similar to the plasma volume (about 40mL/kg), thus demonstrating the limited tissue distribution typically observed in large polar protein therapy. In conclusion, there was no significant difference between the pharmacokinetic parameters of the two antibodies.

Table 2.7.1: pharmacokinetic parameters of antibody a in male cynomolgus monkeys (N = 3/dose) after single intravenous doses of 1mg/kg and 10 mg/kg.

Dosage (mg/kg)	CLp(mL/hr/kg)	Vss(mL/kg)	AUC(μM.hr)	T1/2 (Tian)	MRT (sky)
						1	0.8±0.03	41±6	8.0±0.3	0.9±0.2	2.1±0.2
10	0.10±0.02	42±6	660±92	12.6±0.5	17.5±0.3

Table 2.7.2: pharmacokinetic parameters of antibody B in male cynomolgus monkeys (N = 3/dose) after single intravenous doses of 1mg/kg and 10 mg/kg.

Dosage (mg/kg)	CLp(mL/hr/kg)	Vss(mL/kg)	AUC(μM.hr)	T1/2 (Tian)	MRT (sky)
						1	0.7±0.16	40±2	10.1±2.7	1.5±0.2	2.6±0.860 -->
10	0.09±0.01	41±6	744±55	13.3±3.0	19.3±4.2

B. In vitro pharmacodynamic study

As part of the PK study described above, the pharmacodynamic effects of anti-CD 40 antibodies were analyzed. To this end, whole blood samples were incubated overnight with recombinant CD40L and the increase in CD86 expression on B cells was determined by flow cytometry. Samples were analyzed on day 0 (pre-treatment), day 2, day 7 and day 14 post-dose. Although the increase in CD86 expression was relatively small (about 5-20%), a dose-dependent effect was observed (shown in figure 9). In the group of animals given 10mg/kg of antibody a and antibody B, CD86 induction was completely inhibited on days 2, 7 and 14, which is consistent with sustained exposure at this dose. Animals given 1mg/kg showed complete inhibition on day 2, partial inhibition on day 7, and no inhibition on day 14. Loss of pharmacodynamic effects over time in the low dose group correlates with faster clearance of the antibody.

Example 4: toxicology-related studies: CD40 on platelets

CD40 is constitutively expressed on human platelets (Henn et al, 2001) and (Inwald et al, 2003), whereas CD40L is rapidly transiently expressed on the cell surface of activated platelets (Henn et al, 2001). Although it is expected that the anti-CD 40 antibody, which is free of Fc γ R binding, will not have an effect on platelets, it is important to directly confirm that this is true. Flow cytometry studies were performed to confirm the binding of anti-CD 40 lead candidates to human and cynomolgus platelets.

G28.5 and mAb89 anti-CD 40mAb were previously shown to bind to resting human platelets by flow cytometry (Henn et al, 2001). This was confirmed using FITC-labeled G28.5 antibody. 5-fold serial dilutions of G28.5 were prepared and incubated at room temperature for 30 min in 100. mu.l platelets from humans (2 donors) or cynomolgus monkeys (3 donors) in the range of 0.5. mu.g/ml to 0.32 ng/ml. In addition, anti-CD 45mAb labeled with APC was used to identify antibodies to other CD40⁺Cell type-bound platelets exclude the cells from the assay. After antibody staining, platelets were washed and fixed with OptilyseC and flow cytometry was performed. The Mean Fluorescence Intensity (MFI) was determined as the ratio to CD45^-A measure of platelet-bound antibodies.

FITC labeling was performed on commercially available 5c3 and a selected antibody anti-CD 40 mouse mAb of the invention. Binding to Ramos cells was confirmed. The number of FITC molecules per antibody molecule ranges from 2 to 4 FITC molecules per antibody molecule. 5-fold serial dilutions (ranging from 0.5 μ g/ml to 0.32 ng/ml) of commercial and candidate anti-CD 40 mAbs were prepared and incubated with human (3 donors) and cynomolgus (2 donors) platelets for 30 minutes at room temperature.

A representative graph showing binding of mouse candidate anti-CD 40mAb to human platelets is shown in figure 11. The four candidate monoclonal antibodies showed specific binding to human platelets compared to the FITC-labeled isotype control antibody. 10F2, 2H11, 19B10, and 20E2 showed binding comparable to platelets. A similar trend was observed for cynomolgus platelets (data not shown).

In addition to the studies, the ability of directly labeled antibodies B and 4D11 to bind to platelets and B cells was compared in human and cynomolgus whole blood samples (shown in figure 12). In human and cynomolgus monkey blood samples, 4D11 showed similar binding to both B cells and platelets (as exemplified by EC 50). Antibody B showed a similar pattern but the binding potency was much weaker.

Example 5: in vivo pharmacological study in NSG mouse model

The efficacy of the humanized antibody (antibody a) was evaluated in an antibody production model in which human PBMCs were injected into immunodeficient NSG mice to generate a graft-versus-host response. Significant production of human igm (hlm) and igg (higg) was detectable at the first 2 weeks after graft implantation. Treatment with antibody A at 5mg/kg and 1mg/kg significantly inhibited both hIgG and hIgM responses at weeks 2 and 3 after graft implantation. The competing antibody (4D11) was evaluated at a single dose of 5mg/kg and was also shown to abrogate the response. In a second study, all antibodies (antibody a, antibody B and antibody C) were tested in a single dose of 1mg/kg and showed complete inhibition of IgM and IgG responses at week 2 (fig. 10).

Example 6: biomarker analysis

Receptor up-regulation: CD 40L-induced receptor upregulation can be measured by flow cytometry. Human whole blood can be stimulated with optimal concentrations of soluble CD40L, and the overall percentage of CD20+ receptor + cells can be measured by flow cytometry. In parallel with the cynomolgus pk study evaluating antibodies a and B, the percent change in CD86 expression on CD20 positive cells was measured (fig. 9). The data show inhibition of CD86 upregulation at time points consistent with antibody exposure.

Targeted proteomics: the increase in protein secretion in whole blood following CD40 stimulation can be used as a potential biomarker. The Luminex multiplex bead platform for detection of MDC/CCL22 and several other secreted proteins was used to establish optimal concentrations and stimulation times for soluble CD 40L. Clinical samples from human whole blood will be evaluated with a full dose range of anti-CD 40 mAb.

Receptor occupancy: CD40 receptor occupancy can be determined in an in vitro or ex vivo assay based on a B-cell flow cytometry analysis in human whole blood. Receptor occupancy assays will be quantified using the current candidate antibody of the invention and the non-competitive anti-CD 40 antibody 5C 3.

Example 7: anti-tumor Activity of humanized anti-CD 40 antibodies

In some cases, it may be desirable to determine the anti-tumor properties of the antibodies of the invention. The assay can be performed by analyzing the anti-tumor activity of humanized anti-CD 40 antibody in a SCID mouse lymphoma xenograft model. The SCID model can be injected with cancer cells to present a tumor, e.g., 5x10 can be administered 13 days before drug treatment is initiated⁶(million) tumor cells were injected subcutaneously into SCID mice (10/group). The murine anti-CD 40 antibody of the invention or a control antibody (e.g., a control or other humanized antibody) was administered intraperitoneally 3 times per week (4 mg/kg/dose), and 8 or 5 doses were administered. The appearance and growth of tumors was monitored in mice and tumor volumes were measured weekly during selected study periods (e.g., 14 day study period). Preferably, the results will show that the tumor growth in control mice is 2,3, 4,5, 6,7, 8,9, 10 or more times that of mice treated with the antibody of the invention.Preferably, during the treatment phase, tumor growth is negligible in mice treated with the antibody of the invention. The data demonstrate that the tested humanized antibodies are effective in inhibiting tumor growth in this B lymphoma xenograft model.

Example 8: prolongation of survival by humanized anti-CD 40 antibodies

The efficacy of humanized anti-CD 40 antibodies on survival of tumor-bearing mice (such as those described above) can be analyzed in a SCID mouse lymphoma xenograft model.SCID mice (10 mice/group) are inoculated intravenously 3 days prior to antibody treatment with 1 × 10⁶(million) tumor cells. Mice were then treated with the murine or humanized anti-CD 40 antibody of the invention or Ig control, given intraperitoneally twice weekly (4 mg/kg/dose) for a total of 5 doses. Mortality in mouse cages was then examined daily to determine the degree of efficacy of the antibodies in prolonging survival of individuals with cancer.

Various references, including patent applications, patents, and scientific publications, are cited herein, the disclosures of which are incorporated by reference in their entirety. Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.

Preferred aspects of the invention can be illustrated according to the embodiments in the following paragraphs:

a humanized monoclonal antibody, wherein the antibody specifically binds human CD40, and which has antagonist activity with an IC50 of less than 1nM in B cell proliferation and no agonism at concentrations up to 100 μ g/ml, and wherein the antibody is further characterized by an in vivo half-life of at least 10 days in a non-human primate.

Paragraph 2 the humanized monoclonal antibody of paragraph 1, wherein the antibody has a half-life in cynomolgus monkey of greater than 8 days at a dose of less than 30 mg/kg.

Paragraph 3. the antibody of paragraph 1, wherein the antibody comprises a heavy chain sequence selected from any one of SEQ ID NO:1 to SEQ ID NO:4 and a light chain sequence selected from any one of SEQ ID NO:5 to SEQ ID NO: 8.

The antibody of paragraph 4. the antibody of paragraph 1, wherein the antibody is a humanized antibody or an antigen-binding fragment of an antibody having a heavy chain variable region amino acid sequence of any one of: SEQ ID NO. 1 to 4, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, or SEQ ID NO. 73.

The antibody of paragraph 5. the antibody of paragraph 1, wherein the antibody is a humanized antibody or an antigen-binding fragment of an antibody comprising the following light chain variable domain amino acid sequence: SEQ ID NO. 5 to SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 74, SEQ ID NO. 75, or SEQ ID NO. 76.

Paragraph 6. the monoclonal antibody of paragraph 1, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain CDR1 sequence is selected from seq id No. 9 to seq id No. 11, the heavy chain CDR2 sequence is selected from seq id No. 12 to seq id No. 15, and the heavy chain CDR3 sequence is selected from seq id No. 16 to seq id No. 17; and wherein the light chain CDR1 sequence is selected from SEQ ID NO:18 to SEQ ID NO:21, the light chain CDR2 sequence is SEQ ID NO:22 to SEQ ID NO:23, and the light chain CDR3 sequence is selected from SEQ ID NO:24 to SEQ ID NO: 25.

Paragraph 7. the monoclonal antibody of paragraph 1, wherein the antibody comprises heavy chain CDR1 sequence seq id No. 10, heavy chain CDR2 sequence seq id No. 13 and heavy chain CDR3 sequence seq id No. 16, and wherein the antibody comprises light chain CDR1 sequence seq id No. 19, light chain CDR2 sequence seq id No. 22 and light chain CDR3 sequence seq id No. 24.

Paragraph 8. the monoclonal antibody of paragraph 1, wherein the antibody comprises heavy chain CDR1 sequence seq id No. 9, heavy chain CDR2 sequence seq id No. 14 and heavy chain CDR3 sequence seq id No. 16, and wherein the antibody comprises light chain CDR1 sequence seq id No. 20, light chain CDR2 sequence seq id No. 22 and light chain CDR3 sequence seq id No. 24.

Paragraph 9 an anti-CD 40 antibody comprising the heavy chain variable domain sequence of any one of SEQ ID NOs 1 to 4.

Paragraph 10 an anti-CD 40 antibody comprising the light chain variable domain sequence of any one of seq id No. 5 to seq id No. 8.

Paragraph 12 a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31; SEQ ID NO. 37 and SEQ ID NO. 36; SEQ ID NO. 38 and SEQ ID NO. 36; SEQ ID NO:39 and SEQ ID NO: 36; SEQ ID NO. 40 and SEQ ID NO. 36.

Paragraph 13 an isolated antibody or antigen-binding fragment that specifically binds human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence at least 90% identical to the framework region amino acid sequence in human variable domain heavy chain amino acid sequence seq id No. 27, seq id No. 28, seq id No. 29, or seq id No. 30; and comprises a light chain amino acid sequence at least 90% identical to the corresponding light chain variable domain of SEQ ID NO. 26.

Paragraph 14. an isolated antibody or antigen-binding fragment that specifically binds human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence at least 90% identical to the framework region amino acid sequence in human variable domain heavy chain amino acid sequence seq id No. 32, seq id No. 33, seq id No. 34, or seq id No. 35; and comprises a light chain amino acid sequence at least 90% identical to the corresponding light chain variable domain of SEQ ID NO. 31.

Paragraph 15. an isolated antibody or antigen-binding fragment that specifically binds human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence at least 90% identical to the framework region amino acid sequence in human variable domain heavy chain amino acid sequence of seq id No. 37, seq id No. 38, seq id No. 39, or seq id No. 40; and comprises a light chain amino acid sequence at least 90% identical to the corresponding light chain of SEQ ID NO. 36.

Paragraph 16 the antibody of paragraph 1, wherein the antibody is incapable of stimulating cytokine production by B cells in the absence of CD 40L.

Paragraph 17 the antibody of paragraph 1, wherein the antibody binds to human CD40 in the presence of 50% human serum and the rate of binding is reduced by less than two-fold.

Paragraph 18 the antibody of paragraph 1, wherein the antibody inhibits IgM and IgG production in the mammal at a concentration of 1 mg/kg.

Paragraph 19 a method of blocking the function of human CD40 in a mammal comprising administering to the mammal a composition comprising the antibody of paragraph 1 in an amount sufficient to block a CD 40-mediated immune response in the mammal.

Paragraph 20 a method of treating or ameliorating graft-versus-host disease in a mammal comprising administering to the mammal a composition comprising the antibody of paragraph 1 in an amount sufficient to reduce one or more symptoms of graft-versus-host disease in the animal.

Paragraph 21 the method of paragraph 20, wherein the mammal has an autoimmune or inflammatory disease selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, proliferative lupus glomerulonephritis, Inflammatory Bowel Disease (IBD), psoriasis, Idiopathic Thrombocytopenic Purpura (ITP), crohn's disease and Systemic Lupus Erythematosus (SLE), hashimoto's thyroiditis, primary mucoedema, thyrotoxicosis/graves 'disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune myocarditis, addison's disease, premature menopause, type I diabetes, goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, chronic active hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus and systemic vasculitis.

Paragraph 22 the method of paragraph 19, wherein the mammal has rheumatoid arthritis.

Paragraph 23. the method of paragraph 20, further comprising administering a second therapeutic agent selected from the group consisting of: TNF antagonists, disease modifying antirheumatics, CTLA4 antagonists, anti-IL-6 receptor mAb and anti-CD 20 mAb.

Paragraph 24. the method of paragraph 20, wherein the inflammatory disease or autoimmune disease is an inflammatory disease or autoimmune disease associated with cells expressing both CD40 and CD 20.

Paragraph 25 the method of paragraph 19, wherein the anti-CD 40 antibody is administered by a parenteral route of administration.

Paragraph 26 the method of paragraph 19, wherein the anti-CD 40 antibody is administered intravenously or subcutaneously.

Paragraph 27 a method of inhibiting antibody production by B cells in a human patient comprising administering to the human patient an effective amount of the anti-CD 40 antibody of paragraph 1.

Paragraph 28 the method of paragraph 27, wherein the human patient has an inflammatory disease or an autoimmune disease associated with cells expressing CD 40.

Paragraph 29 the method of paragraph 27, wherein the human patient has an autoimmune disease selected from the group consisting of an autoimmune or inflammatory disease selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, proliferative lupus glomerulonephritis, Inflammatory Bowel Disease (IBD), psoriasis, Idiopathic Thrombocytopenic Purpura (ITP), crohn's disease and Systemic Lupus Erythematosus (SLE), hashimoto's thyroiditis, primary mucoedema, thyrotoxicosis/graves 'disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune myocarditis, addison's disease, premature menopause, type I diabetes, goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, chronic active hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus and systemic vasculitis.

Paragraph 30 a method of inhibiting growth of a cell expressing human CD40 antigen comprising administering to said cell the antibody or antigen binding fragment of paragraph 1 that specifically binds to human cell surface CD40 antigen, wherein binding of the antibody or antigen binding fragment to the CD40 antigen inhibits growth or differentiation of said cell.

Paragraph 31 a method of treating an individual having a CD 40-associated disorder, comprising administering to the individual an antibody or antigen-binding fragment of paragraph 1 that specifically binds to human CD40, wherein binding of the antibody or antigen-binding fragment to CD40 inhibits growth or differentiation of cells of the CD 40-associated disorder.

Paragraph 32 the method of paragraph 31, wherein the cells in the CD 40-associated disorder are B lymphoid stem cells, pancreatic cells, lung cells, breast cells, ovarian cells, colon cells, prostate cells, skin cells, head and neck cells, bladder cells, bone cells, or kidney cells.

Paragraph 33 the method of paragraph 31, wherein the CD 40-associated disorder is chronic lymphocytic leukemia, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's disease, Waldenstrom's macroglobulinemia or Kaposi's sarcoma.

Paragraph 34. a method of inducing clearance of peripheral B cells, comprising administering to said cells the antibody or antigen-binding fragment of paragraph 1 that specifically binds to human cell surface CD40 antigen, wherein binding of the antibody or antigen-binding fragment to the CD40 antigen induces clearance of said cells.

Paragraph 35 the method of paragraph 34, wherein the antibody or antigen binding fragment is administered to an individual suffering from an immune disorder.

Paragraph 36. the method of paragraph 34, wherein the immune disorder is rheumatoid arthritis or systemic lupus erythematosus.

Paragraph 37. a method of treating rheumatoid arthritis in an individual comprising administering to the individual the antibody of paragraph 1, wherein the antibody is an antagonist antibody that blocks CD40 function in the individual.

Paragraph 38 the method of paragraph 37, wherein the antibody is administered in an amount effective to inhibit B cell differentiation and antibody isotype switching in the individual.

Paragraph 39 the method of paragraph 37, wherein the antibody is administered in an amount effective to inhibit cytokine and chemokine production and adhesion molecule upregulation in T cells and macrophages of the individual.

Paragraph 40 the method of paragraph 37, wherein the antibody is administered in an amount effective to inhibit dendritic cell activation in the individual.

Paragraph 41 the method of paragraph 37, wherein the antibody is administered in an amount effective to inhibit down-regulation of pro-inflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandin production and adhesion molecules in non-immune cells of the individual.

Paragraph 42 the method of paragraph 37, wherein the antibody is administered in combination with a regimen comprising the administration of methotrexate and/or the administration of Enbrel/Humira.

Paragraph 43 the method of paragraph 37, wherein the subject is a subject with rheumatoid arthritis and is non-responsive to methotrexate treatment alone.

Paragraph 44. the method of paragraph 43, wherein the method comprises treating the subject with a regimen comprising administration of methotrexate and/or administration of Enbrel/Humira.

Paragraph 45 the method of paragraph 37, wherein the efficacy of treating the subject with the antagonistic anti-CD 40 antibody is superior to treatment with methotrexate alone, Enbrel alone, a combination of Enbrel + methotrexate.

Paragraph 46. the method of paragraph 43, wherein the efficacy of treating the subject with the antagonist anti-CD 40 antibody is superior to treatment with Enbrel + MTX in patients who do not respond adequately to methotrexate.

Paragraph 47. the method of paragraph 37, wherein the antibody is administered in combination with a regimen comprising an anti-TNF agent.

Paragraph 48. the method of paragraph 37, wherein the subject is a subject having rheumatoid arthritis and is non-responsive to treatment with an anti-TNF drug alone.

Paragraph 49 the method of paragraph 48, wherein the method comprises treating the subject with a regimen comprising treatment with an anti-TNF drug and the antagonistic anti-CD 40 antibody.

Paragraph 50 the method of paragraph 37, wherein the efficacy of treating the subject with the antagonistic anti-CD 40 antibody is superior to treatment with an anti-TNF drug.

Paragraph 51. the method of paragraph 48, wherein the efficacy of treating the subject with the antagonist anti-CD 40 antibody is superior to treatment with Orencia or Rituxan in patients who do not adequately respond to the anti-TNF drug alone.

Paragraph 52 a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment as in paragraph 8; and (ii) a pharmaceutically acceptable excipient.

Paragraph 53 the pharmaceutical composition of paragraph 52, wherein the antibody or antigen binding fragment thereof is conjugated to a second drug.

Paragraph 54 the pharmaceutical composition of paragraph 52, wherein the second drug is a cytotoxic agent, a PEG carrier, an enzyme, or a label.

An isolated polynucleotide encoding the heavy chain variable region amino acid sequence of any one of: SEQ ID NO. 1 to 4, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 53, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, or SEQ ID NO. 73.

Paragraph 56 an isolated polynucleotide encoding the light chain variable region amino acid sequence of any one of: SEQ ID NO. 5 to SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 74, SEQ ID NO. 75, or SEQ ID NO. 76.

Paragraph 57 use of the antibody of paragraph 1 in the preparation of a medicament for blocking the function of human CD40 in a mammal, wherein the medicament blocks a CD 40-mediated immune response in the mammal.

Paragraph 58. use of the antibody of paragraph 1 in the preparation of a medicament for treating or ameliorating graft-versus-host disease in a mammal.

Paragraph 59 the use of paragraph 58, wherein the medicament is prepared for treating an autoimmune or inflammatory disease selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, proliferative lupus glomerulonephritis, Inflammatory Bowel Disease (IBD), psoriasis, Idiopathic Thrombocytopenic Purpura (ITP), crohn's disease and Systemic Lupus Erythematosus (SLE), hashimoto's thyroiditis, primary mucoedema, thyrotoxicosis/graves 'disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune myocarditis, addison's disease, premature menopause, type I diabetes, goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, chronic active hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus and systemic vasculitis.

Paragraph 60 the use of paragraph 58, wherein the medicament further comprises a second therapeutic agent selected from the group consisting of: TNF antagonists, disease modifying antirheumatics, CTLA4 antagonists, anti-IL-6 receptor mAb and anti-CD 20 mAb.

Paragraph 61 the use of paragraph 57, wherein the medicament is prepared for parenteral administration.

Paragraph 62 the use of paragraph 57, wherein the medicament is prepared for intravenous or subcutaneous use.

Paragraph 63 use of the antibody of paragraph 1 in the preparation of a medicament for inhibiting production of antibodies by B cells in a human patient.

Paragraph 64 use of the antibody of paragraph 1 in the preparation of a medicament for inhibiting growth and/or differentiation of cells expressing human CD40 antigen.

Paragraph 65 use of the antibody of paragraph 1 for the preparation of a medicament for treating an individual having a CD 40-associated disorder, wherein binding of the antibody or antigen-binding fragment to CD40 in the medicament inhibits growth or differentiation of cells of the CD 40-associated disorder.

Paragraph 66 the use of paragraph 65, wherein the medicament is for treating a CD 40-associated disorder selected from the group consisting of: b lymphoid stem cells, pancreatic cells, lung cells, breast cells, ovarian cells, colon cells, prostate cells, skin cells, head and neck cells, bladder cells, bone cells, or kidney cells.

Paragraph 67. the use of paragraph 65, wherein the medicament is for the treatment of chronic lymphocytic leukemia, burkitt's lymphoma, multiple myeloma, T-cell lymphoma, non-hodgkin's lymphoma, hodgkin's disease, waldenstrom's macroglobulinemia, or kaposi's sarcoma.

Paragraph 68 use of the antibody of paragraph 1 in the preparation of a medicament for inducing clearance of peripheral B cells, wherein the antibody or antigen binding fragment specifically binds to human cell surface CD40 antigen, wherein binding of the antibody or antigen binding fragment to the CD40 antigen induces clearance of said cells.

Paragraph 69 the use of paragraph 68, wherein the medicament is for treating an individual having an immune disorder.

Paragraph 70 the use of paragraph 68 wherein the medicament is for the treatment of rheumatoid arthritis or systemic lupus erythematosus.

Paragraph 71 use of the antibody of paragraph 1 in the manufacture of a medicament for treating rheumatoid arthritis in an individual.

Paragraph 72 the use of paragraph 71, wherein the medicament is for inhibiting B cell differentiation and antibody isotype switching in the individual.

Paragraph 73. the use of paragraph 71, wherein the medicament is for inhibiting cytokine and chemokine production and adhesion molecule upregulation in T cells and macrophages of the individual.

Paragraph 74 the use of paragraph 71, wherein the medicament is for inhibiting dendritic cell activation in the individual.

Paragraph 75. the use of paragraph 71, wherein the medicament is for inhibiting the production of pro-inflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandins and down-regulating adhesion molecules in non-immune cells of the individual.

Paragraph 76 the use of paragraph 71, wherein the medicament is a combination medicament to be administered in combination with a regimen comprising the administration of methotrexate and/or the administration of Enbrel/Humira.

Paragraph 77 the use of paragraph 71, wherein the medicament further comprises an anti-TNF drug.

The application of the teachings disclosed herein is not limited to the specific embodiments described herein. Indeed, various modifications may be made by those skilled in the art in light of the teachings contained herein and the appended embodiments. Such modifications are intended to fall within the scope of the appended claims.

Claims (9)

1. A humanized monoclonal antibody or antibody fragment having a heavy chain variable domain and a light chain variable domain each comprising the amino acid sequences: SEQ ID NO. 27 and SEQ ID NO. 26; SEQ ID NO. 28 and SEQ ID NO. 26; SEQ ID NO. 29 and SEQ ID NO. 26; SEQ ID NO. 30 and SEQ ID NO. 26; SEQ ID NO. 32 and SEQ ID NO. 31; SEQ ID NO. 33 and SEQ ID NO. 31; SEQ ID NO. 34 and SEQ ID NO. 31; SEQ ID NO. 35 and SEQ ID NO. 31.

2. An isolated antibody or antigen-binding fragment that specifically binds human CD40, comprising

A humanized heavy chain variable domain comprising a framework region having an amino acid sequence identical to the amino acid sequence of the framework region in human variable domain heavy chain amino acid sequence SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35, and comprising a light chain amino acid sequence identical to the corresponding light chain variable domain of SEQ ID NO: 31.

3. The antibody of any one of claims 1 to 2, wherein the antibody or antigen-binding fragment of the antibody specifically binds human CD40, has an antagonist activity of IC50 of less than 1nM in B cell proliferation and no agonism at concentrations up to 100 μ g/ml, and wherein the antibody or antigen-binding fragment of the antibody is further characterized by an in vivo half-life of the antibody or antigen-binding fragment of the antibody in a non-human primate of at least 10 days; and wherein the half-life of said antibody or antigen-binding fragment of said antibody in a cynomolgus monkey at a dose of less than 30mg/kg is optionally greater than 8 days.

4. Use of the antibody of any one of claims 1-2 in the manufacture of a medicament for a method of blocking the function of human CD40 in a mammal, the method comprising administering to the mammal the medicament comprising the antibody of any one of claims 1-2 in an amount sufficient to block a CD 40-mediated immune response in the mammal.

5. Use of an antibody of any one of claims 1-2 in the manufacture of a medicament for use in a method of treating or ameliorating a disease or disorder in a mammal, the method comprising administering the medicament to the mammal, the medicament comprising the antibody of any one of claims 1-2 in an amount sufficient to reduce one or more symptoms of the disease or disorder in the mammal, wherein the disease or disorder is selected from lupus nephritis and rheumatoid arthritis.

6. The use of claim 5, further comprising administering a second therapeutic agent of choice.

7. The use of claim 5, wherein the antibody is administered by a parenteral route, intravenous route, or subcutaneous route.

8. A pharmaceutical composition comprising (i) the antibody of any one of claims 1-3; and (ii) a pharmaceutically acceptable excipient; and wherein the antibody or antigen-binding fragment thereof is optionally conjugated to a second drug.

9. An isolated polynucleotide encoding a heavy chain variable region amino acid sequence or a light chain variable region, wherein the heavy chain variable region amino acid sequence comprises any one of: 27, 28, 29, 30, 32, 33, 34, 35; and the light chain variable region amino acid sequence comprises any one of: SEQ ID NO. 26 and SEQ ID NO. 31.